Development of lectins using protein engineering methods

Abstract

Provided are modified glycosylating enzymes with altered enzymatic activity and / or altered glycan binding properties, such as reduced enzymatic activity but stronger glycan binding properties than unmodified proteins or proteins of the same type with different modifications, and enzymes with greater activity than natural occurring entities. The modified glycan binding proteins and enzymes can be provided as fusion proteins. A process for generating and identifying new glycan binding proteins and novel glycosylating enzymes is also provided.

Description

Attorney Docket No.: 011520.01969DEVELOPMENT OF LECTINS USING PROTEIN ENGINEERING METHODSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. provisional application no. 63 / 684,349, filed August 17, 2024, from which the entire disclosure is incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under grant numbers GM139160 and HL103411 awarded by National Institutes of Health. The government has certain rights in the invention.BACKGROUND OF THE DISCLOSURE

[0003] Glycosylation is a ubiquitous post-translational modification that regulates diverse biological processes including molecular recognition, cell adhesion, and signaling1,2. Glycan structure changes also serve as biomarkers of cell differentiation and disease3,4. Due to these basic science and translational roles, the measurement of cell surface glycans has broad biomedical importance. Such measurements are, however, complicated due to the stereochemistry and branched nature of glycans. While mass spectrometry is well suited to identify glycan compositions and topologies particularly for abundant carbohydrate entities, detailed structural analysis and linkage specification is challenging, and low abundant entities are missed. Complementary molecular tools that recognize specific glycan epitopes or substructures associated with biological transformation are thus needed.

[0004] Lectins derived from prokaryotes and eukaryotes have been used for glycan recognition in biochemical assays5. These glycan-binding proteins (GBPs), however, often lack binding specificity in that they commonly bind degenerate terminal epitopes, and currently available lectins do not cover all known glycan epitopes. Carbohydrate-binding modules (CBMs) are well-known GBPs that are part of glycan- processing enzymes that bring enzymes and substrates together to promote reactions. However, the CBMs usually recognize glyco-homopolymers or terminal monosaccharides, and they lack the ability to bind more complex structures6,7. Anti-carbohydrate antibodies have been developed as an alternative, but low immunogenicity of carbohydrate antigens makes it challenging to derive monoclonal antibodies (mAbs) against arbitrary glycan epitopes8,9. Protein engineering approaches for GBPs have been attempted to engineer nature-derived GBPs to increase affinity or to convert specificity to related glycan epitopes10,11. However, these attempts havesuffered from promiscuous binding to other glycan structures and reduced affinity. Shallow interaction interfaces of these nature-derived GBPs can also promote rapid dissociation of bound glycans due to competition with other molecules such as water. Finally, bacterial adhesin containing Siglec-like domains have been prepared as sialoglycans, and these broadly recognize Neu5Ac(a2-3)Gal epitopes and related epitopes12,13.

[0005] Besides naturally occurring lectins, attempts have been made to endow other generic proteins with glycan-binding properties. For example, the DNA-binding protein, Sso7d, was engineered through directed evolution to bind Thomsen-Fridenreich (TF) antigen. However, the engineered protein exhibited cross reactivity with unknown ligands14. Type B lamprey variable lymphocyte receptors (VLRBs), lambodies, were also engineered with some success to develop GBPs, but the ability of the leucine-rich zipper to bind arbitrary human glycan structures remains unknown15,16, particularly as these are products of broad screening efforts rather than rational, predictive design. Overall, as the binding pockets of generic proteins are not naturally optimized for carbohydrate recognition, engineering them as GBPs can result in low affinity, non-specific binders. Thus, additional scaffolds with deep and extended glycan binding pockets would be desired. The present disclosure is pertinent to these and related needs.BRIEF SUMMARY

[0006] The present disclosure relates to engineered GBPs, method of making engineered (e.g., mutated GBPs), methods of screening engineered GBPs to identify engineered GBPs that exhibit desirable target binding properties, fusion proteins comprising mutated GBPs, and methods of using the GBPs for a variety of diagnostic and therapeutic purposes.

[0007] The disclosure relates in part to a fusion protein that was created with human IgGl Fc N-terminally linked to pig ST3Gall (abbreviated PSI). This protein exhibited enzyme activity but only weak binding to O-glycans. Introduction of mutation in this construct resulted in loss of enzymatic activity but strong binding preference for sialoglycans in multiple cell types. By performing a broad CRISPR-Cas9 based library screen, studies with a panel of isogenic glycogene knockouts, and glycan microarray studies, we determined that a mutation described herein at H243 A (histidine to alanine at position 243) specifically binds a(2-3)sialylated core-2 O-glycans. Additionally, its binding specificity was distinct from other known sialic acid binding lectins. To expand the repertoire of lectins, a novel mammalian cell surface display platform was developed, and this was used to screen foradditional H243A variants. Using a rationally designed mutant library with 1680 candidates, we identified a triple mutant of PSI, sCore2, containing H243A, A253I and F254S mutations that displayed better binding properties than H243 A for sialyl-core 2 O-glycans. In spectral flow cytometry studies that analyzed glycan profiles of 35 immune cell types, sCore2 exhibited strong binding for neutrophils, basophils, monocytes, and terminally differentiated NK- and T-cells, suggesting that sialyl-core 2 O-glycan expression is abundant in terminal effector cell types. In human tissue microarrays, sCore2 stained normal spleen and breast cancer tissue, confirming the reagent utility in paraffin embedded sections. Thus, the disclosure includes mutated version of ST3Gall, and other described proteins, as a component of a fusion protein, and a streamlined approach to generate glycan binding proteins based on the known substrate specificity of the starting glycosyltransferases, as well as the aforementioned methods for using the described proteins for diagnostic and therapeutic purposes.

[0008] As extension of the above the disclosure also describes that the surface displayed enzymes can enable the screening of the glycosylating enzymes, allowed for the screening of glycoenzymes from multiple families, multiple species and the identification of protein engineered enzymes with higher activity than naturally occurring counterparts. Thus the technology and methodology described in this disclosure could be used for detailed structure-function analysis of mammalian glycoenzymes in high-throughput manner.BRIEF DESCRIPTION OF THE FIGURES

[0009] For a fuller understanding of the nature and objects of the disclosure, reference should be made to the following detailed description taken in conjunction with the accompanying figures.

[0010] Fig. 1 displays that Fc-pST3Gall H243 mutant exhibits sialic acid dependent binding but lacks enzyme activity, (a) Fc-pST3Gall (‘PSI’) has four structural motifs. Amino acids shown in cyan (Q49A, Y174A, and Y210F) and magenta (H243A) were mutated in ‘Dead’ and ‘H243A’ mutants, respectively, (b) pST3Gall co-crystalized with CMP and Gaipi,3GalNAca-pNP (PDB: 2WNB). Protein is in green, with selected residues and ligands shown as sticks. CMP is orange, Gaipi,3GalNAca-pNP is yellow, amino acids mutated in Dead mutant are cyan, and H243A is magenta, (c) Western blots of Fc-fusion proteins expressed using HEK293T cells under reducing and non-reducing conditions. Detection was done using anti-human Fc specific IgG. (d) LC-MS / MS analysis of ST3Gall enzymatic activity. Commercial rhST3Gall served as positive control. H243A and dead mutants did notyield product, (e) Lectin binding to HEK293T (gray bars), COLO357-FG (green bars) or Calu-3 (blue bars) cells, with or without sialidase treatment, measured using flow cytometry. Cells were incubated with 5pg / mL fluorescent MALII, PNA, or I pg / mL purified Fc-fusion proteins pre-complexed with AF647- conjugated anti-human Fc specific IgG. Data are mean + STD (N=3). *p<0.05 with respect to all other samples; Jp<O.O5 with respect to all other samples except samples marked by { are not different from each other, (f) Dose dependent binding of PSI and H243A pre-complexed with AF488-conjugated anti-human Fc specific IgG. HEK293T cells treated with or without sialidase were used. Data are mean + STD (N=2).

[0011] Fig. 2 displays that H243A binds sialylated core-2 O-linked glycans. (a) Isogenic HEK293T KO cells were produced lacking selected gly can-processing enzymes or transporters, (b) Next-generation sequencing (NGS) results for isogenic clones lacking GCNT1, SLC35A1, and ST3Gall. Wildtype DNA and amino acid sequence corresponding to sense strand are shown above, with gene edited sequence below. Exons and introns appear in uppercase and lowercase, respectively. As two sgRNA were added simultaneously to create KOs, red and green represent the first and second editing regions, and blue presents the protospacer adjacent motif (PAM) on sense (GCNTl and ST3Gall KO) or anti-sense strands (SLC35A1 KO). The entire sequence between the sgRNA cut sites was excised in the GCNT1 and SLC35A1 KO. This resulted in a few different closely- resembling read sequences. The frequency of occurrence of each of these edit patterns is shown on the right side of the DNA sequence (underlined), (c) Flow cytometry binding assay using 5pg / mL fluorescent-lectins (MALII and PNA for O-glycans, PHA-L and ECL for A-glycans). HEK293T WT cells and panel of isogenic HEK293T KO cells were used, (d) Flow cytometry binding assay using Ipg / mL purified Fc-fusion proteins pre- complexed with AF488-conjugated anti-human Fc specific IgG. Data are mean + STD (N=5-6). Data show that H243 A binds sialylated core-2 O-linked glycans. *p<0.05 with respect to all other samples; ip<0.05 with respect to all other samples except that samples marked by1are not different from each other; tp<0.05 with respect to WT cells.

[0012] Fig. 3 displays that H243 A displays strict binding specificity for sialylated core-2 motif, (a) Schematic of glycan microarray study performed using CFG 5.5 with 562 immobilized glycans. PSI or H243A were added to microarray at either 5pg / mL (supplemental data) or 50pg / mL, followed by detection using fluorescent anti- human Fc specific IgG (‘FL-anti Fc’). (b-c) Lectin binding quantified using relative fluorescence units (RFU) for PSI (panel b) and H243 A (panel c). Data are mean + STD (N=6). Red arrows withglycan symbol and number represent strong binders. * Glycan 107 is non-specific binder, as its binding is Fc-protein dose independent, (d) Non-binders of H243A and PSI are shown. Neither Fc-fusi on proteins recognized a2,3-sialyl core-1 O- glycans (219 and 220), a2,6- sialyl core-1 O-glycans (134, 135, 238, and 239), or core-2 O-glycan with a2,3- sialyl LacNAc chains (281, 309, 326 and 560), or a host of N-glycan and glycolipid structures, (e) HL60 WT cells were cultured with sodium chlorate to prevent sulfation, prior to measuring the binding of Ipg / mL Fc- fusion protein, pre-complexed with AF647-conjugated anti-human Fc specific IgG. P-selectin Fc binding, but not H243A or PSI binding, was chlorate sensitive. Results confirm sialylated core-2 glycan binding specificity of H243A. Data are mean + STD (N=3). tp<0.05 with respect to no sodium chlorate treatment.

[0013] Fig. 4 displays that forward genetic CRISPR-screen identifies glycoEnzymes regulating PSI and H243A binding, (a) Anti-human Fc antibodies were covalently coupled to epoxy-functionalized magnetic beads. The beads were added to HL60 glycoCRISPR library cells that were pre-incubated with PSI or H243A. Cells binding the Fc- proteins were captured onto magnetic beads whereas the non-binders remained in solution. These unbound cells were isolated, expanded, and subjected to two more round of magnetic enrichment. Genomic DNA from the third round was isolated and sequenced to determine sgRNA and corresponding genes regulating Fc- protein binding, (b, c) Flow cytometry histograms on left side show the HL60 glycoCRISPR library binding profiles for H243A (panel b) and PSI (panel c) before 1st sort, after 3rd sort and negative control without Fc- protein. SgRNA enrichment plots on the right side identify genes whose depletion by CRISPR-Cas9 also reduced cell-lectin conjugate capture by magnetic beads for H243A (panel b) and PSI (panel c). Glycogenes critical for lectin binding (FDR<0.005) were labeled in the figure, along with the prototypic glycan they synthesize.

[0014] Fig. 5 displays that novel human surface display platform enriches H243 A binding properties, (a, b) Fusing the cytoplasmic and transmembrane domain of type-II transmembrane protein DPP4 at the A-terminus of Fc- proteins (H243 A in this example) enabled a novel mammalian surface display platform. Amino acids mutated to create Libi and Lib2 were shown in cyan and magenta, respectively in panel a. (c, d) HEK293T WT cells were transfected to surface display TM-PS1, TM-H243A and TM-Dead. Cells were treated with sialidase to eliminate cv.s-in teractions (panel c). AF647-conjugated goat anti-human Fc specific IgG was used to monitor Fc expression level. Cells with high Fc expression were gated and used in the histogram in panel d as a measure of lectin binding with sialyl core-2 PAA-FITC. (e) Amino acid residues mutated in Libi and Lib2 are shown in Alphafold modelof pST3Gall (green) and its crystal structure (PDB: 2WNB, gray) co-crystalized with CMP (purple stick) and Gaip i ,3GalNAca- NP (yellow stick). These residues, which are represented as cyan (Libi) and magenta (Lib2) sticks, were mutated for optimizing the glycan binding properties of H243A. Neu5Ac superimposed from 5FRE were shown as orange sticks, (f) Sialyl core-2 PAA-FITC binding to HEK293T SLC35A1-KO- cells transduced to express TM-H243A, Libi, and Lib2. (g) Identification of top-6 mutants in Lib2 that are superior binders of sialyl core-2 PAA-FITC. (h) Western blots of expressed mutants identified by library screening. Three of them expressed well, (i) 5pg / mL purified Fc-fusion proteins pre-complexed with AF488-conjugated anti-human Fc specific IgG were incubated with a panel of HEK293T cells (wild-type and KO). All Fc-proteins bound in a sialyl core-2 dependent manner, (j) 5pg / mL purified Fc-fusion proteins were pre-complexed with AF488- conjugated anti-human Fc specific IgG, and incubated with HEK293T cells, with or without sialidase treatment. H243A / A253I / F254S (‘sCore2’) bound HEK293T cells at levels 3-fold greater than H243A. (k) Dose dependent binding of H243A and H243A / A253I / F254S (sCore2) pre-complexed with AF488- conjugated anti-human Fc specific IgG to HEK293T cells. sCore2 showed stronger sialic acid binding preference. Dotted line represents the ratio of 1. All data are mean + STD (N=3 for all, except N=2 for dose dependence study). *p<0.05 with respect to all other samples; ^<0.05 with respect to all other samples except that samples marked by1are not significantly different from each other.

[0015] Fig. 6 displays application to cell-surface glycan detection of human peripheral blood samples and tissue sections from a variety of organs, (a) tSNE plots showing the binding of PNA, MALII and sCore2 to different human blood cell types, immunoprofiled using 23 antibodies. Representative data are shown for a single donor. Legends show numbers and color labels for each cell population in tSNE plot, (b) Heatmap showing the average normalized binding of each lectin to different human blood cell types, based on studies with two donors. Heatmaps are arranged based on cell lineage. Some cell types from heatmap do not appear in tSNE plots as these are rare populations, with small number of cells, (c) Human normal tissue microarray and breast cancer tissue analysis using sCore2. Staining of selected cores are shown, along with higher magnification tissue sections. Data for negative controls using ‘Dead’ Fc-protein and upon sialidase treatment are presented in Supplemental Material. Results show cell specific staining properties of sCore2.

[0016] Fig. 7. Displays large binding interface of glycosyltransferases, (a-j). Cartoon representation of glycan- related proteins co-crystalized with glycan ligands and molecular surface area calculation of binding interface between proteins and glycan ligands. Ligandsused for defining binding interfaces were represented as yellow sticks, except for overlayed sialoglycan in panel b with blue sticks. Whole protein was shown as green cartoons. Binding interfaces were shown as surface. Since pST3Gall possesses an undetermined disordered chain that covers bound glycans, our calculations likely under-estimate the binding surface area of pST3Gall. Although Neu2 also had a deep binding pocket for DANA, the pocket is optimized to recognize the monosaccharide rather than longer sugar chains, (k). Table summarizes the measured surface area / binding interface between the depicted mammalian proteins and their glycan ligands.

[0017] Fig. 8 displays an Fc-protein sequence for Fc-pST3Gall WT (‘PSI’), Fc- CBM40, Fc-diCBM40, and cell- surface displayed Fc-pST3Gall WT (‘TM-PS1’). (a) the VWF signal peptide (yellow), 6xhis tag (blue), 19 amino acids of PSGL-1 (green), TEV cleavage site (plain) and human Fc (gray) preceded the target protein. Truncated pST3Gall (A59) appears in magenta in panel a. Amino acids substituted in H243 A and Dead (Q49A / Y174A / Y210F) are shown using underlined gray and yellow letters, respectively, (b)- (c). CBM40 sequence in single (panel b) or concatenated form (panel c) are highlighted in red. (d) In ‘TM-PSF, the DPP4 cytoplasmic tail (yellow) and transmembrane region (green) precede human Fc (gray) and truncated pST3Gall wild-type (A59, magenta). Amino acids mutated in Libi and Lib2 are shown using yellow and gray underlined fonts (within magenta highlights).

[0018] Fig. 9 displays Fc-protein expression, enzyme activity and sialic acid dependence (a) Expression of PSI and variants in HEK293T wild-type versus CIGalTl-KO. HEK293T WT (wild-type) and HEK293T CIGalTl-KO were used to express PSI, H243A and Dead. Flow cytometry FLIC A assay was used to quantify Fc-fusion protein production in culture supernatant, (b) MS / MS spectra of donor (CMP- Neu5 Ac, top), acceptor substrate (Gaipi,3GalNAca- / ?NP, middle), and product (Neu5AcaGaipi,3GalNAca- / ?NP, bottom), (c) Flow cytometry measured the binding of fluorescent lectins or Fc-fusion protein (precomplexed with fluorescent anti-human Fc specific IgG) to HEK293T, COLO357- FG, or Calu-3 cells, in the presence or absence of sialidase. (d) While studies in panel c used AF647 conjugated anti-human Fc, similar studies were performed with AF488-conjugated secondary Ab for reconfirmation. Strong sialic acid dependent binding was observed for H243 A using both secondary Abs. Data are mean ± STD (N=3). * p< 0.05 with respect to all other samples. ^<0.05 with respect to all other samples except that samples marked by1are not significantly different from each other; tp<0.05 with respect to the same Fc-pST3Gall variant samples expressed with HEK293T WT cells.

[0019] Fig. 10 displays CFG microarray binding data for 5 pg / mL Fc-fusion proteins. (a)-(b). Lectin binding quantified using relative fluorescence units (RFU) for 5 pg / mL PSI (panel a) and H243 A (panel b). Data are mean ± STD (N=6). Red arrows with glycan symbol and number represent strong binders. * Glycan 107 exhibits non-specific binding as its signal does not increase upon increasing Fc-protein concentrations, (c)- (f). Glycan microarray analysis of biotinylated MALII (panels c, e at two concentrations) and PNA (panels d, f) using CFG glycan microarray (Data source'. National Center for Functional Glycomics (NCFG) website). Strong binders, detected using AF488-conjugated streptavidin, are marked by blue arrows. Binding properties of PSI and H243A are vastly different from the commercial lectins. Note that different glycan microarrays were used for a-b vs. c-f, and thus glycans and chart ID values do not necessarily match.

[0020] Fig. 11 displays heterotypic cell binding between ligand and surface-display cells. HEK293T WT cells were labeled with CellTracker Green, and either sialidase treated (third column) or not treated (second column). HEK293T WT cells were transfected with TM-PS1, TM-H243A, or TM-Dead and labeled with CellTracker Orange (along rows). These were either sialidase treated or not treated. Upon mixing the same number of two cell populations for 20min at RT, heterotypic green-orange cell aggregates were only observed upon mixing HEK cells without sialidase (labeled green) with HEK cells bearing TM-PS1 or TM-H243A that were sialidase treated. In the case of TM-PS1 background 1.7% binding was increased to 3.9% upon sialidase treatment. For TM-H243A binding increased from 2.5% (without sialidase) to 6% (with sialidase). In negative controls, neither TM-Dead nor Mock transfected cells displayed such increase. Additionally, removing sialic acid from HEK293T / green cells reduced heterotypic cellular interactions to 1.5% (baseline). Thus, cis- interaction occur between TM-PS1 / H243A and host sial ogly cans. This prevents heterotypic cell interactions unless cells are sialidase treated. Similar to this, cv.s-in teractions may prevent surface-displayed Fc-proteins from binding the sialyl core-2 PAA-FITC probe. This is prevented by expressing surface display proteins on SLC35A1-KO- cells that lack sialic acids.

[0021] Fig. 12 displays lectin surface display library construction, (a) Cloning strategy for constructing Libi and Lib2. Gene of interest is shown in green. Vector was first linearized using PCR primers that flank the insert site (red and blue). Two-step PCR was performed to introduce mutations. In the 1st PCR, the forward primer contained NNK mutations (shown as green, pink, yellow stars) while the reverse primer overlapped with the linearized plasmid. This product was used as a megaprimer in the 2nd PCR along with aforward primer that overlapped with the linearized vector. The final PCR product was then ligated with the linearized vector using Hifi cloning. As all mutagenized primers have 1-2 NNK sites, resulting plasmid is the mixture of plasmids with 1 or 2 mutations, (b) Mutation profiles of Libi and Lib2. The amplicon was generated from the mutagenized plasmids and subjected to NGS (next-generation sequencing). Nucleotides corresponding from L76 to SI 47 were included in the amplicon of Libi, and ones from L204 and T269 were in the amplicon of Lib2. Each nucleotide (A, T, C, and G) was shown in green, red, blue, and yellow, respectively. Mutagenized residues were pointed with arrows and yellow highlights. Because Libi and Lib2 are the mixture of plasmids with one or two mutations, each component was extracted in NGS read processing by fixing the mutagenized nucleotides to the original nucleotide sequences except for the mutations of interest. Logos of extracted reads were shown below the blue arrow.

[0022] Fig. 13 displays construction, sorting, and characterization of selected mutants using TM-H243A Libi and Lib2. (a) Lentivirus transduction and FACS sorting of HEK293T Libi and Lib2. Histogram demonstrates that less than 30% of the cell population was lentivirally transduced ensuring M.O.I. (multiplicity of infection) < 1. FACS sort enrichment of transduced cells was performed based on cell-surface Fc-protein detection, (b) FACS sorting strategy using sialyl-core 2 PAA-FITC for HEK293T Libi and Lib2. Sorting gates were shown in contour plots of Libi 1stsort and Lib2 1stand 2ndsorts based on biding of Fc positive cells to sialyl core-2-PAA. Also shown is the negative sort which encompasses the non-binders. Percentage of quadrant 2 (Q2) was shown for each plot, (c) Positive enrichment scores of mutants in Libi. Only one positive sort was performed for Libi as the binding of sialyl core-2-PAA to these cells was low, whereas two positive sorts were done for Lib2. (d) Sialidase dependence of selected mutants. Flow cytometry histograms showing binding of purified Fc-proteins, pre-complexed with AF488-conjugated anti-human Fc specific IgG to HEK293T cells treated with or without sialidase. Data are representative of triplicate runs.

[0023] Fig. 14 displays spectral flow cytometry analysis of sCore2 and lectins, (a) Lack of competition between commercial lectins and sCore2. Histograms confirm lack of competition between PNA, MALII and sCore2 / Dead in cell binding assay using peripheral blood cells. Histograms are shown for granulocytes, lymphocytes and monocytes based on FSC and SSC gating and for blood donor 1. Note the presence of multiple sCore2 binding populations in the lymphocyte gate. Representative data from donor 1 is shown, (b) tSNE plots showing the different binding patterns of PNA, MALII and sCore2 for different human peripheral blood cells. Representative data are shown for blood cells from donor 2,immunoprofiled using 23 antibodies against human antigens. Numbers and colors in legends correspond to individual labeled cell populations in the tSNE plots. PNA bound to myeloid cell types except for basophils, MALII showed higher binding to lymphoid cell types than myeloid cell types, and sCore2 bound to myeloid cell types and terminally differentiated lymphoid cells, (c) Heatmaps showing the binding of each lectin for human peripheral blood cells from individual donors. Results from donor 1 only, donor 2 only, and both donors were visualized. The Order of heatmaps are aligned based on the cell type (donor 1 or 2 only) or the binding intensity of sCore2 (combined), (d) Binding of Dead to peripheral blood cells was low. tSNE plots show low binding of Dead to different human peripheral blood cell types.

[0024] Fig. 15 display human normal tissue microarray stained using sCore2 or Dead, (a) Human normal tissue microarray and breast cancer tissue analysis was performed using Dead or sCore2. Treatment of tissue with sialidase prevented sCore2 binding, while strong sCore2 binding was noted in absence of sialidase in Fig 6 (main manuscript). Binding using the ‘Dead’ negative control reagent was also low. Note that red stains in seminal vesicle preexists even before deparaffinization. (b) Human normal tissue microarray analysis. Several tissues with moderate staining by sCore2-sialidase were shown individually along with negative controls.

[0025] Fig. 16 displays a cartoon representation of pST3Gall and effects of specific amino acids, mutations, and overlayed ligands, (a) Y135 in pST3Gall in proximity to carboxyl group in superimposed Neu5 Ac in sialoglycan ligand, (b) Superimposed core 2- GlcNAc accepted in the pocket of pST3Gall. (c) Superimposed a2,6-Neu5Ac. Glycerol-like side chain was hidden by pST3Gall shown as surface, indicating that steric hinderance may be caused, (d) R50 and R209 potentially interacting with sulfate group of sulfated sialyl TF- antigen identified in glycan microarray. These Arginines are considered to be flexible, and so they can be directed towards the proximal sulfate group, (e) Unmutated F254 in sCore2 is located near Neu5 Ac. (f) F254S potentially interacting with C5 acetamido group in Neu5 Ac. pST3Gall co-crystalized with CMP and Gaipi,3GalNAca- / ?NP (PDB: 2WNB) and predicted by Alphafold were shown in cyan and green cartoons, respectively. In panels b-d, crystal structure of pST3Gall was shown as surface. Y135, A312, and F313 were shown as orange sticks. Gaipi,3GalNAca- / ?NP, Neu5Aca2,3Gal superimposed from 5FRE, and (6S)GalNAc superimposed from 6S20 were shown as cyan, magenta, and yellow sticks, respectively. GlcNAcpi,6GalNAc superimposed from 6K2N and Neu5Aca2,6Gal superimposed from 3PHZ were shown as white sticks. Potential interactions between atoms in residues andligands are shown as yellow dotted lines. In Fig. b-d, dihedral angles of several bonds were adjusted so the ligand fits in pST3Gall with better conformation.

[0026] Fig. 17 displays ST3Gall activity measurement using surface displayed enzyme, (a) Cytoplasmic and transmembrane domains of CD94 and DPP4 were fused at the N-terminus of Fc-pST3Gall to enable surface display of pST3Gall. C-terminal FLAG tag was included in some cases, (b) Flow cytometry analysis of HEK293T WT and ST3Gall KO cells stained with MALII and PNA. Knocking out ST3Gal-I decreases MALII binding and augments PNA recognition as seen in the shift of cells relative to the fixed diagonal line, (c) Three different TM-Fc-pST3Gall fusion proteins were transiently surface-displayed on HEK293T ST3Gall KOs. Pl - P4 gates were set based on human IgG Fc or FLAG expression, with P4 including the top 20% of cells. TM(DPP4)-PS1 and TM(DPP4)-PS1- FLAG were active as they restored MAL-II binding and decreased PNA binding to levels seen in wild-type cells. TM(DPP4)-H243 A was catalytically inactive, (d) Quantitative comparison of enzymatic activity in different Fc-pST3Gall variants on MALII (purple bars) and PNA (yellow bars) binding. All data except Mock are based on P4 gate, (e) Quantitation of enzymatic activity based on MALII binding for P1-P4 cell populations with different levels of Fc or FLAG expression. Data are mean + standard deviation for n=3-6. ip<0.05 with respect to all other samples except that samples marked by1are not significantly different from each other.

[0027] Fig. 18 displays activity measurement of surface-displayed ST3Gall using click-chemistry, (a) Cells displaying TM-PS1 and variants on the surface were treated with sialidase to expose the acceptor substrates. The donor CMP-Neu5Ac,9Az was generated in situ using recombinant CMP-sialic acid synthase (NmCSS), CTP and Neu5Ac,9Az. Neu5Ac,9Az from donor was transferred to cell-surface acceptors by surface displayed enzymes. Azido groups are then functionalized using either DBCO-AF488 or DBCO-biotin, with the latter being detected using fluorescent anti-biotin. (b) Activity of surface displayed TM-PS1 and variants measured using flow cytometry using either DBCO-AF488 (top) or DBCO-biotin followed by AF488-anti-biotin (bottom). Only the active enzyme TM(DPP4)- PS1 showed increased DBCO-AF488 and higher anti -biotin AF488 binding. Cell fraction with high human IgG Fc expression are colored in blue, (c) Quantitation of enzymatic activity of surface displayed pST3Gall variants. All data except for Mock are quantified using Q1+Q2 quadrants with high Fc expression, (d) All reagents are necessary to observe AF488 signal derived from enzymatic activity. Cells with high Fc expression were used forquantitation. In all panels, data are mean ± standard deviation of triplicate. *p<0.05 with respect to all other samples.

[0028] Fig. 19 displays that pST3Gall mutant screening identifies mutations that either enhance or abrogate ST3Gall enzyme activity, (a) Libi and Lib2 mutations were introduced around the ligand-binding pocket of TM(DPP4)- PSI -FLAG. After lentiviral transduction into ST3Gall-KO at low MOI, FACS sorting was performed on Fc- positive cells, using either MALII or click chemistry-based methods with DBCO-biotin and antibiotin for selection of specific sub-populations. Genomic DNA from parent cells, activitynegative cells from 1stFACS (1stNeg), and activity-positive cells from 1stand 2ndFACS (1stPos and 2ndPos) were subjected to NGS analysis. Enrichment value (EV) for each cell population was calculated, and these were combined into Enrichment score (ES) to evaluate the impact of site-specific mutations. MALII and click chemistry experiment data were independently evaluated to check for consistency across methods, (b) ES heatmap of Libi (left) and Lib2 (right) sorted using MALII (top) or DBCO-biotin + anti-biotin (bottom). Mutations that enhance enzyme activity have ES>0 (red shades), while inactivating mutations have ES<0 (blue shades). Mutants which were completely depleted in 1stPos, 2ndPos, or 1stNeg were shown in white. Amino acids shown on the left axis correspond to X shown on the bottom axis label, (c) Heatmap showing Pearson correlation coefficients between Enrichment values and Enrichment scores, (d) Volcano plot of TM-PS1 mutant library. Fraction of reads of parent cells and 2ndPos cells were used to calculate fold changes and / ?-values. Six K256X-related mutants showing high fold changes and p- values were selected for further investigation as potentially better mutants. Note: numbering of animo acids in Fig. 19 is based on full length ST3Gall. To obtain numbering based on SEQ. 02, subtract 59 bases.

[0029] Fig. 20 displays the characterization of selected pST3Gall mutants for high enzymatic activity, (a) Western blot analysis of pST3Gall mutants identified from pST3Gall library before purification, (b) Silver stain analysis of pST3Gall mutants following purification, (c) Phosphate production plots of selected pST3Gall mutants. The concentration of either donor (on left) or acceptor (on right) was titrated, while the other was fixed at 50 pM. Thick magenta line indicates the theoretically maximum yield of the glycosyltransferase reaction at that experimental condition. Phosphate calibration curve is presented in Supplemental Material. K256Q lacks enzyme activity and serves as negative control, (d) Double reciprocal plots of selected pST3Gall mutants, (e) Kinetic parameter fit of selected ST3Gall mutants that showed good data fit (R-square >0.8 for both donor and acceptor titrations), (f) Cartoon representation of Alphafold-predicted pST3Gall (green) superimposedwith CMP and Gal01,3GalNAca- / ?NP from 2WNB. Disordered loop and K256 are colored in magenta and orange, respectively.

[0030] Fig. 21 displays quantitative studies of different human sialyltransferases enabled by click chemistry signal enhancement methods, (a) Two methods were used to augment click chemistry-signal used to measure sialyltransferase activity: i) Instead of using a single fluorescent mouse anti-biotin Ab (‘ 1-step’), a ‘2-step’ method was employed using non-fluorescent mouse anti-biotin Ab followed by a second AF594-conjugated goat antimouse IgG secondary Ab. ii) Concentrations of reactants (CTP, Neu5Ac,9Az, DBCO-biotin) was increased from original IX to 2X and 4X (table), (b) Flow cytometry analysis of HEK293T WT cells expressing TM-PS1 using click chemistry -based enzymatic activity measurements with increased substrate and 2-step detection. The signal derived from enzymatic activity was amplified with higher concentrations of substrates and 2-step detection, (c) Quantitation of enzymatic activity of surface displayed TM-PS1 with increased substrates and 2-step detection methods, (d) Enzymatic activity measurement of surface displayed pST3Gall with different incubation time of sialylation and biotinylation, (e) Quantitation of enzymatic activity of surface displayed hST3Gal4, hST6Gall, and hST6GalNAc2 with 2-step detection methods. Measured signal was enhanced upon addition of more reactants and using the 2-step method. Negative controls include point mutations in the original enzyme. Q1+Q2 quadrant data with high Fc expression were used for quantitation in panels c-e. Data are presented as mean ± standard deviation of n=3-6. *p<0.05 with respect to all other samples.

[0031] Fig. 22 displays using surface display to characterize CAZy sialyltransferases from diverse species, (a) Primary sequence alignment of SP-ST3Gall (Strongylocentrotus purpuratus, sea urchin), CB-ST3Gall (Coregorius sp. ‘balchen’, salmon), XT-ST3Gall (Xenopus tropicalis. frog), MM-ST3Gall (Macaca mulaUa. rhesus macaque), human ST3Gall, and pST3Gall. All motifs (L, S, III, and VS) are highly conserved among these six ST3Galls, including amino acids forming disulfide bonds (yellow), essential interactions with donor (green), and acceptor substrate (blue). Catalytic base is shown using red letters, (b) Phylogenic tree of the ST3Galls. (c) Alphafold-predicted structures of SP-ST3Gall (magenta), CB-ST3Gall (yellow), XT-ST3Gall (purple) and MM-ST3Gall (gray) aligned with the crystal structure of pST3Gall (PDB: 2WNB, green). CMP and Gal 1,3GalNAca- NP co-crystalized with pST3Gall were shown using spheres, (d) Quantitation of enzymatic activity of surface displayed ST3Gall from different species using click chemistry -based 2- step method. Q1+Q2 quadrant data with high Fc expression were used for quantitation. SP-and CB-ST3Gall display low activity on cell surface. XT-ST3Gall displays moderate activity, (e) Quantitation of enzymatic activity of ST3Galls based on measurement of MALII and PNA binding to cells in Q1+Q2 quadrants that are high Fc expressors. CB-ST3Gall display low MALII and high PNA binding. Moderate activity of XT-ST3Gall was measured by PNA binding but not by MALII binding, (f) Simultaneous quantitation of enzymatic activity (in gray bars) and C-terminal FLAG-tag expression (in light blue bars) of surface displayed ST3 Gall -FLAG variants. pST3Gall WT (PSI), SP-ST3Gall WT (SP31), and SP- ST3Gall Q79C / R83C (SP31(CC)) were used. Enzymatic activity was measured using both MALII and click chemistry-based measurement. Q1+Q2 quadrants with high Fc expression were used for quantitation except for Mock transfection samples. Data are presented as mean ± standard deviation (n=3). ^<0.05 with respect to all other samples except that samples marked by1are not significantly different from each other. *p<0.05 with respect to all other samples.

[0032] Fig. 23 displays amino acid sequences of surface display form of Fc-fusion proteins and recombinant CMP-sialic acid synthase, (a) Amino acid sequence of Fc- pST3Gall with transmembrane domain of either DPP4 or CD94. Cytoplasmic tail (yellow) and transmembrane region (green) precede human Fc (gray) and truncated pST3Gall wildtype (A59, magenta). FLAG tag (cyan) is attached to C-terminus in some cases. Amino acids mutated in Dead mutant, Libi, and Lib2 are shown using white, yellow, and gray underlined fonts (within magenta highlights of TM(DPP4)-PS1 or TM(DPP4)-PS1-FLAG). (b) Amino acid sequence of his-tagged NmCSS.

[0033] Fig. 24 displays a formation of CMP-Neu5Ac,9Az using CMP-sialic acid synthase (NmCSS) and its transfer using hST3Gall. (a) Silver stain of recombinantly expressed NmCSS after IMAC purification, (b) Reaction scheme of CMP-Neu5Ac,9Az generation by recombinant NmCSS and subsequent sialylation by commercial recombinant hST3Gall. LC-MS / MS profile for studies with only one enzyme (NmCSS or hST3Gall only) or both enzymes (at two time points). Product formation in the presence of hST3Gall is accompanied by consumption of acceptor substrate and reactant Neu5 Ac, 9Az.

[0034] Fig. 25 displays an MS / MS spectra. MS / MS spectra of Neu5Ac,9Az (a - top), Gaipi,3GalNAca-pNP (b - middle), and Neu5Ac,9Aza2,3Gaipi,3GalNAca-pNP (c -bottom).

[0035] Fig. 26 displays that cell-surface sialylation requires all reaction components. Flow cytometry analysis of HEK293T WT cells with surface displayed TM(DPP4)-PS1. Only one condition with all reaction components (cell-surface sialidase treatment, CTP,Neu5Ac,9Az, NmCSS and DBCO-biotin) showed prominent shift of AF488 signals in high human IgG Fc expressing cell population (blue).

[0036] Fig. 27 displays a mutation profile for Libi and Lib2 measured using NGS. Mutation profiles of pCSCG-TM(DPP4)-Fc-pST3Gall WT-FLAG Libl (panel a) and Lib2 (panel b) plasmids were measured. Nucleotides from L76-S147 were PCR amplified to measure mutations in Libi, and L204-T269 was amplified for Lib2. Mutagenized residues are indicated using arrows and yellow highlights on the top of each panel, with mutation rates of target nucleotides shown just below (mutation rate = % of modified nucleic acid residues at each position of the library). Mutation rates are zero at non-target sites (blue) and they differ among the target site (red) as this depends on the proportion of mixed constituent plasmids. Three plasmids were mixed to form Libi while five were included in Lib 2 (bottom right of each panel). Prevalence of nucleotide at each position due to NNK incorporation is shown using Logo (bottom left of each panel). Only regions proximal to the mutation target site are shown.

[0037] Fig. 28 displays a flow cytometry analysis of HEK293T ST3Gall-KO cells displaying TM(DPP4)- PSI -FLAG, Libi or Lib2 . TM(DPP4)-PS1-FLAG expression on cells was measured using PE-conjugated anti-human Fc Ab in all assays, (a) Cell surface sialylation was measured using click-chemistry method in Fig 2., with DBCO-biotin followed by AF488 anti-biotin being used for detection. While a majority of Fc positive cells exhibited detectable levels of surface sialylation upon expressing wild-type TM(DPP4)-PS1- FLAG(Q2), the fraction of cells in Q2 was reduced for Libi and Lib2. (b) Human Fc expression correlated well with FLAG expression suggesting that mutations did not affect protein expression on cells.

[0038] Fig. 29 displays sorting Libi and Lib2 cells based on MAL-II and DBCO- biotin. FACS sorting strategy of Libi and Lib2 using either MALII-AF488 (left side) or DBCO-biotin along with anti -biotin AF488 (right side). Strategy for 1st sort (top panel) and 2nd sort (bottom panel) were shown. The percentage of Q2 (top right of the quadrant) and the gates used for sorting were shown in each plot.

[0039] Fig. 30 displays a heatmap of TM(DPP4)-PS1-FLAG Libi (left) and Lib2 (right) sorted using MALII or DBCO-biotin + anti-biotin. Log2(Enrichment value) of each sorted population (1stPos, 2ndPos, or 1stNeg) were visualized with potentially active mutants shown in red and potentially inactive mutants shown in blue. Mutants which were completely depleted in 1stPos, 2ndPos, or 1stNeg were shown in white. Target amino acids in individuallibraries are shown along bottom / horizontal-axis, with amino acids that are substituted by X depicted along vertical-axis.

[0040] Fig. 31 displays a calibration curve for malachite green phosphate assay. Free phosphate standard in the kit was used to draw the curve. Calibration curve was drawn for each experiment, but only one representative figure is shown.

[0041] Fig. 32 displays a temperature effect on biotinylation reaction. Enzymatic activity measurement of surface displayed pST3Gall at room temperature or 37 °C during biotinylation.

[0042] Fig. 33 displays amino acid sequences of surface display form of Fc- hST6Gall, Fc-hST3Gal4, and Fc-hST6GalNAc2. Figures show cytoplasmic tail (yellow), transmembrane region (green), human Fc (gray) and truncated human STs that includes only the full catalytic domain (A89-hST6Gall, A42-hST3Gal4, A62-hST6GalNAc2, magenta). Amino acids essential for hydrogen bond formation with phosphate group of donor substrate are shown using yellow font with underlining.

[0043] Fig. 34 displays studies with different sialyltransferases. (a) Structure alignment of hST6Gall (PDB: 4JS2, orange), hST3Gal4 (Alphafold2-predicted, green), and hST6GalNAc2 (PDB: 6APL, yellow) with pST3Gall (PDB: 2WNB, cyan). CMP and residues important for hydrogen bond formation with CMP are represented as stick, (b) Flow cytometry analysis of HEK293T WT cells with surface display form of hST3Gal4 and hST6GalNAc2 using click chemistry-based measurement with 2- step detection and increasing reactant concentrations. hST3Gal4 H294A and hST6GalNAc2 H336A were enzymatically inactive, (c) Flow cytometry analysis of HEK293T WT cells with surface displayed form of hST6Gall using click chemistry -based measurement with 2-step detection. The effect of Y354A mutation was only partial, demonstrating that Y354 is only partially important for enzymatic activity.

[0044] Fig. 35 displays studies with ST3Gall from various species, (a) Phylogenic tree of GT29 ST3Gall uncharacterized in CAZy database, (b) Alphafold-predicted cartoon structures of ST3Gall from Strongylocentrotus purpuratus (magenta), Coregonus sp. ‘balchen’ (yellow), Xenopus tropicalis (purple), Macaca mulatta (gray), Bos Taurus (Cyan), Callorhinchus milii (red), Danio rerio (orange), Oncorhynchus mykiss (brown), Oryzias latipes (blue), Pan troglodytes (pink), Takifugu rubripes (dark gray), Tetraodon nigroviridis (pale yellow) aligned with the crystal structure of pST3Gall (PDB: 2WNB, green). CMP and Gaipi,3GalNAca- / ?NP co-crystalized with pST3Gall were shown in spheres, (c) Flow cytometry analysis of HEK293T WT cells with surface display form of ST3Gall from avariety of organisms using click chemistry- based measurement with 2-step detection, (d) Flow cytometry analysis of HEK293T WT cells with surface display form of ST3Gall from a variety of organisms using MALII and PNA. Q1+Q2 quadrants with high Fc expression were gated prior to plotting MALII and PNA binding.

[0045] Fig. 36 displays amino acid sequences of surface display form of ST3Gall from various organisms, (a) All proteins had DPP4 cytoplasmic tail (yellow), DPP4 transmembrane region (green), human Fc (gray) and flexible Gly-Ser link. Following this was truncated form of the ST3Galls (A76-SP_ST3Gall, A54-CB_ST3Gall, A54-XT_ST3Gall, A59-MM_ST3Gall, or A59-hST3Gall, magenta), (b) Amino acid sequence of TM(DPP4)- SP31-FLAG. FLAG tag (cyan) is attached to C-terminus. Q79 and R83 are shown.

[0046] Fig. 37 displays lacking SP-ST3Gall catalytic domain during surface display, (a) Western blots of HEK293T cell lysate transfected with various ST3Gall or hST3Gal4. Detection was done using goat anti- human IgG Fc. These bands represent Fc-fusion proteins either on the cell surface or inside the cells while processing or being degraded proteolytically. (b, c) Flow cytometry analysis of HEK293T WT cells with surface display form of pST3 Gal 1-FLAG, SP-ST3Gall-FLAG, and SP-ST3Gall Q79C / R83C-FL AG using click chemistry-based measurement or MALII along with anti-FLAG. FLAG expression was measured simultaneously with (b) intracellular sialylation or (c) extracellular sialylation. Cell population with high human Fc expression is colored in blue. Catalytic domain of SP- ST3Gall was not displayed on the cell surface.DETAILED DESCRIPTION OF THE DISCLOSURE

[0047] Although claimed subject matter will be described in terms of certain embodiments, other embodiments, including embodiments that do not provide all of the benefits and features set forth herein, are also within the scope of this disclosure. Various structural, logical, process step, and other changes may be made without departing from the scope of the disclosure.

[0048] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0049] Every numerical range given throughout this specification includes its upper and lower values, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein. When such a range is expressed, another example includes from the one particular value and / or to the other particular value.Similarly, when values are expressed as approximations, by the use of the antecedent “about” it will be understood that the particular value forms another example. The term “about” in relation to a numerical value encompasses variations of + / - 10%, + / - 5%, or + / - 1%.

[0050] As used in the specification and the appended claims, the singular forms “a” "and” and “the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value.

[0051] This disclosure includes every amino acid sequence described herein and all nucleotide sequences encoding the amino acid sequences. The disclosure includes all polynucleotide sequences encoding every protein described herein.

[0052] The disclosure includes all methods steps described herein and as depicted on the accompanying figures. In examples, a described process comprises or consists of the described steps. The steps may be performed in any combination, including but not necessarily sequentially.

[0053] The disclosure includes all methods of making the described proteins as described herein. The disclosure includes polynucleotides encoding the described proteins, including but not limited to use of any suitable expression vector, including those described herein, and other expression vectors, such as plasmids, retroviral vectors, adeno and adeno- associated viral vectors, and the like. The disclosure includes all cells modified to express a described protein. The disclosure includes described proteins that are isolated from cells that have been programmed to produce such proteins.

[0054] This disclosure includes Part 1 and Part 2. Part 1 and Part 2 are related to each other. Citations for Part 1 and Part 2 are provided as indicated.

[0055] In examples, the disclosure provides modified proteins, and methods of producing, screening, and selecting modified proteins with altered target binding properties.

[0056] In examples, modified proteins of the disclosure include mutations, relative to their unmodified counterparts. In examples, the disclosure includes modifying any mammalian glycosyltransferase (GT).

[0057] In examples, the disclosure includes a novel mammalian cell-surface display platform that can be used to identify mutated glycosyltransferases via a display and selection process, as further described below. In examples, by screening a plurality of mutated proteins, the disclosure includes identification of GTs that have been converted to gly canbinding proteins (GBPs). In examples, a library of mutated proteins is screened against a panel of targets to determine improved target binding and / or enzymatic activity exhibited bymembers of the library. In an example, improved target binding proteins are further mutated and screened against a panel of targets to identify proteins with further improved binding and / or enzymatic activity. Improved glycosyltransferases, improved sialyltransferases, and improved CAZymes (Carbohydrate-Active enZYmes) can be identified using the described approaches. In examples, a described protein may be provided as a fusion protein as described herein, and may include transmembrane and cytoplasmic domains at the N- terminus of Fc-fusion glycosyltransferases. This approach permits both glycosyltransferase presentation in the ER / Golgi lumen, and cell surface display. Click chemistry with azido derivatized nucleotide-sugars is then used to quantify enzyme activity using flow cytometry.

[0058] In examples, any protein of this disclosure can exhibit improved target binding properties relative to binding properties of a control protein. In examples, a mutated GBP that is generated and / or identified by a described screening process exhibits different target binding characteristics than a starting GT, i.e., a non-mutated or only partially mutated starting GT. In examples, the disclosure provides a protein-based directed evolution process, as further described in Part 2. Part 2 includes improved click chemistry approaches used to identify proteins with improved enzymatic activity, such as by using transfer of a clickchemistry derivatized sialic acid to cellular acceptors which may be analyzed by flow cytometry or other methods.

[0059] In examples, any described protein may include a secretable signal peptide, a non-limiting and representative example of which is provided herein, and may include any suitable purification tag, representative sequences of which are provided herein. Any described protein may also include a transmembrane domain and as such may be displayed on the surface of cells.

[0060] In examples, mutated proteins, such as those identified herein with or without using the described screening process, are provided. In examples, the amino acid location of mutations of a described protein are based on the following representative sequence, which is the pST3Gall WT (‘PSI’) sequence without the Ig leader sequence that is present in certain examples of amino acid sequences described below:RPCTCTRCIEEQRVSAWFDERFNRSMQPLLTAKNAHLEEDTYKWWLRLQREKQPNN LNDTIRELFQVVPGNVDPLLEKRLVSCRRCAVVGNSGNLKESYYGPQIDSHDFVLRM NKAPTEGFEADVGSKTTHHFVYPESFRELAQEVSMILVPFKTTDLEWVISATTTGRIS HTYVPVPAKIKVKKEKILIYHPAFIKYVFDRWLQGHGRYPSTGILSVIFSLHICDEVDLYGFGADSKGNWH / / YWENNPSAGAFRKTGVHDGDFESNVTTILASINKIRIFKGR (SEQ ID NO: 1).

[0061] In examples, SEQ ID NO: 1 may comprise a change of H243; or a change of amino acid A253; or a change of amino acid F254; or a combination of these amino acid changes. In examples, the mutation at H243 is any amino acid other than H; the mutation at A253 is any amino acid other than A; and the mutation at F254 is any amino acid other than F. All single described mutations, and all combinations of described mutations, are included within the scope of this disclosure. In an example, for SEQ ID NO: 1, the mutation may comprise one or any combination of H243A, A253I, and F254S.

[0062] In an example, a representative mutated protein comprises the following sequence which includes the stated of H243A, A253I, and F254S mutations:RPCTCTRCIEEQRVSAWFDERFNRSMQPLLTAKNAHLEEDTYKWWLRLQREKQPNN LNDTIRELFQVVPGNVDPLLEKRLVSCRRCAVVGNSGNLKESYYGPQIDSHDFVLRM NKAPTEGFEADVGSKTTHHFVYPESFRELAQEVSMILVPFKTTDLEWVISATTTGRIS HTYVPVPAKIKVKKEKILIYHPAFIKYVFDRWLQGHGRYPSTGILSVIFSLHICDEVDL YGFGADSKGNWHAYWENNPSAGZSRKTGVHDGDFESNVTTILASINKIRIFKGR (SEQ ID NO:2) wherein the stated mutations are in bold and italics.

[0063] In examples, a described protein comprises any mutation or combination of mutations described in Part 2 that improves or otherwise enhances desirable target binding and / or enzymatic activity. Those skilled in the art will be able to resolve any differences in amino acid numbering as between SEQ ID NO: 1, SEQ ID NO:2, and the other amino acid sequences provided herein using routine protein alignment tools. In particular, any reference to residues H243, A253 and F254 in the description means residues H243, A253 and F254, respectively, in relation to SEQ ID NO: 1 and SEQ ID NO:2. In SEQ ID NO:2, these mutations are H243A, A253I, and F254S. The same relative positions for all amino acid sequences discussed herein applies and as depicted on the figures and in the tables of this disclosure applies. Thus, by comparing any amino acid sequence where H243, A253 and F254 are indicated, the residue number can be adjusted by simple comparison to EQ ID NO: 1 and SEQ ID NO:2. The same approach applies to other proteins described herein.

[0064] In examples, a described protein comprise an immunoglobulin (Fc) segment, a representative sequence of which is: TCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<ALPAPIEI<TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID N0:3).

[0065] In examples, a described protein may include an amino acid linking sequence that separates defined sequences. In an example, said linking sequence is located between the sequence of SEQ ID NO: 1 and SEQ ID NO:2. The linker sequence optionally comprises the sequence GSGSGSGSGS (SEQ ID NO:4).

[0066] In examples, a described protein comprises any suitable secretion sequence, a representative secretion sequence comprising the sequence MIPARFAGVLLALALILPGTLC (SEQ ID NO:5).

[0067] In examples, a described protein comprises any suitable purification tag, which may be a sequence of H residues. The H residues may be present at the N- or C- terminus of the protein, or elsewhere provided the sequence can be used for purification. In an example, the purification tag comprises the sequence HHHHHH (SEQ ID NO:6).

[0068] In an example, a described protein comprises the sequence:MIPARFAGVLLALALILPGTLCHHHHHHTCPPCPAPELAGAPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSRPCTCTRCIEEQRVSAWFDERFNR SMQPLLTAI<NAHLEEDTYI<WWLRLQREI<QPNNLNDTIRELFQVVPGNVDPLLEI<RL VSCRRCAVVGNSGNLKESYYGPQIDSHDFVLRMNKAPTEGFEADVGSKTTHHFVYP ESFRELAQEVSMILVPFKTTDLEWVISATTTGRISHTYVPVPAKIKVKKEKILIYHPAFI KYVFDRWLQGHGRYPSTGILSVIFSLHICDEVDLYGFGADSKGNWHAYWENNPSAG ISRKTGVHDGDFESNVTTILASINKIRIFKGR (SEQ ID NO:7). This sequence is related to a sequence that is also be referred to herein or in the priority document as Fc-pST3Gall H243A / A253I / F254S (‘sCore2’). When reference to Fc-pST3Gall H243A / A253I / F254S and ‘sCore2’ is made, it refers to SEQ ID NO:7, which in turn includes SEQ ID NO:2.

[0069] The disclosure includes all amino acid sequences that have 80-99% sequence identity with SEQ ID NO:2 and / or with any other source of mammalian GTs that are described herein. As such, in examples, the disclosure includes modifications of other, related proteins, that are derived from diverse species, as shown in Supplementary Tables S2 and S4 of Part 2, and otherwise described in Part 2.

[0070] In examples, a fusion protein of this disclosure includes a first segment comprising a sequence that is at least 80% identical to SEQ ID NO:2, along with any one or combination of described mutations, and a segment of segment of another protein, and / or a segment of a third protein, and may include additional amino acids. Additional segments of other proteins, or duplicated segments of the same protein, may be included. “Segment” as used herein with respect to fusion proteins includes full length proteins, and fragments thereof. In examples, the second and / or third segments of a fusion protein comprises a cytoplasmic and / or transmembrane (TM) domain of one or more proteins which may not be SEQ ID NO:2. In an example, a fusion protein of this disclosure comprises SEQ ID NO:2 or a sequence that is at least 80% identical to SEQ ID NO:2, and cytoplasmic and / or transmembrane domains of, for instance, a Type II protein, such as CD94 and DPP4, the amino acid sequences of which are known in the art and are provided as segments of amino acid sequences described herein. Second and third segments (or more segments if desired) may be placed in any orientation. In an example, a cytoplasmic and transmembrane domain may be positioned at the N-terminus of any described protein, but other orientations are not necessarily excluded. In examples, a fusion protein of this disclosure may further include any suitable tag segment, such as for detecting surface display of a described fusion protein. In a non-limiting example, a human Fc tag may be used. In examples, any described protein may include a secretory signal, a protease fusion cleavage site, a purification tag, or any combination thereof. Any described segments of a fusion protein described herein may be linked to one another by any suitable linking amino acid sequence, which typically comprise Glycine and / or Serine. The linker may include 2-20, or more amino acids. In examples, one or more pluralities of protein are provided, such as for use in a described screening process. In examples, a plurality of mutated proteins may be described as a protein library.

[0071] The disclosure includes cells that express described proteins, and pluralities of cells that express different proteins, such as those described in a screen of this disclosure.

[0072] In examples a described fusion protein comprises, in addition to a segment that is at least 80% identical to SEQ ID NO:2, a detectable protein segment, including but not necessarily limited to GPF, EGPF, mCherry, and the like. Accordingly a protein of this disclosure may be detectably labeled using the aforementioned Fc tag, or any other suitable detectable tag, or for example, a radioisotope. In examples, a described fusion protein includes an antigen that can be recognized by a detectably labeled binding partner, such as an antibody or antigen-binding fragment thereof. Those skilled in the art will recognize that Fcfusion proteins are engineered proteins made by fusing the Fc (fragment crystallizable) region of an antibody to another protein.

[0073] In examples, a protein and / or a fusion protein of this disclosure may function akin to an antibody that exhibits specificity for a particular target. As such, a protein and / or a fusion protein of this disclosure may be used alone, or connected to a drug akin to an antibody-drug conjugate (ADC), or a toxin. In an example, a described protein may be a segment of a chimeric antigen receptor (CAR), or may be connected to any other agent that is designed to detect or otherwise specifically bind to any target displayed by any type of cell. As such, the disclosure incudes administering to an individual an effective amount of any described protein that could have a prophylactic or therapeutic effect, for use with any disease or disorder. In examples, a described protein and / or fusion protein specifically binds to specific to sialylated core-2 O-linked glycans, or specifically binds to only one or only several sialylated core-2 O-linked glycans, non-limiting examples of which are discussed further below. In examples, a described protein binds to a cancer cell, representative examples of such binding being described further below.

[0074] All polynucleotides encoding the described protein are included in this disclosure, whether in isolated form, or when present in, for example, any suitable expression vector. In examples, the disclosure includes all methods of making a described protein, such as by expressing the protein in a plurality of cells, and isolating the proteins from the cells. The isolated proteins can be purified to any desired degree of purity.

[0075] The disclosure provides examples of enhancing activity of described proteins. In an example, the disclosure provides a modified protein comprising SEQ ID NO: 1 with a K256V mutation, and wherein the modified protein has improved enzymatic activity relative to a protein comprising SEQ ID NO: 1 that does not contain the K256V mutation.

[0076] The following parts / examples are presented to illustrate the present disclosure. They are not intended to be limiting in any matter.PART 1: Engineering glycosyltransferases into glycan binding proteins using a novel mammalian surface display platform

[0077] This Example provides an analysis conducted to determine if GTs can be engineered to yield GBPs. In this regard, the binding pocket of mammalian GTs have been evolutionarily optimized to accept specific glycans in order to synthesize a range of mammalian N-glycans, O-glycans, and glycolipids17. Modification of this interface mayresult in loss of enzyme catalytic activity but may favor either substrate or product binding. To test this, we engineered porcine ST3Gall (pST3Gall, P- galactoside a2,3-sialyltransferase 1), a member of the GT29 CAZy family, whose crystal structure is known18. We chose pST3Gall as it has a large binding pocket that can simultaneously accommodate the donor CMP-Neu5Ac (Cytidine-5-monophospho-N-acetylneuraminic acid) and TF-antigen disaccharide acceptor (Gaipi-3GalNAca). We observed that the introduction of an H243A mutation (SEQ ID NO:2) into pST3Gall through rational design led to a lectin that specifically binds sialyl core-2 O-glycans. A new mammalian surface display platform was developed to screen for additional variants, wherein said display surface platform is an aspect of this disclosure. This resulted in a mutant H243A / A253I / F254S (‘sCore2’ lectin) (SEQ ID NO:2) that displayed even stronger binding for the sialyl core-2 epitope. Compared to traditional GBPs, this lectin displayed unique binding patterns to different human peripheral blood cell types, normal human tissue, and tumor sections. In examples, this disclosure provides a generalizable, rational design and high-throughput screening strategy to convert GTs into GBPs. This strategy may be applied to other enzymes from diverse species.

[0078] Conversion of Fc-pST3Gall (‘PSI’) into the glycan-binding proteinH243A / H243: Upon comparing the molecular recognition surface area of pST3Gall with respect to other GBPs and glycosidases, we determined that the pST3Gall ligand-binding interfacial area (~ 540 A2) is larger compared to the other entities (283 - 502 A2) (Fig. 7). This is consistent with the notion that lectins and glycosidases have broad binding specificity mostly towards terminal residues, whereas GTs display a more intricate binding interface.

[0079] We analyzed whether the large GT binding interface may afford opportunities to generate lectins with novel binding properties that recognize the natural reaction substrate or product of the parent enzyme (Fig. 1). To test this, we created a set of three Fc-fusion proteins with the GT catalytic domain expressed at the C-terminal to mimic their presentation in the Golgi (Fig. la, Fig. 8a): i) The truncated pST3Gall-A59 wild-type enzyme lacking TV- terminal cytoplasmic and transmembrane domains (abbreviated ‘PSI’)19, ii) ‘H243A’, a PSI variant that we hypothesized would be inactive due to loss of highly conserved H243 in motif 3. We analyzed whether this would retain binding capacity for acceptor substrate or product as the mutation targets the hydrogen bond formed with the phosphate group of CMP- Neu5Ac20, iii) ‘Dead’ (Q49A / Y174A / Y210F), a non-binding mutant which cannot bind its TF antigen acceptor, Gal(pi-3)GalNAca, due to loss of essential hydrogen bonds and 7t-7t interactions (Fig. lb)21. All proteins were expressed in dimeric form with a flexible Gly-Ser hinge in order to mimic natural lectin oligomerization22'24using a lentiviral vector containinga secretory Von Willebrand factor signal peptide, a 6xhis-tag for purification, and an enterokinase cleavage site. These proteins were expressed along with Fc-CBM40, a Fc-fusion protein containing a sialic acid-binding bacterial carbohydrate-binding module, and its tandem variant Fc-diCBM40 (Fig. 8b, 8c)25. PSI, H243A, Dead, and Fc- CBM40 expressed well upon transient transfection into HEK293T cells and appeared at their expected molecular mass in western blots both under reducing and non-reducing conditions. Fc-diCBM40, however, was not stable (Fig. 1c). Attempts were made to increase the yield of PSI and its variants by expression in HEK293T CIGalTl-KO (knockout) cells harboring truncated O- glycans26, as such structures may act as potential ligands that bind and sequester the release of PSI and related variants (Fig. 9a). However, the expression level was not increased. Thus, all Fc-proteins were expressed using wild-type HEK293T and purified using 6xhis-tag prior to functional assays described below.

[0080] Enzyme activity and binding studies were performed to characterize PSI, H243A and Dead. In LC- MS / MS investigations, PSI afforded the reaction product, Neu5Ac(a2-3)Gal(pi-3)GalNAc-p-nitrophenol (pNP), using acceptor substrate Gal(pi- 3)GalNAc-pNP (TF-antigen), and donor substrate, CMP-Neu5Ac, similar to commercial human ST3Gall (Fig. Id, Fig. 9b). The enzymatic activity was ablated in H243A and Dead. In cytometry studies, H243 A displayed strong sialic acid-dependent binding to multiple epithelial cell types: kidney HEK293T, pancreatic COLO357-FG, and lung Calu-3, with particularly strong binding to the highly metastatic pancreatic cancer cells (Fig. le, Fig. 9c- d). Compared to H243 A, Fc-CBM40 exhibited smaller sialic acid dependence and Dead was a non-binder as predicted. The sialic acid-binding property of H243A was similar to that of MALII, which binds a2,3-sialylated O-glycans27, and it was distinct from PNA, which recognizes the TF-antigen. Compared to H243 A, the sialic acid-dependent binding property of PSI was weaker, with this dependence being more prominent at high protein concentrations (Fig. le, f). H243A and PSI binding increased monotonically with lectin concentration up to 20pg / mL (Fig. If). Overall, the introduction of the H243 A mutation into pST3Gall led to a novel GBP that prefers to bind the product of the original enzymatic reaction.

[0081] Specific binding of H243A to sialyl core-2 O-glycan: To better define the binding specificity of H243A, a panel of isogenic HEK293T knockouts were created using CRISPR-Cas9 technology. This includes previously established MGAT1-K0 and CIGalTl- KO cells lacking complex N-glycans and core-1 O- glycans respectively26, SLC35A1-KO lacking the CMP-Neu5Ac transporter necessary for synthesis of sial ogly cans, GCNT1-KOlacking core-2 O-glycans, and ST3Gall-K0 (Fig. 2a). Two different sgRNA (single-guide RNA) targeting each of these genes were cloned into the pX330 vector, mixed and transfected into HEK293T cells. Isogenic clones derived from this process commonly contained excisions between the two sgRNA target sites (Fig. 2b). MGAT1-K0 reduced PHA-L and ECL binding consistent with the lack of formation of complex N-glycans and the reduced prevalence of the Gal(pi-4)GlcNAcP epitope, respectively (Fig. 2c). CIGalTl-KO had low MAL-II and PNA binding due to the absence of the TF-antigen. ST3Gall-KO had reduced MAL-II binding on O-linked glycans. SLC35A1-KO had high ECL and PNA binding, and low MAL-II binding due to the absence of terminal sialic acid.

[0082] Among Fc-fusion proteins, H243A did not bind CIGalTl-KO, SLC35A1-KO, and ST3Gall-KO but it bound MGAT1-KO similarly to wild-type HEK293T cells (Fig. 2d). Additionally, this lectin displayed reduced binding to GCNT1-KO, which lacks core-2 O- glycan formation. The binding profile of H243A was markedly different from MALII in this core-2 dependence, suggesting that H243A is a novel GBP. PSI exhibited similar binding specificity as H243 A although the sialic acid dependence was only partial. CBM40 only showed weak sialic acid-dependent binding in these studies. Dead did not bind any of the cell types. Overall, H243 A displayed unique binding to sialyl core-2 O-glycans, unlike other conventional GBPs.

[0083] Binding specificity of H243A confirmed using glycan microarray: We performed glycan microarray analysis with the CFG5.5 microarray as it contains a broad spectrum of 562 glycan structures (Fig. 3a). Here, both PSI and H243A were observed to specifically bind only O-glycan epitopes and not epitopes related to N-glycans and glycolipids (Fig. 3b-c, Fig. lOa-b). Whereas PSI predominantly bound non-sialylated core-2 O-glycans, it displayed less binding to non-sialyl core-1 O-glycans. H243A, in contrast, only recognized two glycans, sialyl core-2 O-glycan

[0561] and sialyl 6-sulfated O-glycan

[0237] , The binding of both PSI and H243A was quite specific in that they did not recognize several closely related 3-O-sulfated

[0029] , fucosyl [61, 62], monosialyl [134, 135, 219, 220], disialyl [238, 239] TF antigens, sialylated Tn antigen

[0240] , and core-2 O-glycans with sialic acid cap on core-2 branch [281, 309, 326, 560] (Fig. 3d, Supplementary Table SI). Furthermore, these lectins did not also bind additional sialoglycans [233, 245] that had structures very similar to

[0237] , The binding specificity of H243A and PSI was also distinct from PNA and MALII, which displayed much broader binding specificity, thus highlighting the important advantage of engineering GTs for GBP applications (Fig. lOc-f).

[0084] Given the strong binding of H243A to 6-O-sulfated GalNAc

[0237] , we analyzed if the lectin may bind physiological sulfated glycans. Such structures are synthesized by carbohydrate sulfotransferases belonging to the CHST and Gal3ST families28'30, but none of these enzymes are currently known to have 6-O-sulfotransferase activity towards GalNAc. To examine this in a cellular context, we treated promyelocytic HL60 cells with sodium chlorate, a potent ATP-sulfurylase inhibitor that blocks sulfated epitope biosynthesis (Fig. 3e). This treatment did not impact PSI and H243A binding to leukocytes, but it reduced P-selectin Fc-fusion protein binding, which is consistent with the known role of protein tyrosine sulfation in leukocyte adhesion31. The data suggest that H243A likely only binds sialyl core-2 O-glycans in a cellular context. 6-O-sulfated sialyl O-glycans such as

[0237] likely represent non-physiological glycans present on the CFG5.5 microarray.

[0085] CRISPR screen shows that PSI and H243A bind sialylated core-2 O- glycans: Only a limited repertoire of natural glycans is presented on the CFG microarray, and their presentation on glass is non-physiological. We thus performed forward genetic screens using a previously developed HL60 glycoCRISPR cell library, to determine the glycogene determinants for lectin binding on cells. This knockout library allows study of 347 glycogenes involved in human carbohydrate biosynthesis32,33. To achieve this, anti-human IgG Fc specific antibodies were conjugated onto epoxy-functionalized magnetic beads, and these beads were incubated with HL60 glycoCRISPR library cells pre-incubated with PSI or H243 A (Fig. 4a). Unbound HL60 cells lacking glycan epitopes required for Fc-protein binding were negative-selected in three rounds, followed by next-generation sequencing (NGS) to identify enriched sgRNAs that regulate lectin binding. HL60 cells enriched with H243 A showed a distinct negative population as its initial binding was strong and the decrease in lectin binding caused a population shift (Fig. 4b). This shift was less distinct for PSI as its initial binding was lower (Fig. 4c). NGS analysis of sgRNA enriched in the nonbinding population revealed that all enzymes involved in sialic acid biosynthesis (NANS, GNE, CMAS, and SLC35A1\ core-1 O-glycan biosynthesis (CIGalTICl and ClGalTl)', and core-2 O-glycan biosynthesis (GCNT1) are critical for both H243A and PSI binding (Fig. 4b, c). sgRNAs targeting ST3Gall and ST3Gal4 were also enriched in the PSI screen, consistent with the concept that PSI has different but overlapping binding specificity compared to H243 A for core-2 O-glycans. No conclusion could, however, be reached regarding the impact of lactosamine chain extension on the core-2 arm since multiple Gal and GlcNAc-transferases contribute to such synthesis.

[0086] Mammalian surface display enables a generalizable strategy to select for superior glycan-binding proteins: Phage display34and yeast surface display35are commonly used for protein engineering, and similar high-throughput screening of mammalian GBPs requires the development of analogous mammalian surface display platforms. To anchor proteins on mammalian cell surface, we fused the cytoplasmic and transmembrane domains of the type-2 single-pass transmembrane protein, dipeptidyl peptidase 4 (DPP4), to the N-terminus of our Fc-protein constructs (Fig. 5a, b, sequence in Fig. 8d)

[0087] Using this strategy, TM-PS1, TM-H243A and TM-Dead were robustly expressed on HEK293T surface (Fig. 5c, d). These surface displayed Fc-proteins, however, were unable to bind a fluorescent sialyl core-2-PAA-FITC polymer that was synthesized upon sialylation of commercially available core-2-PAA- FITC polymer using rhST3Gall and CMP-Neu5Ac. We reasoned that this may be due to c / .s-interactions between surface displayed GBPs and endogenous sialoglycans presented on cell surface (Fig. 5c,24>36’37). Indeed, flow cytometry -based dual-color cell-cell interaction assays show that sialidase treatment of HEK293T cells expressing TM-PS1 and TM-H243A increases heterotypic binding of surface display cells to wild-type HEK293T ligand cells that bear their counterreceptor (Fig. 11). This binding is blocked by sialidase treatment of the ligand bearing cells, and also upon surface expression of TM-Dead (instead of TM-PS1 and TM-H243A). Consistent with this notion, sialyl core-2-PAA-FITC displayed robust binding to cells bearing TM-PS1 and TM-H243A upon sialidase treatment (Fig. 5d). This sialylated polymer, however, could not bind TM-Dead cells as this is a non-binder.

[0088] To screen for TM-H243A variants with improved binding to sialyl core-2- PAA-FITC, two plasmid libraries were created, each targeting selected residues proximal to the sialic acid binding pocket of pST3Gall based on an AlphaFold molecular modeling (Fig. 5e, Fig. 12a). Libi constitutes a 440-member mutation library focused on amino acids prior to motif 2 in the protein primary sequence, while Lib2 contains 1240-m ember mutations in later residues (Fig. 5a). While a vast majority of constructs contained the H243A mutation, the library also includes members where H243 is modified to other amino acids besides alanine. Amino acid representation in all libraries was verified using NGS (Fig. 12b). Lentivirus corresponding to each of these libraries was produced, and these were used to transduce HEK293T SLC35A1-KO cells that lack endogenous sialoglycans. Virus was applied at low multiplicity of infection (MOI < 0.25) in order to achieve ~1 mutant / cell (Fig. 13a). In cytometry binding assays, the Lib2 cells exhibited greater binding to sialyl core-2-PAA-FITC, compared to either TM-H243A or Libi cells (Fig. 5f). Two rounds of FACS sorting were thus performed with Lib2 to select for cells with superior sialyl core-2-PAA- FITC binding properties (Fig. 13b). One round of sorting was performed for Libi. NGS analysis revealed several mutations in Lib2 with high positive enrichment scores (Fig. 5g, Supplementary Table S2), while Libi selection did not reveal any candidate hits (Fig. 13c). The Lib2 mutations with enhanced sialyl core-2 binding were centered at residues S212 - T213 and A253 - F254 of the full protein.

[0089] The top six mutation candidates were individually cloned and expressed as soluble Fc-proteins. In western blots, all H243A / A253X / F254X mutants expressed well but the H243A / S212X / T213X clones did not express (Fig. 5h). Studies were performed with the panel of isogenic HEK293T-KOs to characterize lectin binding properties. These data showed that all H243A / A253X / F254X mutants retained the sialylated core-2 O-glycan binding properties of H243A (Fig. 5i). Among the mutants, H243A / A253I / F254S displayed stronger sialic acid-dependent binding compared to the original H243 A (Fig. 5j, Fig. 13d). This was further confirmed by performing dose dependence binding assays (Fig. 5k). Due to distinct binding to the sialyl core-2 epitope, the H243A / A253I / F254S pST3Gall Fc-protein was named ‘sCore2’.

[0090] High expression of sialyl core-2 O-glycans on myeloid cells, terminally differentiated T-cells and selected normal and cancer tissue: sCore2 was applied in spectral flow cytometry studies to investigate the O-glycan profiles of human blood cell types. To this end, the Cytek immunoprofiling kit was modified to include channels for fluorescent lectins and to expand the coverage of myeloid sub-populations that were otherwise missed. Three related O-glycan specific lectins, sCore2, MALII, and PNA, were applied. Control studies confirmed minimal competition among these reagents for different blood cell populations, likely because only a small fraction of glycan epitopes on cells are bound by these lectins (Fig. 14a). Using this approach, we discerned the lectin binding profiles of 24 different blood cell subtypes as depicted using tSNE (t-distributed stochastic neighbor embedding) plots (Fig. 6a, Fig. 14b). Here, PNA binding was largely restricted to classical and non-classical monocytes, neutrophils, and eosinophils, but not basophils and lymphoid cells. MAL-II binding was almost inverse, suggesting that the extent of terminal sialylation of the Neu5Aca2-3Gaipi-3GalNAca arm may be less on myeloid cell populations compared to lymphoid cells. Only naive B-cells were low for MAL-II and PNA, and these findings were consistent across donors (Fig. 14c).

[0091] The binding profile of sCore2 was different from other lectins in that it bound myeloid cells and terminal effector T-cells rather than immature T- or B-cells. Besides terminal CD4+, and CD8+ T-cells, increased binding was also noted for mature and terminal NK cells compared to early NK populations. As MAL-II binding decreased with such cell differentiation, the data suggest a transition from mono- and di- sialyl O-glycans to sialyl core-2 structures upon lymphocyte differentiation. Low binding of sCore2 to naive and central memory T-cells, and high binding to effector T-cells is consistent with observations of increased GCNT1 expression upon T cell maturation38,39. To the best of our knowledge, this is the first report that NK-cell differentiation is also correlated with the sialyl core-2 O-glycan levels. With regard to B-cells, it is proposed that GCNT1 mRNA levels are reduced upon B- cell maturation from naive to memory subtypes40. However, our data with sCore2 suggest that these transcript level changes may not quantitatively contribute to core2 branching patterns on B-cells. As expected, Dead showed low binding compared to the binding of sCore2 to all peripheral blood cell populations (Fig. 14d).

[0092] We used sCore2 to detect the sialyl core-2 O-glycan epitope on 40 different normal human tissue on a tissue microarray and a limited number of cancer tissue (Fig. 6c, Fig. 15a, b). In all cases, negative control studies performed using the Dead mutant and upon sialidase treatment showed minimal signal. In comparison to normal breast tissue, staining was moderate to strong (greater than 50%) in invasive breast ductal carcinoma within the glandular or follicular regions, particularly in the apical lumen and associated microlumens. Additionally, the sialyl core-2 O-glycan expression was observed in the glandular cytoplasm of the normal endometrium, apical cytoplasm of fallopian tubes, and cytoplasm of liver and pancreas. In the case of spleen, staining was weak in the cytoplasmic lymphoid white pulp, but stronger in the red pulp sinusoidal cells (>80%). sCore2 staining was also weakly observed in thymus and synovium, in -50% of capillary endothelium of the cardiac muscles, and 70% of the granular neuropils of the brain. sCore2 staining on tumor cells, blood cells and specific cell types from selected normal organs. However, staining was not observed in cells from the oral and salivary regions (salivary glands, tonsils), lungs, soft tissue (cartilage, skeletal muscles, intestines, uterus), gastrointestinal (gastric and colon mucosa) and urological tract (kidney, bladder).DISCUSSION

[0093] This Part 1 of this disclosure introduces protein engineering strategies to convert mammalian GTs into GBPs that recognize the substrate, product or relatedcarbohydrates. The disclosure demonstrates production and identification of lectins that bind sialylated core-2 glycans on a number of cell and tissue types. This overcomes a challenge with raising antibodies that cannot be easily generated against core-2 O-glycans as they are naturally occurring in mammalian cells. Indeed, while a mouse anti-CD43 mAb, 1B11, has been proposed to bind core-2 O- glycans41, its protein dependence restricts its use to blood cells which express CD43.

[0094] In studies that contrast PSI with H243A, we observed that both Fc-fusion proteins function as lectins that bind core-2 O-glycans. These lectins prefer to bind core-2 O- glycans rather than linear core-1 chains, consistent with the known substrate preference for ST3Gall which transforms core-2 acceptors (KM = 8pM) more readily compared to core-1 substrates (KM = 50pM)42. In cell-based assays, PSI preferentially bound to sial ogly cans at high concentrations, but it also bound non-sialylated carbohydrates at lower concentrations and in glycan microarray studies. Additionally, in forward genetic screens, the lectin appeared to bind structures formed by both ST3Gall and ST3Gal4. In contrast, H243 A displayed more strict binding preference for sialyl core-2 O-glycans, a product of ST3Gall- catalyzed reactions. These observations suggest the importance of His243 in regulating lectin binding. Whereas His residue naturally interacts with CMP-Neu5Ac to catalyze chemical reactions, mutating it to alanine abolishes this transformation. This modification may then alter the side chain orientations other amino acids in the binding pocket, like Y135, promoting hydrogen bonds with the carboxyl residue of sialic acid (Fig. 16a). As His is highly conserved in many mammalian GT29 family sialyltransferases, the disclosure includes use of the same or similar mutations on other sialyltransferases also, as this may afford additional specific, sialic acid-binding lectins.

[0095] The broad binding interface of PSI and H243A is different from that of other sialoglycan-binding proteins like MAL-II, SNA (Sambucus nigra agglutinin), influenza hemagglutinins43,44and Siglecs45, none of which display core-2 dependent ligand recognition. This is consistent with the observation that H243 A requires two different carbohydrate motifs for high affinity binding: Neu5Ac(a2-3)Gal and Core-2. The crystal structure of pST3Gall affords space to accept core-2 GlcNAc, including forming potential hydrogen bond interactions (Fig. 16b). However, the bulky nature of Neu5Ac may prevent inclusion of Neu5Aca2,6GalNAc due to amino acid clashes (Fig. 16c). Additionally, two arginine residues, R50 and R209, around the C6 hydroxy group of GalNAc may form electrostatic interactions if a sulfate substituent replaces GlcNAc, and this may explain H243 A binding to 6-O-sulfated sialyl TF-antigen in the glycan microarray studies (Fig. 16d). However, suchstructures are not yet reported in mammals, and human sulfotransferases were not identified to be critical in our CRISPR genetic screen. Indeed, several other sialoglycans such as MALII and Siglec families also recognize sulfated glycans5, 45, 46, which may also be driven by similar electrostatic interactions. Thus, mutations in R50 and R209 may allow further tuning of H243 specificity. While this example established the binding preference of H243A for sialyl core2 O-glycans, the repertoire of glycans on the glycan microarray was insufficient to explain the types of LacNAc chain extensions on the core-2 arm that are tolerated by this lectin. The core-2 O-glycan binding preference of PSI and H243A and enhanced binding specificity of H243A for a(2-3) sialylated glycans are consistent with available structural evidence and enzyme kinetics data.

[0096] We established a mammalian cell surface display platform for high-throughput screening of GT variants. Although the expression of mammalian sialyltransferases in yeast and E. coli has been reported47'49, we experienced low yield when expressing wild-type sialyltransferases in these systems and difficulty producing mutants due to protein misfolding (data not shown). Thus, mammalian cell platforms are preferred for the study of these enzymes. In such studies, we focused on residues proximal to the sialic acid binding pocket including the disordered loop18, which upon folding interacts with the bound ligand. These studies identified H243A / A253I / F254S (sCore2) as a superior sialyl core-2 binder compared to H243A alone. As A253 and F254 are missing in the original crystal structure18, AlphaFold modeling was performed and this suggests that the large phenyl group of F254 may clash with the bound ligand. The substitution F254S may reduce bulkiness, promoting hydrogen bond formation with Neu5Ac residues, such as C5-acetamide (Fig. 16e, f). The development of sCore2 validates the experimental focus on GTs, including the rationale for the directed evolution process. The disclosure includes expression of any mammalian GTs using this system, which is expected to be a useful platform for both engineering new GBPs and high- throughput screening of GTs. Whereas standard site-directed mutagenesis is used in our screening approach, more modern mammalian cell-based directed evolution such as TRACE50, CRISPR-X51, and adenovirus-based continuous evolution52may also be used to further enhance this approach.

[0097] New and specific lectins are needed to recognize clinically significant carbohydrate epitopes, as glycan transformation commonly accompanies metabolic disorders2, 4’53, as gene expression alone does not directly correlate with carbohydrate alterations1and as glycoscience mass spectrometry techniques lag behind clinical practice54. In this context, sCore2 binds sialyl core-2 O-glycans on peripheral blood cells efficiently,particularly selected myeloid cell and effector lymphoid cell populations. This reagent also binds distinct cell types in normal human tissue microarrays, with initial data suggesting predilection to bind cancer cells. This was observed for breast cancer tissue compared to normal breast tissue, and also the highly metastatic COLO357-FG pancreatic cells. Indeed, levels of some pi-6GlcNActransf erases like GCNT1 are significantly upregulated in a number of TCGA (The cancer genome atlas) cancers like kidney chromophobe (10.7-fold, adjusted-p<10'9), uterine corpus endometrial carcinoma (5.6-fold, p<l O’8), prostate adenocarcinoma (3.7-fold, p<10’7), colon carcinoma (1.5-fold, p<10’3) etc.55. Besides solid tumors, there is now evidence that cell-surface carbohydrates are altered during myelodysplastic neoplasms56, and studies with sCore2 may add new dimensions to our understanding of such hematologic malignancies. The expanded use of sCore2 for such clinical investigations is encompassed by this disclosure.

[0098] In conclusion of this Part 1, the disclosure provides a novel sial ogly canbinding protein by engineering GTs, and this lectin displays glycan binding profiles different from other known lectins and antibodies. We also developed a new platform technology for screening GT function. Extension of this approach provides a rational strategy for generating new families of GBPs, based on the carbohydrate acceptor preference of the parent enzyme. This is expected to also result in new GBPs for profiling healthy and disease human tissue, and related expansions to uncover the human glycome.MATERIALS AND METHODS

[0099] Materials. A 25-color immunoprofiling assay cFluor kit (Product code: R7- 40002) was purchased from Cytek Biosciences (Fremont, CA). Monoclonal antibodies (mAbs) against human IgM, CD38, PD-1, CD14, CD16, and CD123 were removed from this panel, in order to accommodate additional fluorescent lectins and to expand the number of gated cell types. These mAbs were replaced by anti-CD14 (Clone: 63D3-BV711, Product code: 367139), anti-CD16 (Clone: 3G8- BV785, Product code: 243045), and anti-CD123 (Clone: 6H6-BV510, Product code: 306021) from BioLegend (San Diego, CA), and anti- CD15 (Clone: HI98-BUV395, Product code: 563872) from BD Biosciences (Franklin Lakes, NJ). Goat anti-human Fey fragment-specific IgG in unconjugated, Horseradish Peroxidase (HRP)-conjugated, AF488 (AlexaFluor 488)-conjugated, and AF647- conjugated forms (Product codes: 109-005-190, 109-035-098, 109-545-098, and 109-605-098, respectively) were from Jackson ImmunoResearch (West Grove, PA). Recombinant human ST3Gall (rhST3Gall, Product code: 6905-GT-020) and Recombinant human P-selectin Fc chimera (P-selectin-Fc, Product code: 137-PS-050) were from R&D Systems (Minneapolis, MN). Unconjugated Peanut Agglutinin (PNA, Product code: L-1070), Maackia Amurensis Lectin II (MALII, Product code: L-1260), Erythrina Cristagalli Lectin (ECL, Product code: L-l 140), Phaseolus Vulgaris Leucoagglutinin (PHA-L, Product code: L-l 110), and H.O.H (Human on Human) Immunodetection kit (Product code: HOH-3000) were from Vector Laboratories (Newark, CA). Gaipi,3GalNAca-pNP (para-nitrophenol) was available from previous work42. CMP-Neu5Ac (cytidine 5 ’-monophospho P-D-N-acetylneuraminic acid) sodium salt (Product code: MC04391) was from Biosynth (Staad, Switzerland). a2-3, 6, 8, 9 Neuraminidase A (sialidase, Product code: P0722) was from New England Biolabs (Ipswich, MA). Sulfo-NHS (N-hydroxysuccinimide) (Product code: 24510), CellTracker Green CMFDA dye (Product code: C2925), and CellTracker Orange CMTMR dye (Product code: C2927) were from ThermoFisher (Waltham, MA). EDC (1- Ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride, Product code: BC25) was from G- biosciences (St. Louis, MO). AZDye 488 NHS (N-hydroxysuccinimidyl) ester (Product code: 1013) and AZDye 594 NHS ester (Product code: 1101) were from Fluoroprobe (Scottsdale, AZ). CF532 Dye SE / TFP Ester, CF594 Dye SE / TFP Ester, and CF532 Dye SE / TFP Ester (Product code: 92104, 92132, and 96067, respectively) were from Biotium (Fremont, CA). Hematoxylin solution (Mayer’s, Modified, Product code: ab220365) was from Abeam (Waltham, MA). Permount mounting medium (Product code: 17986-05) was from Electron Microscopy Sciences (Hatfield, PA). All other chemicals were from Sigma, cell culture reagents from LifeTechnologies / ThermoFisher and molecular biology reagents from New England Biolabs, unless otherwise specified.

[0100] Fluorescent labeling of lectins and antibodies. The buffer used for all lectins or antibodies (PNA, MALII, ECL, PHA-L, and goat anti-human Fey fragment-specific IgG) was exchanged to PBS (phosphate-buffered saline) using Zeba™ Spin desalting columns (7K MWCO, 0.5mL). For labeling, 5-fold molar excess AZDye or CF dye was added to lectins or Abs for Ih at RT in the dark. Reaction was then quenched with 1 / 10 volume of IM Tris, followed by buffer exchange to PBS.

[0101] Molecular Biology. - For constructing pCSCG-Fc-pST3Gall WT, human Fc and pST3Gall were amplified and connected via 5xGS linker sequence by overlap extension PCR. PCR product and pCSCG plasmid from previous study were digested with Agel / BstBI, followed by gel purification and ligation57. For pCSCG-Fc-pST3Gall mutant (H243A, Dead, and individual mutants from Lib2, pCSCG-Fc- CBM40 and pCSCG-Fc-diCBM40), PCR amplification was conducted using primers with 15-20 base pair overlaps and assembledusing NEBuilder Hifi Assembly kit (New England Biolabs) or In-fusion Snap Assembly kit (Takara Bio). pET45b-CBM40 plasmid was kindly provided by Dr. Anne Imberty (University Grenoble Alpes, CNRS, CERMAV)25.

[0102] For constructing surface display form of pCSCG-Fc-pST3Gall, PCR amplification of cytoplasmic tail and transmembrane domain portion of human DPP4 was done using TEMP2 oligonucleotide as a template (Supplementary Table S4). The PCR amplicon was inserted into pCSGC-Fc-pST3Gall using shared Nhel / Xbal sites.

[0103] For constructing pCSCG-Fc-pST3Gall H243A mutant library, PCR amplification of vector backbone was done using 3 IMutaVec FWD and 3 IMutaVec REV (Supplementary Table S4). PCR amplification of mutagenic insert was done in 2 steps. In the 1st step, 3 IMutalns FWD and mutagenic reverse primers were used for Libi followed by gel extraction. Similarly, 3 IMutalns REV and mutagenic forward primers were used for Lib2. In the 2nd step, 3 IMutalns REV and megaprimers from the 1st step were used for Libi construction. Similarly, 31MutaIns_FWD and megaprimers from the 1st step were used for Lib2. After gel extraction of each mutagenic fragment for insert, 3 fragments for Libi and 5 fragments for Lib2 were pooled and subjected to assembly with vector backbone using Hifi Assembly kit. Products from Hifi assembly were electroporated into NEB 10-beta competent E. coli (New England Biolabs) following manufacturer’s instructions. The number of colonies formed on the agar plate was checked so that there was at least lOOx colony representation for each mutant in the library.

[0104] Cell culture. Human embryonic kidney 293T cells (HEK293T) were cultured in Dulbecco’s Modified Eagle Medium (DMEM). HL-60 cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM). COLO357-FG cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium. Calu-3 cells were cultured in DMEM supplemented with 1% MEM non-essential amino acid solution. All media were supplemented with 10 % fetal bovine serum (FBS), 1 % Antibiotic-Antimycotic and 1 % GlutaMAX supplement. All cells were cultured at 37 °C in humidified 5 % CO2 atmosphere.

[0105] CRISPR-Cas9 isogenic clones. HEK293T lacking the human a(l-3)- mannosyl-gly coprotein 2- beta-N-acetylglucosaminyltransf erase MGAT1 (MGAT1-KO) and core-1 synthase CIGalTl (CIGalTl-KO) were previously established26. To make isogenic HEK293T clones lacking glucosaminyl (N-acetyl) transferase- 1 GCNT1 (GCNT-KO), solute carrier family 35 member Al SLC35A1 (SLC35A1-KO), and ST3 P-galactose a(2- 3)sialyltransferase-l ST3Gall (ST3Gall- KO), single-guide RNAs (sgRNAs) targeting these genes were cloned into pX330-U6- Chimeric_BB-CBh-hSpCas9 vector (Addgene, plasmid#42230). A mixture of two plasmids containing sgRNAs targeting distinct sites on the target gene were pooled and transfected into HEK293T wildtype cells using the calcium phosphate method. Isogenic clones for each KO cell were obtained by single-cell FACS sorting. Fluorescently labeled MALII and PNA were used to assist sorting of SLC35A1-KO and ST3Gall-KO cells. For GCNT1, single- cell sorting did not use any markers. After scale-up, genomic DNA was extracted from each cell lysate using PureLink™ Genomic DNA Mini Kit (Invitrogen). Target gene editing sites were PCR amplified, Nextera dual index adaptors were appended, and then 150bp paired-end amplicon sequencing (NGS) was performed using Illumina sequencers to confirm gene editing.

[0106] Molecular surface area calculations. Sialoglycan ligand from 5FRE was overlayed into the original acceptor ligand of 2WNB to predict the sialic acid-binding pocket of pST3Gall. Atoms within 4 A from the ligand were defined as the binding interface between protein and glycan ligands. Crystal structures of all glycan-related proteins co- crystalized with naturally derived glycan ligands except for DANA, a transition state analogue of sialic acid, were from the protein data bank. These were visualized using PyMOL. Molecular surface areas of defined binding interfaces were calculated by get area command. Sampling density was set to 4.

[0107] Fc-fusion protein expression and purification. Plasmids encoding for soluble Fc-fusion proteins were transiently transfected into HEK293T cells using the calcium phosphate method. To this end, cells were plated in 100 mm or 150 mm cell culture petri dishes. After overnight culture, medium was replaced with fresh DMEM 1 h before transfection. 1 / 10 cell culture volume of transfection mixture was prepared by mixing calcium chloride (final concentration; 125 mM) with plasmid DNA followed by addition of 2x HBS buffer (concentrations: 50 mM HEPES, 10 mM KC1, 140 mM NaCl, 1.5 mM Na2HPO4, pH 7.05). 25 or 55 pg plasmid DNA were used for 100 mm and 150 mm cell culture petri dishes, respectively. After treating cells with 25 pM chloroquine, transfection mixtures were added dropwise onto cells. Six hours post-transfection, cell culture medium was replaced with serum-free DMEM lacking phenol red, but with 1 % MEM non-essential amino acid solution, 1 % Insulin-Transferrin-Selenium-ethanolamine, 0.4 g / L AlbuMAX™ Lipid- rich BSA, 1 % Antibiotic-Antimycotic and 1 % GlutaMAX supplement.

[0108] Culture supernatant (10 mL for 100 mm dish and 20 mL for 150 mm dish) was collected 3 days post-transfection, cell debris was removed by centrifugation (3000 g, 3 min) and then the material was concentrated using Amicon Ultra centrifugal filter units (30 kDa MWCO) by 20~40-fold. Concentrated supernatant was dissolved in 10 mL PBS and mixedwith 1 mL NEB Express Ni resin (New England Biolabs) pre-equilibrated with 20 mL PBS. The mixture of resins and supernatant was incubated end-over-end at 4 °C for 15 min. Following centrifugation (800 g, 1 min), the resin was washed with 30 mL PBS containing 10 mM imidazole. Fc-fusion proteins were then eluted with 10 mL PBS containing 200 mM imidazole. The elution fraction was immediately concentrated using Amicon Ultra centrifugal filter units (30 kDa MWCO) and buffer-exchanged using Zeba™ Spin desalting columns (7 K MWCO, 0.5 mL) to PBS.

[0109] Western blot. Cell culture supernatant was mixed with SDS loading buffer. 33 mM DTT was added for runs conducted under reducing conditions. All samples were denatured by heating at 95 °C for 5 min, loaded onto 4-12 % Tris-glycine SDS-PAGE gels, resolved and transferred onto nitrocellulose membranes. Following blocking with 5 % non-fat milk in TBST (20 mM Tris-HCl, 100 mM NaCl, 0.1 % Tween-20) for 1 h at RT, the membrane was incubated at 4 °C overnight in a TBST solution containing 1 :5000 HRP- conjugated goat anti-human Fey fragment-specific IgG and 2 % non-fat milk. Following additional washes, signal was developed using SuperSignal™ West Pico PLUS Chemiluminescent substrate. Images were acquired using ChemiDoc Imaging system (BioRad).

[0110] Fc-protein quantitation using cytometry FLICA. A fluorescence linked immune-coupled assay (‘FLICA’) was used to quantify Fc-protein concentration. To generate microspheres bearing anti- Fc Ab, 80 * 106 carboxylate microsphere beads (3 pm size, Product code: 09850, Polysciences, Warrington, PA) were washed twice using PBS and dissolved into 300 pL MOPS buffer (20 mM MOPS, pH 6.0), followed by addition of 0.25 M EDC and 0.25 M sulfo-NHS (total reaction volume: 500 pL). After 30min incubation at RT, the microspheres were washed five times using PBS, and supplemented with 150 pL goat anti-human Fey fragment-specific IgG (stock: 1.3 mg / mL) in 1 mL PBS. After 3 h at RT, the beads were centrifuged (14000 rpm, 6 min) and resuspended in PBS containing 40 mM ethanolamine at RT for 30 min. The final beads were washed and stored in 1 mL PBS containing 1% BSA at 4 °C prior to use.[OHl] Efalizumab (humanized anti-CDl la, Genentech) was used as a standard for Fc-fusion protein quantification. To this end, Efalizumab serially diluted standards (6.25-200 ng / mL) or Fc-proteins were added to 2 pL FLICA beads in PBS containing 1 % BSA. Following 20 min incubation on ice, the beads were washed and resuspended into PBS containing 1 % BSA and 1 :200 AF488- conjugated goat anti-human Fey fragment-specific IgG (3.75 pg / mL). The samples were placed on ice for 20 min, washed and analyzed using aflow cytometer. Calibration curves created using serial dilution of Efalizumab were used to determine Fc-fusion protein concentrations.

[0112] LC-MS / MS enzymatic analysis. 1 pL PBS (negative control), 1 pL commercial rhST3Gall (positive control), or 1.5 pg / mL PSI, H243A, or Dead were added to the mixture of 15 pL 100 mM cacodylate buffer (pH 6.0), 2 pL Gaip i ,3GalNAca- / NP (5 mM), and 2 pL CMP-Neu5Ac sodium salt (5 mM). Following overnight incubation at 37 °C, the reaction was quenched by addition of 80 pL acetonitrile to each sample at 4 °C for 30 min. These samples were then centrifuged at 4 °C (13000 g, 10 min), 90 pL of the supernatant was collected and then dried using Savant™ SPD131DDA SpeedVac Concentrator (Thermo). All samples were stored at -20 °C prior to LC- MS / MS analysis. Prior to injection, dried samples were dissolved in 50 % methanol (0.1 % formic acid). Each sample was separated on XSelect C18 column (3.5 pm, 4.6 mm * 150 mm) before being subjected to MS / MS analysis using Q-Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo) in positive mode. Flow rate was 0.2 mL / min and the column was maintained at 40 °C while running samples. Mobile phases were (A) MilliQ (0.1 % formic acid) and (B) acetonitrile (0.1 % formic acid). The gradient of liquid chromatography was as follows: (i) 0-40 % B (0-20 min), (ii) 40-100 % B (20-25 min), and (iii) 100-0 % B (25-30 min). Molecular weights used for obtaining XICs (extracted ion chromatogram) were as follows: donor substrate ([M+H]+ = 615.1551), acceptor substrate ([M+H]+ = 505.1670), and product ([M+H]+ = 796.2624).

[0113] Flow cytometry. All flow cytometry assays using live cells were performed in the mileu of HEPES buffer (30 mM HEPES, 110 mM NaCl, 10 mM KC1, 2 mM MgCh, 10 mM glucose, 1.5 mM CaC12, 1 % BSA, pH 7.4) with some of the steps being semi-automated using an OT-2 robot (Opentrons, Brooklyn, NY). In case of sialidase treatment, these cells in 200 pL volume were incubated with 2 pL sialidase (200 units / mL) or 2 pL PBS control (without sialidase) at 37 °C for 1 h, prior to resuspending in 200 pL HEPES buffer. Prior to cell staining, 2 pg / mL Fc-fusion proteins were pre- complexed with AF488- or AF647- conjugated goat anti-human Fey fragment-specific IgG (3 pg / mL) in HEPES buffer on ice for 10 min. During the labeling step, 5 pL cell suspension (107cells / mL) was mixed with 5 pL fluorescently-labeled lectins (5 pg / mL) or pre-complexed Fc-fusion proteins (1 pg / mL) on ice for 20 min. Cell samples were diluted by addition of 90 pL HEPES buffer, washed and analyzed using a BD LSRFortessa™ X-20 flow cytometer (BD Biosciences). For binding assay using Fc-pST3Gall H243A mutants identified from Lib2, Fc-fusion proteins (finalconcentration: 5 pg / mL) and AF488-conjugated goat anti-human Fey fragment-specific IgG (final concentration: 7.5 pg / mL) were used.

[0114] In variations of the above protocol, in dose-dependence studies, Fc-fusion proteins were pre- complexed with AF488-conjugated anti-human Fey fragment-specific IgG at 2:3 ratio as above and added to cells, prior to fluorescence detection. In other cases, 4* 105HL60 cells were seeded in 6-well plates prior to addition of 50 mM sodium chlorate in HEPES buffer for 2 days in standard tissue culture incubators. Cells were then resuspended at 107 / mL, and their ability to bind 1 pg / mL of various Fc-fusion proteins (PSI, H243A, and P- selectin-Fc) was measured using the above method.

[0115] Glycan microarray. Binding assay using Fc-proteins (PSI and H243A) and glycan microarrays was performed following the protocol described at the NCFG website (‘Glycan binding assay with fusion or epitope tagged proteins’ : https: / / research.bidmc.org / ncfg / protocols / glycan-binding- assay-fusion-or-epitope-tagged- protein). Briefly, hydrated CFG slides were incubated with 50 or 5 pg / mL PSI or H243A in a humidified chamber for 1 h at RT. After washing, the slides were incubated with AF488- conjugated anti-human IgG Fc in a humidified chamber for 1 h at RT. Slides were then scanned to detect fluorescence following washing.

[0116] CRISPR forward genetic screen using magnetic cell sorting. 500 pL M- 450 Epoxy Dynabeads (2 x 108beads, Product code: 14011) were washed three times with 1 mL PBS. 100 pg goat anti- human Fey fragment-specific IgG (200 pg / mL) was then added and incubated end-over-end overnight at RT. The reaction was then quenched using 25 mM ethanolamine for 30 min at RT. Following three additional washes using 1 mL PBS containing 1 % BSA, beads were stored at 4 °C until use.

[0117] 5 X 106HL60 glycoCRISPR library cells, available from previous work33, were suspended in HEPES buffer at 107cells / mL. 10 pg / mL Fc-fusion proteins were added to these cells for 20 min at 4 °C. Following washing with HEPES buffer, 5 x 106Dynabeads bearing anti-Fc Ab were added to the cells in 2.5 mL volume for 30 min at 4 °C. The cellbead mixture was then separated by placing sample tubes in MagnaRack™ -magnetic separation racks (Invitrogen) for 2 min and resulting supernatant was collected. The collected cells were then washed and cultured in IMDM culture medium for scale-up and additional sorting. Three rounds of cell enrichments were conducted in this manner to obtain cell populations that did not bind Fc-proteins.

[0118] Genomic DNA was extracted from these cells (either 5 x io6unsorted or magnetically sorted HL60-lib cells) using PureLink™ Genomic DNA Mini Kit. Thesequences proximal to the sgRNA site were amplified as previously described33, and this was subjected to NGS using Illumina MiSeq kit (2 million reads, 150 bp paired end). sgRNA enrichment analysis was performed using MAGeCK package58.

[0119] Transient surface-display of Fc-pST3Gall proteins. For surface display of Fc-fusion proteins, HEK293T cells plated in 6-well plates were transiently transfected using the above calcium phosphate method, only with 3 pg plasmid DNA / well. At 6 h, the medium was replaced with DMEM with phenol red supplemented with 10 % FBS, 1 % Antibiotic- Antimycotic and 1 % GlutaMAX. Following overnight culture, cells were tripsinized and used for binding assay.

[0120] For cell-cell interaction assay for checking cis-interaction, HEK293T cells untransfected, mock transfected or transfected to display TM-PS1, TM-H243A, or TM-Dead were prepared. While some cells were sialidase treated, others served as PBS control. Here, the transfected cells (at 107cells / mL) were labeled with CellTracker Orange CMTMR dye (20 nM), and untransfected control HEK293T cells were labeled with CellTracker Green CMFDA dye (100 nM) by incubating for 30 min at 37 °C. After washing three times, 5 pL of green cells were mixed with 5 pL orange surface-displaying cells for 20 min at RT. Flow cytometer was then used to measure the formation of green-orange cell adhesion events.

[0121] Fluorescent sialylated core-2 polyacrylamide (PAA) polymer preparation.GlcNAcpi- 6(Gaipi-3)GalNAca-sp3-PAA-fluo (core 2-PAA-FP, MW-30 kDa, Product code: 0078-FP) was purchased from GlycoNZ (Auckland, New Zealand). This polymer was dissolved in PBS at 1 mg / mL. 20 pL of this reagent was then mixed with 1 pL commercial rhST3Gall and 4 pL CMP- Neu5Ac sodium salt (final concentration: 800 pM) overnight at 37 °C. The reaction was stopped by removing excess unreacted CMP-Neu5Ac using Amicon Ultra centrifugal filter units (3 kDa MWCO). PAA-polymer concentration was determined by comparing the absorbance at 488 nm of the original Core-2-PAA-FP, with the reagent following sialylation.

[0122] Surface display and selection of Fc-pST3Gall H243A mutant libraries.Lentivirus pools were produced for stable surface-display of Libi and Lib2 constructs using the pCSCG-Fc-pST3Gall H243A mutant libraries. To make virions, the plasmid libraries were transiently transfected into HEK293T cells using the calcium phosphate method (see above), along with packaging plasmid (psPAX2, Addgene_# 12260) and envelope plasmid encoding for VSV-G (pMD2.G, Addgene_#12259). Transfections were performed in 150 mm cell culture petri dishes using 55 pg total plasmid, with the molar ratio of transfer (pCSCG), packaging, and envelope plasmids varying as 2:2: 1. Six hours post-transfection,the cell culture medium was replaced with 20 mL OptiMEM, with first virus batch being collected after overnight culture. Fresh 20 mL OptiMEM with 10 mM sodium butyrate was then added, with second virus batch was collected the next day. Both viral batches were then pooled, centrifuged (2000 g, 2 min) to remove debris, filtered using polyethersulfone (PES) syringe filters (0.45 pm), and subjected to ultracentrifugation (50,000 g, 2 h, 4 °C). The resulting lentivirus pellet was dissolved in 100 pL OptiMEM, aliquoted and stored at -80 °C.

[0123] For lentiviral transduction, HEK293T SLC35A1 KO cells were plated in 60 mm cell culture petri dishes. Cell culture medium was replaced with DMEM supplemented with 8 pg / mL polybrene for 5 min, and the lentivirus library pool was then serially diluted onto the cells. Cell culture medium was replaced to DMEM after overnight incubation. Following further overnight incubation, the trypsinized transduced cells were mixed with AF647-conjugated goat anti-human Fey fragment- specific IgG for 20 min on ice. Cell surface Fc expression was measured using flow cytometry as a surrogate measure of % viral transduction. The viral titer resulting in 20-30 % Fc-positive cells was scaled up, and FACS sorted to constitute the mutant libraries.

[0124] Mutants in Libi and Lib2 displaying enhanced binding to sialyl core-2-PAA- FP were sorted and sequenced. To this end, 107library cells / mL suspended in HEPES buffer were treated with sialidase as above. These were mixed with sialyl core-2-PAA-FP (5 pg / mL) and AF647- conjugated goat anti-human Fey fragment-specific IgG (0.075 pg / mL) for 20 min on ice in ~ 500pL volume. AF647-positive cells with high sialyl core-2 PAA-FP binding were sorted. Following scale up, genomic DNA was extracted from 5 * 106unsorted or FACS sorted HEK293T Libi and Lib2 cells. The region spanning the pST3Gall mutations was PCR amplified, barcoded and subjected to NGS using Illumina MiSeq Micro kit (4 million reads, 150 bp paired end). Mutant enrichment analysis was performed using Seqkit59. First, the read number of each mutant was divided by the total read number to calculate the read number ratio. Negative, 1st positive and 2nd positive enrichment values were then calculated by dividing the read number ratio of these samples by the read number ratio of sample before sorting. Enrichment scores were calculated by multiplying 1st positive and 2nd positive enrichment values and dividing it by negative enrichment value.

[0125] Spectral flow cytometry. Fresh human blood was obtained from healthy nonsmoking adult volunteers by venipuncture, following protocols approved by the University at Buffalo Health Sciences Institutional Review Board (HSIRB). 5 mM EDTA was used as anticoagulant. Buffy coat was obtained from this blood by centrifugation (1000 g, 12 min). Red blood cells (RBC) were lysed from buffy coat by addition of 30 mL RBC lysis buffer (10mM KHCO3, 155 mM NH4Q, 1 mM Na2EDTA) for 10 min at RT, followed by washing with 5 mL HEPES buffer. The resulting white blood cells were resuspended at 25 * 106cells / mL, and incubated with the 23 anti-human leukocyte fluorescent mAb panel. 1.5 x 106cells were labeled in this manner for 30 min at 4 °C in 125 pL volume, both for the mixed panel and single-stain Ab controls. Labeled cells were washed using HEPES buffer and fixed overnight using 0.5 % paraformaldehyde at 4 °C. The following day, the cells were again washed using HEPES buffer, and incubated with 1 pg / mL CF532-PNA, CF594-MALII, or Fc-proteins precomplexed with 1.5 pg / mL CF700-goat anti-human Fey fragment-specific IgG for 10 min at 4 °C. Cell labeling was performed for 30 min at 4 °C, prior to washing using HEPES buffer and data acquisition on Cytek Aurora spectral flow cytometer. Unmixing was done by SpectroFlo software and compensation correction using FlowJo (BD Biosciences). tSNE plots were created using Bokeh (bohek.org). For heatmaps, the medians for each population of lectins were scaled from 0 to 1 before plotting the results.

[0126] Tissue microarray analysis. Paraffin-embedded deidentified human tissue microarrays were kindly provided by the Cooperative Human Tissue Network (CHTN) at the University of Virginia (CHTN_Norm3 and CHTN_Test3). All tissues were deparaffinized using xylene (1 x 10 min) and rehydrated with 100 %, 95 %, 70 % and 50 % EtOH (1 x 10 min for 100 % EtOH and 1 x 2 min for others) and MilliQ water (1 x 1 min). Following antigen retrieval in 10 mM citrate buffer (pH 6.0) at 96 °C for 45 min and washing with PBS (2 x 5 min), endogenous peroxidase activity was blocked using 3 % hydrogen peroxide at RT for 10 min, and washed with PBS (2 x 5 min). Tissue sections were treated with sialidase (200 U / mL) in PBS (0.1 % BSA) at 37 °C for 1 h. The tissue samples were then washed with PBS (2 x 5 min), before being blocked with protein block solution provided with the H.O.H immunodetection kit. Tissue sections were then incubated with either 20 pg / mL sCore2 or Dead pre-complexed with 15 pg / mL HRP-goat anti -human Fey fragment-specific IgG overnight at 4 °C. Pre-complexing was performed by mixing sCore2 or Dead and HRP-goat anti-human Fey fragment-specific IgG at appropriate ratio in PBS (0.1 % BSA), incubating at RT for 30 min, adding solution-B from H.O.H immunodetection kit, and then incubating at RT for 30 min. Combination of pre-complex method and human-on-human detection kit reduces human Fc- derived background for human tissue60. Following washing with PBS (2 x 5 min), tissues were incubated with ImmPACT DAB (3,3 ’-diaminobenzidine) EqV Reagents in immunodetection kit for 5 min to develop brown-colored precipitates. These sections were then washed with PBS (2 x 5 min), counterstained using 50 % hematoxylin diluted with MilliQ, dehydrated with 95 % EtOH, 100 % EtOH and xylene (1 x 1 min for each) andcovered using permount mounting medium. Whole slides were scanned using Aperio Versa 200 (Leica Biosystems).

[0127] Statistical analysis. All data are presented as mean ± standard deviation (STD). Dual comparisons were performed using two-tailed unpaired Student’s Ltest. Multiple comparisons were performed using one-way ANOVA followed by the Tukey post-test. p<0.05 was considered to be statistically significant. Number of repeats are specified in individual panels.

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Obligatory requirement of sulfation for P-selectin binding to human salivary gland carcinoma Acc-M cells and breast carcinoma ZR-75-30 cells. J Immunol 168, 1690- 1696 (2002). https: / / doi.org: 10.4049 / jimmunol.168.4.169032 Kelkar, A., Groth, T. & Neelamegham, S. Forward Genetic Screens of Human Glycosylation Pathways Using the GlycoGene CRISPR Library. Curr Protoc 2, e402 (2022). https: / / doi.org: 10.1002 / cpzl .40233 Zhu, Y., Groth, T., Kelkar, A., Zhou, Y. & Neelamegham, S. A GlycoGene CRISPR- Cas9 lentiviral library to study lectin binding and human glycan biosynthesis pathways. Glycobiology 31, 173- 180 (2021). https: / / doi.org: 10.1093 / glycob / cwaa07434 Mccafferty, J., Griffiths, A. D., Winter, G. & Chiswell, D. J. Phage Antibodies - Filamentous Phage Displaying Antibody Variable Domains. Nature 348, 552-554 (1990). https: / / doi.org:DOI 10.1038 / 348552a035 Boder, E. T. & Wittrup, K. D. Yeast surface display for screening combinatorial polypeptide libraries. 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Front Immunol 9, 2857 (2018). https: / / doi.org: 10.3389 / fimmu.2018.0285741 Barran, P., Fellinger, W., Warren, C. E., Dennis, J. W. & Ziltener, H. J. Modification of CD43 and other lymphocyte O-glycoproteins by core 2 N- acetylglucosaminyltransferase. Glycobiology 7, 129-136 (1997). https: / / doi.org: 10.1093 / glycob / 7.1.12942 Gupta, R., Matta, K. L. & Neelamegham, S. A systematic analysis of acceptor specificity and reaction kinetics of five human alpha(2,3)sialyltransferases: Product inhibition studies illustrate reaction mechanism for ST3Gal-I. Biochem Biophys Res Commun 469, 606-612 (2016). https: / / doi.org:10.1016 / j.bbrc.2015.11.13043 Chandrasekaran, A. et al. Glycan topology determines human adaptation of avian H5N1 virus hemagglutinin. Nat Biotechnol 26, 107-113 (2008). https: / / doi.org: 10.1038 / nbtl37544 Stevens, J. et al. Glycan microarray analysis of the hemagglutinins from modern and pandemic influenza viruses reveals different receptor specificities. J Mol Biol 355, 1143-1155 (2006). https: / / doi.org: 10.1016 / j.jmb.2005.11.00245 Bull, C. et al. Probing the binding specificities of human Siglecs by cell-based glycan arrays. Proc Natl Acad Sci U S A 118 (2021). https: / / doi.org: 10.1073 / pnas.202610211846 Jung, J. et al. Carbohydrate Sulfation As a Mechanism for Fine-Tuning Siglec Ligands. ACS Chem Biol 16, 2673-2689 (2021). https: / / doi.org: 10.1021 / acschembio.lc0050147 Ortiz-Soto, M. E. & Seibel, J. Expression of Functional Human Sialyltransferases ST3Gall and ST6Gall in Escherichia coli. PLoS One 11, e0155410 (2016). https: / / doi.org: 10.1371 / joumal. pone.015541048 Luley-Goedl, C. et al. Combining expression and process engineering for high-quality production of human sialyltransferase in Pichia pastoris. J Biotechnol 235, 54-60 (2016). https: / / doi.org: 10.1016 / j.jbiotec.2016.03.04649 Skretas, G. et al. Expression of active human sialyltransferase ST6GalNAcI in Escherichia coli. Microb Cell Fact 8, 50 (2009). https: / / doi.org: 10.1186 / 1475-2859-8-5050 Chen, H. et al. Efficient, continuous mutagenesis in human cells using a pseudo-random DNA editor. Nat Biotechnol 38, 165-168 (2020). https: / / doi.org: 10.1038 / s41587-019- 0331-851 Hess, G. T. et al. Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells. Nat Methods 13, 1036-1042 (2016). https: / / doi.org: 10.1038 / nmeth.403852 Berman, C. M. et al. An Adaptable Platform for Directed Evolution in Human Cells. J Am Chem Soc 140, 18093-18103 (2018). https: / / doi.org: 10.1021 / jacs.8bl093753 Reily, C., Stewart, T. J., Renfrow, M. B. & Novak, J. Glycosylation in health and disease. Nat Rev Nephrol 15, 346-366 (2019). https: / / doi.org: 10.1038 / s41581-019- 0129-454 Kawahara, R. et al. Community evaluation of glycoproteomics informatics solutions reveals high- performance search strategies for serum glycopeptide analysis. Nat Methods 18, 1304-1316 (2021). https: / / doi.org: 10.1038 / s41592-021-01309-x55 Groth, T., Diehl, A. D., Gunawan, R. & Neelamegham, S. GlycoEnzOnto: a GlycoEnzyme pathway and molecular function ontology. Bioinformatics 38, 5413-5420 (2022). https: / / doi.org: 10.1093 / bioinformatics / btac70456 van Spronsen, M. F. et al. Dysregulation of developmental and cell type-specific expression of glycoconjugates on hematopoietic cells: a new characteristic of myelodysplastic neoplasms (MDS). Leukemia 37, 702-707 (2023). https: / / doi.org: 10.1038 / s41375-022-01784-x57 Lo, C. Y. et al. Cell surface glycoengineering improves selectin-mediated adhesion of mesenchymal stem cells (MSCs) and cardiosphere-derived cells (CDCs): Pilot validation in porcine ischemia-reperfusion model. Biomaterials 74, 19-30 (2016). https: / / doi.org: 10.1016 / j. biomaterials.2015.09.02658 Li, W. et al. MAGeCK enables robust identification of essential genes from genomescale CRISPR / Cas9 knockout screens. Genome Biol 15, 554 (2014). https: / / doi.org:10.1186 / sl3059-014-0554-459 Shen, W., Le, S., Li, Y. & Hu, F. SeqKit: A Cross-Platform and Ultrafast Toolkit for FASTA / Q File Manipulation. PLoS One 11, e0163962 (2016). https: / / doi.org: 10.1371 / joumal. pone.016396260 Goodpaster, T. & Randolph-Habecker, J. A flexible mouse-on-mouse immunohistochemical staining technique adaptable to biotin-free reagents, immunofluorescence, and multiple antibody staining. J Histochem Cytochem 62, 197- 204 (2014). https: / / doi.org: 10.1369 / 0022155413511620PART 2Mammalian cell surface display for protein engineering of glycosyltransferase activity

[0129] As discussed above in Part 1, high-throughput methods have been needed to study structure-function relationships of mammalian glycosyltransferases due to their essential role in assembling the glycocalyx. Mutations in these enzymes result in Congenital Disorders of Glycosylation, and improvements of enzyme properties could yield new biocatalysts for biotechnology applications. This Part 2 expands the description of the novel cell-based glycosyltransferase activity assay using mammalian surface display that was also discussed in Part 1. The disclosure demonstrates that coupling this approach with strain-promoted click chemistry enables rapid quantification of glycosyltransferase activity. Screening of 1680 different pig ST3Gall (pST3Gall) mutants yielded a(2,3)sialyltransferases with improved enzymatic properties. Using endogenous cell-surface substrates, the method is expected to be extendable to diverse human sialyltransferases including but not necessarily limited to ST6Gall, ST3Gal4 and ST6GalNAc-II. The described approach could also be used to screen putative CAZymes (Carbohydrate-Active enZYmes) predicted based on informatics analysis. The disclosure thus provides a provides a facile, robust, high-throughput, low-cost method to study glycosyltransferase.

[0130] As discussed above in Part 1, glycosylation is an essential post-translational modification in mammals that mediates molecular recognition1,2, immune response3, and additional fundamental biological processes4. Glycan structures are complex due to their branched pattern and isomeric configuration which results in the presentation of heterogenous complex carbohydrate structures at single glycosylation sites on protein and lipid scaffolds. Such assembly is regulated by the concerted action of a family of enzymes called glycosyltransferases, primarily in the endoplasmic reticulum (ER) and Golgi compartments5,6. In humans, this is a set of 200+ genes that form a range of complex extracellular carbohydrate structures, and simpler modifications inside cells7. Due to their unique substrate specificity that distinguishes between glycan stereoisomers, engineering these enzymes may result in new avenues for engineering therapeutic proteins and vaccine8’9. Understanding the molecular features regulating glycosyltransferase activity is also important during the diagnosis of congenital disorders of glycosylation (CDG), as some mutations may have only minor effects on enzyme activity whereas other point mutations could result in clinical phenotype10,11. In order to catalog the impact of individual amino acid substitutions or indels on enzyme activity, a preferable assay would be performed in a high-throughput manner. This approach is described herein.

[0131] There are several hurdles that limit the ability to transform conventional glycosyltransferase assay methods for high-throughput structure-function analysis. First, glycosyltransferases have typically been previously expressed in soluble form for activity assays, which means that mutations need to be individually introduced and proteins are separately produced. Additionally, enzyme expression is commonly complicated when using heterologous hosts like bacteria where mammalian glycosyltransferases often appear misfolded within inclusion bodies12'14, and yeast expression systems where the glycosyltransferases may be modified with non-natural oligomannose structures that impact their function15. Second, specialized bioorganic methods were previously needed to obtainthe acceptor substrates in sufficient amounts for such assays. Such complex carbohydrate substrates are often precious, with limited commercial availability and high costs that are out of reach of non-specialist laboratories16. Third, the measurement of glycosyltransferase activity often relies on radioactivity17, colorimetry18,19, or liquid-chromatograph based approaches. While these methods are useful for estimating Michaelis-Menten constant (KM) and related parameters, these are not ideal for high-throughput screens as they rely on individual purified enzymes and acceptor substrates. Some methods have been developed to overcome these limitations. For example, the cell-based selective exoenzymatic labeling (SEEL) method utilizes cell surface glycans as acceptor substrates20'22. However, this method also relies on soluble glycosyltransferases for catalytic activity, which limits scaling to high- throughput format. FRET-based intracellular sialyltransf erase activity measurements have been developed, but it is complex to determine the specific sialyltransferases that contribute to the FRET signal23.

[0132] To overcome the above limitations, this disclosure provides a novel technology to measure mammalian glycosyltransferase activity following surface display on human cells. In order to mimic the natural presentation of glycosyltransferases that express with an N-terminal transmembrane domain and C-terminal catalytic domain within the ER / Golgi lumen, we incorporate the transmembrane and cytoplasmic domains of two Type-II transmembrane proteins, CD94 / NKG2 and DPP4 (dipeptidyl peptidase-4) at the N-terminus of Fc-fusion glycosyltransferases. This enables both glycosyltransferase presentation in the ER / Golgi lumen, and cell surface display. Click chemistry with azido derivatized nucleotidesugars is then used to quantify enzyme activity using flow cytometry. Preferred methods are presented in this Part 2, demonstrating the use of this technology for screening sialyltransferases mutations en mass, assaying sialyltransferases from different organisms reported in the CAZy (Carbohydrate-active enzymes) database, and also diverse sub-types of human sialyltransferases. While this Example focusses on sialyltransferases, the technology is expected to be scalable to other enzyme families also.RESULTS

[0133] Surface displayed pST3Gall (TM-PS1) is enzymatically active within cells: To enable the development of a mammalian surface display platform that mimics the natural presentation of glycosyltransferases, we fused the cytoplasmic and transmembrane domains of two Type II proteins, CD94 and DPP4, at the N-terminus of pig ST3Gall (pST3Gall or PSI). This construct also contained a human Fc tag for quantification ofsurface display, and a Gly-Ser flexible hinge (Fig. 17a, Fig. 23a). The final constructs are abbreviated TM(CD94)-PS1 and TM(DPP4)-PS1, with ‘TM-PS1’ representing the entire class of such surface-displayed proteins. A variant of this also contained C-terminal FLAG. We hypothesized the expression of these TM- PSI constructs would result in cell surface expression with the C-terminal catalytic enzyme facing both the ER / Golgi lumen and the extracellular space24.

[0134] Display of TM-PS 1 catalytic domain in the ER / Golgi lumen would result in sialylation of endogenous cellular proteins. To test this, CRISPR-Cas9 knockouts lacking ST3Gall activity (‘ST3Gall-KO’) were developed on the wild-type HEK293T (‘HEK-WT’) background (Fig. 17b). While HEK-WT bound high levels of the MAL-II due to expression of a(2-3)sialylated O-glycans [i.e. Neu5Ac(a2-3)Gal(pi-3)GalNAcal-Ser / Thr], ST3Gall- KO display reduced MAL-II binding but high PNA recognition as this lectin binds desialylated Gal(pi-3)GalNAcal-Ser / Thr. Different TM- PSI constructs were expressed on the ST3Gall-KO with anti-Fc or anti-FLAG being used to measure surface display and the lectins used to assay for enzyme activity (Fig. 17c). As seen, expression of TM(DPP4)-PS1 and TM(DPP4)-PS1-FLAG both resulted in a(2-3)sialylation of endogenous O-glycans as evinced by increased binding of MAL-II and reduction in PNA binding. This was particularly clear upon gating the top -20% of cells in gate P4, either based on anti-Fc or anti-FLAG antibodies (Abs). This shift in the pattern of lectin binding was observed both upon using DPP4 and CD94 as the anchor peptide (Fig. 17d). Additionally, both mutating a key catalytic histidine residue (H243 A) that binds CMP-Neu5Ac and incorporating multiple mutations in the acceptor substrate binding region (Q49A / Y174A / Y210F in ‘Dead’) abolished enzyme activity (Fig. 17c, 17d). MAL-II binding increases with surface Fc expression (Fig. 17e). Together, these data demonstrate that the catalytic domain of TM-PS 1 is active in intracellular compartments.

[0135] Click chemistry-based one-pot multienzyme (OPME) strategy detects extracellular activity of TM-PS1: We developed a click chemistry strategy to determine if TM-PS 1 displays active enzyme on the cell surface (Fig. 18a). Here, cells transfected with TM-PS 1 or variants were treated with sialidase to expose the acceptor substrates, previously capped with sialic acid on the cell surface (Fig. 18a). These cells were then incubated with Neisseria meningitidis CMP-sialic acid synthase (NmCSS, Fig. 23b), CTP and 9-azido- Neu5Ac (Neu5Ac,9Az) to generate CMP-Neu5Ac,9Az in situ. The presence of active surface displayed TM-PS 1 then enabled the formation of azido derivatized cell-surface sial ogly cans that could be quantified using either Fluorescent DBCO (DBCO-AF488) or DBCO-biotinfollowed by fluorescent anti-biotin antibodies. Fig. 24 confirms the expression and purification strategy for NmCSS, along with the ability to perform OPME upon mixing NmCSS and commercial recombinant hST3Gall with CTP, Neu5Ac,9Az and synthetic acceptor Gaipi,3GalNAca- / ?-nitrophenol. Here, in LC-MS / MS studies, the expected product (Neu5Ac,9Aza2,3Gaipi,3GalNAca- / ?-nitrophenol) was only formed upon addition of both NmCSS and hST3Gall, with the reaction going to near completion withing 1 h. Neu5Ac,9Az was simultaneously consumed. As expected, NmCSS or hST3Gall alone did not generate the reaction product (Fig. 24c, 25).

[0136] Similar observations were made upon expression of TM(DPP4)-PS1 and additional variants on HEK-WT cells. Here, flow cytometry showed increased DBCO-AF488 and AF488-anti -biotin signal in cells expressing high levels of TM(DPP4)-PS1, suggesting the transfer of azido- derivatized sialic acid to cellular acceptors using the immobilized active enzyme. Such signal was, however, absent upon using catalytically dead TM(DPP4)-H243 A and TM(DPP4)-Dead (Fig. 18b, c). Upon comparing DBCO-AF488 and DBCO-biotin + AF488-anti-biotin with respect to wild-type cells (Fig. 18b), the AF488-anti-biotin strategy was superior. This is because the background signal of DBCO-AF488 with respect to the parent cell is higher as the molecule displays non-specific cell permeability. This background is, however, absent when using AF488 anti-biotin as antibodies are not cell permeable. In a final control, all components (cell-surface sialidase treatment, CTP, Neu5Ac,9Az, NmCSS and DBCO-biotin) were found to be necessary to gain sufficient signals with TM(DPP4)-PS1 (Fig. 18d, Fig. 26). The multienzyme click-chemistry strategy provides a facile method to assay the catalytic activity of surface-displayed glycosyltransferases.

[0137] High-throughput screening of sialyltransferase mutant libraries using mammalian surface- display technology: We determined if the surface-display technology could be applied to screen a large number of site-directed mutations in glycosyltransferases, using TM(DPP4)-PS1-FLAG as a case study (Fig. 19a). Thus, NNK mutations were implemented at selected pST3Gall sites in TM(DPP4)-PSl-FLAG to permute the original amino acid to all 20 natural amino acids (Fig. 27). Such mutations were implemented at residues that surround the sialic acid binding pocket. Altogether, a library of 1680 protein mutants were surface-expressed with 440 mutants in Libi that encompass the N91-S138 region of pST3Gall, and 1240 mutants in Lib2 within S212-T257. These proteins were individually expressed on ST3Gall-KO cells by transduction of library lentivirus at low multiplicity of infection (MOI-0.3). After selection, 11.1% of Libi cells expressed active enzyme, while this percent was higher at 23.4% in Lib2 cells (Fig. 28). A strong correlationwas observed between N-terminal Fc and C- terminal FLAG expression in PSI, Libi and Lib2 cells, suggesting that mutations in the pST3Gall catalytic domain do not impact expression of the transmembrane protein on cell surface (Fig. 28b).

[0138] FACS sorting of Libi and Lib2 cells was performed based on either MAL-II lectin binding or DBCO-biotin followed by fluorescent anti-biotin binding. This enabled selection of cell populations that expressed either active or inactive pST3Gall. In general, the percent of cells expressing active pST3Gall as measured based on MAL-II increased from 19.4% in the starting Libi population to 47.9% and 84.9% following either one (1stPos) or two (2ndPos) positive enrichment cycles, respectively (Fig. 29). This same percent increased from 27.9% to 47.7% to 83.1% upon enriching active pST3Gall clones in Lib2. Similar trends were observed upon using anti-biotin for selection (Fig. 29). Genomic DNA was extracted from these sorted cells along with unsorted parent cells and negative selected populations (1stNeg). PCR amplicons were generated for each of these populations to capture the genomic loci containing mutations, and these were subject to NGS. Enrichment values were calculated for each sorted cell population compared to parent cells (Fig. 30).Enrichment scores combined data from multiple sorts to quantify the effect of site-specific mutations on pST3Gall activity (Fig. 19b). Upon visualizing the results in heatmaps, it is apparent that most mutations at N91, N114, Y135, S138, S212, T213 and T257 were depleted in the positive sorts suggesting that they are essential for enzyme activity. Mutations in the pST3Gall disordered loop, R255R / K256X, R255K / K256X and A253X / F254W, were enriched in both the MALII and DBCO-biotin + anti -biotin sorts, and depleted in 1stNeg samples. These analyses suggest that positively charged amino acids are preferred at R255 and bulky aromatic amino acids at F254. Also, the disordered loop may be an attractive mutagenesis site to tune enzyme activity. Finally, a direct correlation was observed upon comparing the enrichment values and enrichment score of results from the MAL-II method with respect to the click-chemistry protocol strategy (Fig. 19c). This is an expected result and it partially validates the DBCO-biotin approach.

[0139] Fc-pST3Gall K256V exhibits improved reaction parameters compared to PSI: (K256 is in relation to SEQ ID NO: 1). The disclosure includes SEQ ID NO: 1 with a mutation of position 256 to an amino acid that is not K. In an example, the mutation is K256V.

[0140] Volcano plot analysis using 2ndPos samples identified several K256X mutants that potentially have higher activity compared to PSI (Fig. 19d, Supplementary Table SI). To validate this finding, the top six hits (K256F, R255K / K256Y, K256Y, K256V, K256T,and K256Q) were expressed as soluble Fc- proteins in HEK293T. Western blots (Fig. 20a) and silver stains (Fig. 20b) verify the size and purity of the expressed glycoproteins. Enzyme activity measurements were performed in ELISA format based on release of free phosphate from CMP, a side product of the sialyltransferase reaction. In some studies, CMP-Neu5Ac concentration was varied while maintaining acceptor Gaipi,3GalNAca-Alloc at 50 pM, whereas acceptor was varied in other studies while keeping CMP-Neu5Ac at 50 pM (Fig. 20c, Fig. 31). Among the mutants, all proteins exhibited similar extents of reaction compared to PSI, except K256F was reduced by half and K256Q did not form product. Reaction parameters were calculated for several mutants K256F, K256V, and K256T. The KM for these enzymes with respect to the acceptor was reduced with respect to PSI according to K256V(17.7 pM) < K256F(30.7 pM) < K256T(43.5 pM) < PS1(58.5 pM) (Fig. 20d, e). The KM with respect to donor was also reduced in several cases: K256V(38.8 pM) < K256T(52.3 pM) < PS1(85.9 pM). These findings suggest that mutations in the disordered loop enhance the affinity of the enzyme for both the donor and acceptor substrates. In this regard, K256 is positioned at the entrance of both substrates into the pocket (Fig. 20f), so hydrophobic amino acids might be more favored to recruit carbohydrate ligands than charged amino acids. These favorable interactions may drive faster overall enzymatic reactions.

[0141] Signal enhancement of click chemistry-based enzyme activity measurement allows profiling of diverse sialyltransferases: We determined if click chemistry-based glycosyltransferase activity measurements can be made more sensitive, as this would be necessary to profile surface displayed sialyltransferases that either have low activity or reduced acceptor prevalence on cell surface. Two strategies were tested: (i) increasing the concentration of reactants used both for sialylation and biotinylation, and (ii) 2-step detection using unconjugated mouse anti-biotin to bind DBCO-biotin followed by addition of fluorescent goat anti- mouse IgG to amplify the signal (Fig. 21a). Both approaches were successful as doubling the concentration of all reactants increased cellsurface sialylation by 3.5-fold, and going from a l- step detection to 2-step detection strategy also increased signal-to-noise by 3.3-fold (Fig. 21b, c). Compared to the 1-step method with the original reactant concentration, thus, the signal-to-noise ratio was increased by 3.5-fold upon using both the 2-step method and doubling reactant amounts. The relation between cellsurface Fc-expression and Neu5Ac,9Az incorporation in cells was linear, confirming that this method can be used for quantitative assays. In negative controls, no increase of baseline signal was observed upon using inactive surface-displayed H243 A enzyme under any of the experimental conditions. Measurable signal was also absent upon omitting the anti-biotinantibody. In further studies on optimal labeling conditions, we noted that 30-60 min reaction time for both sialylation and biotinylation was sufficient to obtain high signal (Fig. 21d). Biotinylation incubation temperature (RT versus 37 °C) did not have a marked effect of the final measurement (Fig. 32).

[0142] We determined if the 2-step strategy would allow us to also profile the enzyme activity of other GT29 human sialyltransferases, hST6Gall, hST3Gal4, and hST6GalNAc2. The catalytic domains of these enzymes (A89-hST6Gall, A42-hST3Gal4, and A62- hST6GalNAc2) were thus expressed as surface-displayed Fc-fusion proteins after truncating the N-terminal transmembrane and cytoplasmic regions (Fig. 33)25. Based on pST3Gall H243A (with residue numbering as discussed above and in context of SEQ ID NO:2) which lacks enzymatic activity, putative inactive mutants were also created for each of these other sialyltransferases to serve as negative controls: hST3Gal4 H294A, hST6GalNAc2 H336A and hST6Gall Y354A. In the last case, as the conserved His is absent in ST6Gal family enzymes, the adjacent Tyr was modified as this may form hydrogen bonds with CMP in donor substrate based on crystal structure (Fig. 34a, Supplementary Table S2)26’27Each of these proteins was surface displayed on wild-type HEK293T cells. Following sialidase treatment to generate cell-surface acceptor substrates, enzyme activity was measured using the 2-step method using different reactant concentrations (Fig. 21e, Fig. 34b, c). As seen, enzyme activity for hST6Gall was readily detected, though the Y354A mutation only partially reduced enzyme activity. Higher concentration of reactants was preferred for facile detection of hST3Gal4 and hST6GalNAc2 activity, with His mutations abolishing enzyme activity. Methods were developed for quantitative sialyltransferase assays using surfacedisplay that could be streamlined to assay diverse glycosyltransferase structure-function relationships.

[0143] Enzymatic activity measurement of ST3Gall from diverse organisms:Due to a variety of activity type involving substrate recognition and bond formation through evolution28, 29, exploring the activity of enzymes classified in the same enzyme family but from different organisms is quite attractive to discover enzymes that display unprecedented types of activity. Although several studies systematically examining expression and activity of soluble glycosyltransferases from multiple organisms have been reported30, the methods of this disclosure are expected to serve as an assay to achieve this type of experiment in a high- throughput manner without a large scale expression, purification, and concern about storing the recombinant proteins properly retaining the enzyme activity. To show the utility of the methods, first we performed bioinformatics analysis of ST3Gall from 12 different organisms,all of which have not been characterized based on Carbohydrate Active enZYmes (CAZy) database31. It was verified that the more evolutionally distant from human ST3Gall (hST3Gall), the less amino acid sequence identity it was compared to hST3Gall, whereas the sequence identity compared to hST3Gal2 and hST6Gall was low independent from the evolution (Fig. 35a and Supplementary Table S3). The amino acid sequence alignment between these ST3Gall enzymes along with pST3Gall and hST3Gall revealed that four motifs in ST3Gall (L, S, III, and VS motifs) as well as residues involved in disulfide bond formation and substrate binding are highly conserved, suggesting the structural similarity among each enzyme (Supplementary Table S4). To visualize the actual 3D structures of these enzymes, Alphafold 2 was employed32, and structure alignment analysis was performed (Fig. 35b). All 3D structures were aligned well, supported by root mean square deviations (RMSD) compared to pST3Gall and hST3Gall, which confirm the structural similarity (Supplementary Table S3).

[0144] To check whether the enzymatic activity is experimentally observed using the developed methods, ST3Gall enzymes from Strongylocentrotus purpuratus (sea urchin, SP_ST3Gall), Coregonus sp. ‘butcher (fish, CB_ST3Gall), Xenopus tropicalis (frog, XT_ST3Gall), Macaca mulatto, (monkey, MM_ST3Gall), and human were selected, N- terminally truncated similar to pST3Gall, and cloned as surface display form of Fc-fusion proteins (Fig. 22a-c, Fig. 36). First, we checked the expression and activity of these surface displayed ST3Gall enzymes using click chemistry -based method. It was uncovered that MM_ST3Gall and hST3Gall, which are evolutionally closely related to pST3Gall, showed comparable sialyltransferase activity with pST3Gall (Fig. 22d, Fig. 35c). XT_ST3Gall showed moderate activity and SP_ST3Gall activity was lower than that of XT_ST3Gall. CB_ST3Gall was not even expressed well in HEK293T cells. This analysis quantifies the sialyltransferase activity of various ST3Gall in the same enzyme family and demonstrates the heterogeneity of expression and activity in mammalian cell system. For further analysis, we performed the activity measurement using MALII and PNA for these ST3Gall enzymes using HEK293T ST3Gall KO cells. CB_ST3Gall, XT_ST3Gall, MM_ST3Gall and hST3Gall showed consistent results compared with click chemistry -based method, and hST3Gal4 expectedly did not show any activity by MALII and PNA due to the detection of different sialoglycan with these lectins (Fig. 22e, Fig. 35d). Interestingly, though SP_ST3Gall showed lower activity than PSI using click chemistry -based method, the activity was comparable to PSI when detected by MALII and PNA. To investigate the reasonfor this phenomenon, we focused on the difference between intracellular activity detection by lectin-based method and extracellular activity detection by click chemistry method.

[0145] Catalytic domain of TM-Fc-SP_ST3Gall is absent during surface display: Because of the substantial difference of measured SP_ST3Gall activity between lectin-based and click chemistry -based method, we hypothesized that catalytic domain of TM-Fc- SP_ST3Gall for sialyltransferase activity is cleaved before surface display. SP_ST3Gall may be active during processing in Golgi independent of whether it is cleaved or intact, while the enzyme is expected to be inactive after surface display given that the cleavage takes place. To check the presence of C-terminal catalytic domain, first of all, western blot of lysate from transfected cells was performed. We found that the bands corresponding to the molecular weight of Fc-fusion ST3Gall were observed for all ST3Gall except for SP_ST3Gall and this was the case for both HEK293T WT and ST3Gall KO cells (Fig. 37a). This suggests that Fc-fusion SP_ST3Gall is lost inside cells and on the cell surface by some mechanism such as proteasomal degradation. To further support this theory, FLAG tag was inserted at the C-terminus of TM-Fc-SP_ST3Gall similar to TM-PS1-FLAG and detected the FLAG expression along with enzymatic activity using lectin-based and click chemistry-based method (Fig. 22f, Fig. 37b, c). It was demonstrated that C-terminal FLAG expression was strongly observed in TM-PS1-FLAG, whereas TM-Fc-SP_ST3Gall-FLAG showed no FLAG expression, indicating the loss of C- terminal part of the surface displayed Fc-protein and this may be the reason why extracellular sialyltransferase activity is not detected.

[0146] If this cleavage of C-terminal SP_ST3Gall is taking place by proteasomal degradation, particular peptide sequence is assumed to be labile to proteasome cleavage. Based on the difference of amino acid sequence between SP_ST3Gall and other ST3Gall, we realized that one disulfide bond formed between C62 and C67 in pST3Gall is lacking in SP_ST3Gall but corresponding disulfide bond exists in other ST3Gall enzymes (Supplementary Table S4). Due to the N- terminal position in ST3Gall connected to the flexible Gly-Ser linker in the construct we used and importance of disulfide bond for protein stabilization, we hypothesized that the lack of this disulfide bond in SP_ST3Gall decreases the stability by increasing the flexibility around the position and making it vulnerable to proteasomal cleavage. To explore whether this hypothesis is valid or not, we created the double mutant of TM-Fc-SP_ST3Gall, Q79C / R83C, by introducing Cys in the amino acids corresponding to C62 and C67 in pST3Gall, expecting the formation of disulfide bond and stabilization of the catalytic domain. However, the expression level of this double mutant wasreduced and no enzymatic activity and FLAG expression were observed (Fig. 22f, Fig. 37b, c).DISCUSSION

[0147] Notably, hST6Gall Y354A, did not completely ablate the enzymatic activity, suggesting that Y354 plays only a partial role in interacting with donor substrate, or surrounding amino acids compensate the loss of Y354. Because of the lack of lectins that can detect sialoglycans produced by hST6GalNAc2, this methodology is quite strong in that it enables to specifically detect the activity of glycosyltransferases synthesizing carbohydrates that cannot be detected by lectins.

[0148] While we were able to perform a number of sialyltransferases reactions on cell-surface, the rates of sialylation were low in some cases. Additionally, the use of pan- sialidase treatment or SLC35A1- KO to create the acceptor substrate may result in non- physiological reaction substrates. One approach to overcome this is to create knockouts of the specific genes before surface displaying the same to create the necessary substrates.

[0149] Such developments may also provide partially sialylated substrates that are needed for enzymes like ST8Sia family members and ST6GalNAc433.

[0150] Here we developed novel strategies to measure sialyltransferase activity using mammalian cell surface display platform using lectins or click chemistry. Considering a number of efforts for purification of soluble enzymes, their proper storage, and difficult normalization of concentrations especially for small scale protein expression using mammalian cell system, surface display platform is beneficial because we can use freshly expressed enzymes for each experiment without purification and it is easy to normalize the concentration of enzymes by using the same gating strategy with a certain level of cell surface Fc-expression for all samples. This platform is suitable for mutagenesis experiments that can be utilized for directed evolution and scanning mutagenesis. In this example, the effect of 1680 different mutations in pST3Gall on the sialyltransferase activity was investigated in a high-throughput manner. Due to the difficult expression of mammalian proteins in prokaryotic systems34and different glycosylation pathway of yeast potentially affecting the property of recombinant proteins15, mammalian cell-based recombinant protein expression and surface display is a fascinating choice especially for research related to mammalian proteins. This mutagenesis study about pST3Gall can be adapted for discovery of systematic gene-to-function relationship of mammalian sialyltransferases and artificial enzymes that can generate carbohydrates not synthesized naturally.

[0151] From both pathological and biochemical perspective, mammalian ST6Gall is the attractive target sialyltransferase. Pathologically, upregulation of mammalian ST6Gall is known to have adverse effects in metastasis, proliferation, and driving oncogenic signaling pathways in a wide range of cancers35, 36. Discovering point mutations that weaken the activity of ST6Gall by scanning mutagenesis using surface displayed ST6Gall could promote the development of novel therapy targeting ST6Gall, as well as obtaining basic knowledge about mutational effect of ST6Gall to its function. Biochemically, ST6Gall has been used to sialylate N-linked glycans in an al,3Man-arm specific manner due to its unique preference in substrate recognition37, 38. Using directed evolution of ST6Gall using mammalian cell surface display platform we developed, it may be possible to engineer ST6Gall to form al,6Man-arm specific N-sialoglycan or N-glycans with both arms sialylated, which potentially reduce the burden for synthesis and increase the yield. Because ST6Gall is also a potential therapeutic agent for autoimmune disease by facilitating sialylation of Fc portion in IgG antibodies around inflammatory spots39, ST6Gall with enhanced enzymatic activity can be a new class of therapy for autoimmune disease that is effective with less dosage.

[0152] Broad applicability of click chemistry-based glycosyltransferase activity measurement is also as aspect of this Part 2. Although we used sialyltransferases as an example to show the validity of this approach, the described method is expected to be applicable to other glycosyltransferases such as fucosyltransferases and galactosyltransferases. Fucosyltransferases can use GDP-6-azido-Fuc to form fucosylated glycans, suggesting that the described methods are applicable40. It was shown that wild-type mammalian galactosyltransferases can recognize UDP-6-azido-Gal and galactosyltransferases can be engineered to enable the recognition of UDP-GalNAz41,42. These exemplary studies about galactosyltransferase engineering demonstrate that galactosyltransferases are attractive targets for protein engineering to utilize the described methods for mutagenesis study and to establish a biorthogonal tagging system. A wide variety of commercially available exoglycosidases such as fucosidase and galactosidase also support broadening the applicability.

[0153] The sialyltransferases we employed in this study all recognize non- sialoglycans as an acceptor substrate. However, some sialyltransferases such as ST6GalNAc4 and ST8Sia family use sialoglycans as acceptors, which influences applicability of the described because exposition of cell surface acceptor substrates by sialidase loses the sialoglycan acceptor. Although one advantage of click chemistry-based method is no need toprepare glycogene knockout cells, specific knockout cells may be used, especially for exploring these sialyltransf erases. For proteomics to know which glycoproteins can be modified with specific glycosyltransferases, surface display platform is not necessary since soluble glycosyltransferases play the same role as surface displayed proteins43. For proximity labeling of proteins interacting with glycoproteins for biological functions, the described method may be modified to take into account the fact that surface displayed glycosyltransferases may be labeled significantly and interfere the proteomics results44.

[0154] By the described lectin- based method, we can detect the specific linkage of attached monosaccharide if the lectin can detect specific carbohydrate epitope, which may be useful to convert the linkage formation specificity of glycosyltransferases45’46. However, it is difficult to achieve this with previously available click chemistry- based methods because any azido-derivatized glycans attached can be detected. Click chemistry- based method is advantageous compared to lectin-based method in (i) broad applicability to a variety of glycosyltransferases due to unnecessity of lectins, which can only detect a limited type of glycans, (ii) no need to prepare glycogene knockout cells in many cases, and (iii) available method for signal amplification. Though metabolic labeling of glycoproteins using precursors of nucleotide sugar is versatile and widely used, it has been previously impossible to analyze the function of a specific glycosyltransferase, which can be achieved by the click chemistrybased method described herein.

[0155] This disclosure provides novel cell-based methods for glycosyltransferase activity measurement that can be applied to mutagenesis study and simple assessment of multiple variants. These methods can be used to reveal mutation-to-function relationship of other glycosyltransferases and explore glycosyltransferases from other organisms with unprecedented activity.MATERIALS AND METHODS

[0156] Materials. Unconjugated Peanut Agglutinin (PNA, Product code: L-1070), Maackia Amurensis Lectin II (MALII, Product code: L-1260), WS (water soluble) DBCO (dibenzocyclooctyne) biotin (Product code: CCT-A116), AZDye 488 DBCO (Product code: CCT-1278), AZDye 488 NHS (N- hydroxysuccinimidyl) ester (Product code: FP-1013) and AZDye 594 NHS ester (Product code: FP-1101) were from Vector Laboratories (Newark, CA). Goat anti-human Fey fragment-specific IgG in Horseradish Peroxidase (HRP), PE (R- Phycoerythrin), AF488 (Al exaFluor 488), and AF647-conjugated forms (Product codes: 109- 035-098, 109-115-190, 109-545-098, and 109- 605-098, respectively), mouse anti-biotin IgGin unconjugated and AF488-conjugated forms (Product code: 200-002-211 and 200-542-211, respectively), and goat anti-mouse IgG in AF594- conjugated form (Product code: 115-585- 062) were from Jackson ImmunoResearch (West Grove, PA). Rabbit anti-FLAG IgG in AF647-conjugated form (Product code: IC8529R) and recombinant human ST3Gall (rhST3Gall, Product code: 6905-GT-020) were from R&D Systems (Minneapolis, MN). CTP (cytidine 5 ’-triphosphate), Gaipi,3GalNAca- / ?NP ( / / ra-nitrophenol), and Gaipi,3GalNAca-Alloc (allyloxycarbonyl) were available from previous work 17,47. CMP- Neu5Ac (cytidine 5 ’-monophospho P-D-N-acetylneuraminic acid) sodium salt (Product code: MC04391) and Neu5Ac,9Az (A-acetyl-9-azido-9-deoxy-neuraminic acid, Product code: MA30919) were from Biosynth (Staad, Switzerland). a2-3, 6, 8, 9 Neuraminidase A (sialidase, Product code: P0722) and rSAP (shrimp alkaline phosphatase, Product code: M0371) were from New England Biolabs (Ipswich, MA). Malachite green phosphate assay kit (Product code: MAK307) was from Sigma. All other chemicals were from Sigma, cell culture reagents from LifeTechnologies / ThermoFisher and molecular biology reagents from New England Biolabs unless otherwise specified.

[0157] Fluorescent labeling of lectins. Lectins (PNA and MALII) were buffer exchanged to phosphate buffered saline (PBS) using Zeba™ Spin desalting columns (7 K MWCO, 0.5 mL). MALII and PNA were fluorescently labeled with AZDye 488 NHS ester and AZDye 594 NHS ester, respectively. 5-fold molar excess dyes were added to MALII and 10-fold molar excess to PNA. After incubation for 1 h at RT in the dark, reaction was quenched with 1 / 10 volume of 1 M Tris, followed by buffer exchange to PBS.

[0158] Molecular biology. pCSCG lentiviral plasmid containing human hST3Gall17and hST6GalNAc248gene were available from previous work. Sialyltransferases from different organisms (SP_ST3Gall, CB_ST3Gall, XT_ST3Gall, and MM_ST3Gall) were synthesized and cloned into pUC57 vectors by Genscript (Piscataway, NJ). hST6Gall and hST3Gal4 genes were obtained from DNASU plasmid repository (Arizona State University. Briefly, for constructing pCSCG-TM(CD94)-Fc- pST3Gall, synthetic primers (‘TEMPI’) encoding for the cytoplasmic tail and transmembrane portions of human CD94 were annealed and PCR amplified. Nhel / Xbal sites were used for restriction enzyme digestion before inserting the product prior to Fc in pCSCG-Fc-ST3Gall plasmid. A similar strategy was used to clone the transmembrane and cytoplasmic portions of DPP4 prior to Fc using primers ‘TEMP2’.

[0159] pCSCG-TM(DPP4)-PSl-FLAG Libi and Lib2 were constructed by Hifi assembly of PCR-amplified vector backbone and mutagenic inserts. Mutagenic inserts werecreated by 2-step PCR amplification. In the 1st step, 31MutaIns_FWD and mutagenic reverse primers were used for Libi and 3 IMutalns wFLAG Rev and mutagenic forward primers were used for Lib2. In the 2nd step, 3 IMutalns wFLAG Rev and mutagenic megaprimers from the 1st step were used for Libi and 3 IMutalns FWD and mutagenic megaprimers from the 1st step were used for Lib2. After gel extraction of each mutagenic insert, 3 inserts for Libi and 5 inserts for Lib2 were respectively pooled and subjected to Hifi cloning. Assembly mix was electroporated into 10-beta competent E. coli (New England Biolabs). To ensure the library representation, the number of colonies was at least lOOx colonies for each mutant. Sequence representation in these mutant libraries was verified using next-generation sequencing using an Illumina MiSeq Micro kit (4 million reads, 150 bp paired end). For constructing additional mutants, inserting FLAG or his tags, and making modifications to parent glycosyltransferases, primers with 15-20 base pair overlaps were used for PCR amplification before using the NEBuilder Hifi Assembly kit (New England Biolabs) for construct assembly. All products were sequence verified using either Sanger sequencing and / or Oxford nanopore whole plasmid sequencing.

[0160] Cell culture. Human embryonic kidney 293T cells (HEK293T) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), 1 % Antibiotic- Antimycotic and 1 % GlutaMAX supplement. Cells were cultured at 37 °C in a humidified 5 % CO2 atmosphere.

[0161] Surface display of Fc-fusion proteins. 0.4-1 x 106HEK293T cells per well were plated in either 6-well or 12-well plates one day before transfection. Transient transfection with plasmids encoding for surface displayed forms of Fc-fusion proteins was performed using calcium phosphate method with 300 pL of transfection mixture including 3 pg plasmid for 6-well plates or 150 pL of transfection mixture including 1.5 pg for 12-well plates. Briefly, plasmid DNA was diluted with cell culture grade water, mixed with 125 mM calcium chloride (final concentration) followed by addition of 150 pL (6-well plates) or 75 pL (12-well plates) 2 x HBS buffer (50 mM HEPES, 10 mM KC1, 140 mM NaCl, 1.5 mM Na2HPO4, pH 7.05), and added to cells pre-treated with 25 pM chloroquine. After 6 h incubation, the medium was replaced with DMEM supplemented with 10 % FBS, 1 % Antibiotic- Antimycotic and 1 % GlutaMAX. Following overnight culture, cells were trypsinized and used for binding assay.

[0162] Expression and purification of soluble Fc-fusion proteins. Plasmids encoding for soluble Fc- fusion proteins were transiently transfected into HEK293T cells using the calcium phosphate method described above. 107HEK293T cells were typicallyplated in 100 mm cell culture petri dishes and 25 pg plasmids were used for transfection per dish. Six hours post-transfection, cell culture medium was replaced with serum-fee DMEM supplemented with 1 % MEM non-essential amino acid solution, 1 % insulin-transferrin- selenium-ethanolamine, 0.4 g / L AlbuMAX™ Lipid-rich BSA, 1 % Antibiotic-Antimycotic and 1 % GlutaMAX. 10 mL culture supernatant was collected 3 days post-transfection, cell debris was removed by centrifugation (3000 g, 3 min) and then the supernatant was concentrated using Amicon Ultra centrifugal filter units (30 kDa MWCO) by 20- fold. Concentrated supernatant was filled up to 10 mL using PBS and mixed with 1 mL NEBExpress Ni resin (New England Biolabs) pre-equilibrated with 10 mL PBS twice. Supernatant mixed with Ni resins were incubated end-over-end at 4 °C for 30 min. Following centrifugation (800 g, 2 min), the resin was washed with 7.5 mL PBS containing 10 mM imidazole thrice. Fc- fusion proteins were then eluted using 5 mL PBS with 200 mM imidazole twice. Elution fractions were immediately concentrated using Amicon Ultra centrifugal filter units (30 kDa MWCO) and buffer-exchanged using Zeba™ Spin desalting columns (7 K MWCO) to PBS. Protein concentrations were determined using A280. Purified proteins were supplemented with 0.1 % BSA, aliquoted, and stored at -80 °C until use.

[0163] Expression and purification of NmCSS. BL21 competent E. coli were transformed with pCWori- NmCSS-6His and a colony from LB agar plate with carbenicillin was picked after overnight culture. Following small scale overnight culture in 5 mL 2xYT medium with 0.1 % carbenicillin, 500 mL cultures were started using 2xYT medium containing 0.1 % carbenicillin at 37 °C until OD600 became 0.76. 0.1 mM IPTG was then added for induction and incubated continued at 22 °C for 19 h. Cells were spun down (4000 rpm, 30 min) and frozen at -80 °C once, followed by dissolving cells in 20 mL PBS supplemented with 2 tablets of EDTA-free cOmplete Mini protease inhibitor cocktail (Sigma), sonication (40 % amplitude, 1 s on and 1 s off, 1 min x 5), and ultracentrifugation (12000 rpm, 15 min, 4 °C). His tagged CMP-sialic acid synthase was then purified from supernatant as described above, using 3 mL NEBExpress Ni resin. Proteins purified and buffer exchanged to PBS were aliquoted and stored at -80 °C for further study.

[0164] Western blot and silver stain. Cell culture supernatant or cell pellets were mixed with SDS loading buffer (Cell Signaling) with 33 mM DTT. All samples were denatured by heating at 95 °C for 5 min, loaded onto 4 - 12 % Tris-glycine SDS-PAGE gels, resolved and transferred onto nitrocellulose membranes. For cell pellets, samples were gently sonicated for 1 min three times before heating. After blocking with 5 % non-fat milk in TBST (20 mM Tris-HCl, 100 mM NaCl, 0.1 % Tween-20) for 1 h at RT, membranes wereincubated at 4 °C overnight in a TBST solution with 1 : 5000 HRP-conjugated goat antihuman Fey fragment-specific IgG and 2 % non-fat milk. Following additional washes, signal was developed using SuperSignal™ West Pico PLUS Chemiluminescent substrate (ThermoFisher). Images were acquired using ChemiDoc Imaging system (Bio-Rad). Silver staining was performed by running purified proteins on SDS-PAGE gels and then following the instruction of the Pierce silver stain kit (ThermoFisher).

[0165] LC-MS / MS analysis. The reaction mixture contained 100 pM Gaipi,3GalNAca- NP, 200 pM CTP, 100 pM Neu5Ac,9Az, and 20 mM MgCh in 19 pL PBS. 1 pL rhST3Gall (>1000 pmol / min / pg) + 100 pg / mL NmCSS, only rhST3Gall, or only NmCSS was added to initiate reaction at 37 °C for 1 h or 20 h. Reaction was quenched by adding 80 pL acetonitrile to each sample at the end point at 4 °C for 30 min. Following centrifugation (13000 g, 10 min), 90 pL of the supernatant was collected and dried using Savant™ SPD131DDA SpeedVac Concentrator (Thermo). All samples were stored at -20 °C until LC-MS / MS analysis. Dried samples were dissolved in 50 % methanol (0.1 % formic acid) before injection. XSelect C18 column (3.5 pm, 4.6 mm * 150 mm) was used for separation and Q-Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo) was used in positive mode for subsequent MS / MS analysis. MilliQ (0.1 % formic acid) and acetonitrile (0.1 % formic acid) were used as mobile phase A and B. Flow rate was 0.2 mL / min. The column was maintained at 40 °C during separation. The gradient of LC was as follows: (i) 0-40 % B (0-20 min), (ii) 40-100 % B (20-25 min), and (iii) 100-0 % B (25-30 min). Molecular weights used for obtaining XICs were as follows: donor precursor ([M+H]+ = 335.1203), acceptor ([M+H]+= 505.1670), and product ([M+H]+= 821.2689).

[0166] Click chemistry-based cell surface glycoengineering. HEK293T cells with surface displayed form of Fc-fusion proteins were trypsinized and dissolved in HEPES buffer (30 mM HEPES, 110 mM NaCl, 10 mM KC1, 2 mM MgCh, 10 mM glucose, 1.5 mM CaCh, 1 % BSA, pH 7.4). Cells were treated with sialidase (200 units / mL) at 37 °C for 1 h with shaking to cleave all sialic acids on the cell surface, washed twice, and then resuspended in HEPES buffer. For sialylation on the cell surface, the reaction mixture containing 667 pM CTP, 333 pM Neu5Ac,9Az, 67 mM MgCh, and 333 pg / mL NmCSS in HEPES buffer. The mix was added to cell suspension at 3:7 volume ratio (final concentration: 200 pM CTP, 100 pM Neu5Ac,9Az, 20 mM MgCh, and 100 pg / mL NmCSS) and incubated at 37 °C for 1 h with shaking. Cells were then washed twice to stop the reaction and suspended in HEPES buffer. Fluorescent labeling and biotinylation were performed using 100 pM DBCO-AF488 or DBCO-biotin (0.2 % DMSO) at room temperature for 30 min, followed by washing with5-fold volume of PBS twice, and suspension in HEPES buffer. In some studies, the concentrations of CTP, Neu5Ac,9Az, and / or DBCO-biotin was increased by 2-4-fold. DMSO (0.4-0.8%) was added in vehicle control for biotinylation studies. Cells were kept on ice during cell staining and analyzed using flow cytometry.

[0167] Flow cytometry. 1 :2000 diluted PE-, AF488-, or AF647-conjugated antihuman IgG Fc were premixed with 5 pg / mL fluorescently-labeled lectins, 1 pg / mL antibiotin, or 1 :500 diluted anti- FLAG for multicolor measurement. For lectin-based enzymatic activity measurements, 5 pL cell suspension (107 cells / mL in HEPES buffer) was mixed with 5 pL anti -Fc+1 ectin mixture at 4 °C for 20 min with shaking. For one-step click chemistrybased enzymatic activity measurement, 18 pL cell suspension (107cells / mL) was mixed with 2 pL anti-Fc+AF488 anti-biotin mixture. In two-step detection in click chemistry-based activity measurement, after 20 min incubation of 18 pL cell suspension (107cells / mL) with 2 pL mixture of fluorescent goat anti-human IgG Fc and unconjugated mouse anti-biotin (1 pg / mL), 1 pL goat anti-mouse AF594 (3 pg / mL) was directly added to each suspension sample and further incubated at 4 °C for 10 min. The volume ratio of secondary antibody solution and cell suspension should be sufficiently low (e.g. < 1 :20) so the primary antibodies existing in cell suspension are not diluted. Cell samples were washed once and analyzed using a BD LSRFortessa™ X-20 flow cytometer (BD Biosciences).

[0168] Surface display PSI mutant libraries. Lentivirus was produced for stable expression of TM- PSI Libi and Lib2 encoded in pCSCG plasmid vector. To this end, 20* 106cells were plated into 150mm petri dishes. The next day these cells were transfected with 22 pg pCSCG-TM(DPP4)- PSI Libi and Lib2 plasmid , 22 pg packaging plasmid (psPAX2, Addgene_# 12260) and 11 pg envelope plasmid encoding for VSV-G (pMD2.G, Addgene # 12259) using the calcium phosphate method. Culture medium was replaced with OptiMEM six hours post-transfection with the first virus batch being collected after overnight culture. Culture medium was then replaced with OptiMEM containing 10 mM sodium butyrate, and a second virus batch was collected the next day. Both batches were pooled, centrifuged (2000 g, 2 min), filtered using 0.45 pm PES syringe filters, and ultracentrifuged (50000 g, 2 h, 4 °C) to obtain lentivirus pellet. This pellet was dissolved in 100 pL OptiMEM, aliquoted and stored at -80 °C until use.

[0169] The above lentivirus was used to transduce HEK293T ST3Gall KO cells plated in 60 mm cell culture petri dishes. To achieve this, cell culture medium was replaced with DMEM supplemented with 8 pg / mL polybrene for 5 min before addition of serial- diluted lentivirus Libi or Lib2. After overnight incubation, medium was replaced to DMEM.Following another day, transduced cells were trypsinized and expression of surface displayed Fc-fusion proteins was measured using AF647-conjugated goat anti-human IgG Fc antibody in conjunction with flow cytometry. Cells transduced at MOI < 0.3 were FACS sorted based on Fc-fusion protein expression and these were scaled up. These are the HEK293T ST3Gall KO Libi and Lib2 library cells.

[0170] Either the MALII lectin or click chemistry -based DBCO-biotin was used for selection of ST3Gall KO Libi and Lib2 cells based on their sialyltransferase activity. For sorting with MALII, trypsinized cells (107 / mL in 500 pL HEPES) were incubated with AF647-conjugated goat anti-human IgG Fc (0.075 pg / mL) and AF488-MALII (5 pg / mL) for 20 min on ice. For sorting with DBCO-biotin, click chemistry, as described above was used to couple DBCO-biotin to Neu5Az expressing cells. These cells were then stained using PE- conjugated goat anti-human IgG Fc (1 :2000) and AF488- conjugated mouse anti-biotin (2 pg / mL) for 20 min on ice. In the first sort, cell populations with Fc-positive / sialylati on- negative (1stNeg) and Fc-positive / sialylati on-positive (1stPos) were sorted and scaled up. In the second sort, the 1stPos cells were then further FACS sorted to further enrich Fc- positive / sialylati on-positive cells (2ndPos). Genomic DNA from sorted cells along with parent population before sorting were extracted. PCR amplification was performed around the mutation region, and the product was sequenced using an Illumina MiSeq Micro kit (4 million reads, 150 bp paired end). Mutant enrichment analysis was performed using the Seqkit platform50. First, the read number of each mutant in each cell type was divided by the total read number to calculate the ‘normalized read number’. 1stNeg, 1stPos, and 2ndPos ‘enrichment values’ (E) were then calculated by dividing the normalized read number of these samples by the normalized read number of the parent population before sorting. Finally, ‘enrichment scores’ (ES) were calculated by multiplying 1stPos, and 2ndPos enrichment values and dividing it by 1stNeg enrichment value. This allowed us to assemble all experimental data for each mutant in a single metric.

[0171] Plate-based sialyltransferase activity measurement. All soluble Fc- pST3Gall mutants were buffer-exchanged to Tris buffer (25 mM Tris-HCl, 10 mM CaCh, 10 mM MgCh, pH 7.5) using Zeba spin desalting columns (7 K MWCO). In some studies, the donor CMP-Neu5Ac was serially diluted in the range 6.25-200 pM, while the substrate Gaipi,3GalNAca-Alloc was fixed at 50 pM. In other studies, Gaipi,3GalNAca-Alloc was varied in the range 6.25-200 pM while CMP-Neu5Ac was fixed at 50 pM. To each reaction well, 1 unit / mL rSAP was added along with 2 pg / mL Fc- pST3Gall mutants, PSI or vehicle control. After 30 min incubation at 37 °C, malachite green mixture was added to each wellfor additional 15 min at RT following manufacturer’s instructions. Plate absorbance was then measured at 600 - 660 nm using BioTek Synergy 4 plate reader (Agilent Technologies). Phosphate standard solution in malachite green phosphate assay kit was used to draw the calibration curve. Double reciprocal plots were created to obtain KM and kcat.

[0172] Statistical analysis. All data are presented as mean ± standard deviation (STD). Multiple comparisons were performed using one-way ANOVA followed by the Tukey post-test, p < 0.05 was considered to be statistically significant. Number of repeats are specified in individual panels.

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PLoS One 11, e0163962 (2016). https: / / doi.org: 10.1371 / joumal. pone.0163962PART 2 SUPPLEMENTARY TABLES

[0174] Supplementary Table SI

[0175] Supplementary Table S2

[0176] Supplementary Table S3Table S3. ST3Gall enzymes from various organisms that have not been characterized in CAZy database.

[0177] Supplementary Table S4

[0178] Although the present disclosure has been described with respect to one or more particular embodiments and / or examples, it will be understood that other embodiments and / or examples of the present disclosure may be made without departing from the scope of the present disclosure.

[0179] REPRESENTATIVE SEQUENCES THAT ARE INCLUDED IN THIS DISCLOSURE

[0180] The disclosure includes the proviso that any described protein may exclude or include any of the following sequences: QATEYEYLDYDFLPETEPPRP (SEQ ID NO: 8), MMDDDDKSR (SEQ ID NO: 9), DYKDDDDK (SEQ ID NO: 10), and DDDDK (SEQ ID NO: 11). The sequence “TG” may also be excluded.

[0181] Fc-pST3Gall WT (‘PSI’)MIPARFAGVLLALALILPGTLCTGHHHHHHQATEYEYLDYDFLPETEPPRPMMDDDDKSRTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSRPCTCTRCIEEQRVSAWFDERFNRSMQPLLTAKNAHLEEDTYKWWLRLQREKQPNNLNDTIRELFQVVPGNVDPLLEKRLVSCRRCAVVGNSGNLKESYYGPQIDSHDFVLRMNKAPTEGFEADVGSKTTHHFVYPESFRELAQEVSMILVPFKTTDLEWVISATTTGRISHTYVPVPAKIKVKKEKILIYHPAFIKYVFDRWLQGHGRYPSTGILSVIFSLHICDEVDLYGFGADSKGNWHHYWENNPSAGAFRKTGVHDGDFESNVTTILASINKIRIFKGR (SEQ ID NO: 12)

[0182] Fc-pST3Gall H243A / A253I / F254S (‘sCore2’)MIPARFAGVLLALALILPGTLCTGHHHHHHQATEYEYLDYDFLPETEPPRPMMDDDDKSRTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSRPCTCTRCIEEQRVSAWFDERFNRSMQPLLTAKNAHLEEDTYKWWLRLQREKQPNNLNDTIRELFQVVPGNVDPLLEKRLVSCRRCAVVGNSGNLKESYYGPQIDSHDFVLRMNKAPTEGFEADVGSKTTHHFVYPESFRELAQEVSMILVPFKTTDLEWVISATTTGRISHTYVPVPAKIKVKKEKILIYHPAFIKYVFDRWLQGHGRYPSTGILSVIFSLHICDEVDLYGFGADSKGNWHAYWENNPSAGISRKTGVHDGDFESNVTTILASINKIRIFKGR (SEQ ID NO: 13)

[0183] Fc-pST3Gall H243A (‘H243A’)MIPARFAGVLLALALILPGTLCTGHHHHHHQATEYEYLDYDFLPETEPPRPMMDDDDKSRTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSRPCTCTRCIEEQRVSAWFDERFNRSMQPLLTAKNAHLEEDTYKWWLRLQREKQPNNLNDTIRELFQVVPGNVDPLLEKRLVSCRRCAVVGNSGNLKESYYGPQIDSHDFVLRMNKAPTEGFEADVGSKTTHHFVYPESFRELAQEVSMILVPFKTTDLEWVISATTTGRISHTYVPVPAKIKVKKEKILIYHPAFIKYVFDRWLQGHGRYPSTGILSVIFSLHICDEVDLYGFGADSKGNWHAYWENNPSAGAFRKTGVHDGDFESNVTTILASINKIRIFKGR (SEQ ID NO: 14)

[0184] Fc-pST3Gall Q49A / Y174A / Y210F (‘Dead’)MIPARFAGVLLALALILPGTLCTGHHHHHHQATEYEYLDYDFLPETEPPRPMMDDDDKSRTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSRPCTCTRCIEEQRVSAWFDERFNRSMQPLLTAKNAHLEEDTYKWWLRLAREKQPNNLNDTIRELFQVVPGNVDPLLEKRLVSCRRCAVVGNSGNLKESYYGPQIDSHDFVLRMNKAPTEGFEADVGSKTTHHFVYPESFRELAQEVSMILVPFKTTDLEWVISATTTGRISHTAVPVPAKIKVKKEKILIYHPAFIKYVFDRWLQGHGRFPSTGILSVIFSLHICDEVDLYGFGADSKGNWHHYWENNPSAGAFRKTGVHDGDFESNVTTILASINKIRIFKGR (SEQ ID NO: 15)

[0185] TM(DPP4)-Fc-pST3Gall WT (‘TM-PS1’)MKTPWKTGVLLGLLGAAALVTIITVPVVLLSRTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSRPCTCTRCIEEQRVSAWFDERFNRSMQPLLTAI<NAHLEEDTYI<WWLRLQREI<QPNNLNDTIRELFQVVPGNVDPLLEKRLVSCRRCAVVGNSGNLKESYYGPQIDSHDFVLRMNKAPTEGFEADVGSKTTHHFVYPESFRELAQEVSMILVPFKTTDLEWVISATTTGRISHTYVPVPAKIKVKKEKILIYHPAFIKYVFDRWLQGHGRYPSTGILSVIFSLHICDEVDLYGFGADSKGNWHHYWENNPSAGAFRKTGVHDGDFESNVTTILASINKIRIFKGR (SEQ ID NO: 16)

[0186] TM(DPP4)-Fc-pST3Gall H243A (‘TM-H243A’)MKTPWKTGVLLGLLGAAALVTIITVPVVLLSRTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSRPCTCTRCIEEQRVSAWFDE RFNRSMQPLLTAI<NAHLEEDTYI<WWLRLQREI<QPNNLNDTIRELFQVVPGNVDPLL EKRLVSCRRCAVVGNSGNLKESYYGPQIDSHDFVLRMNKAPTEGFEADVGSKTTHH FVYPESFRELAQEVSMILVPFKTTDLEWVISATTTGRISHTYVPVPAKIKVKKEKILIY HPAFIKYVFDRWLQGHGRYPSTGILSVIFSLHICDEVDLYGFGADSKGNWHAYWENNPSAGAFRKTGVHDGDFESNVTTILASINKIRIFKGR (SEQ ID NO: 17)

[0187] TM(DPP4)-Fc-pST3Gall Q49A / Y174A / Y210F (‘TM-Dead’)MKTPWKTGVLLGLLGAAALVTIITVPVVLLSRTCPPCPAPELAGAPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSRPCTCTRCIEEQRVSAWFDE RFNRSMQPLLTAI<NAHLEEDTYI<WWLRLAREI<QPNNLNDTIRELFQVVPGNVDPLLEKRLVSCRRCAVVGNSGNLKESYYGPQIDSHDFVLRMNKAPTEGFEADVGSKTTHH FVYPESFRELAQEVSMILVPFKTTDLEWVISATTTGRISHTAVPVPAKIKVKKEKILIY HPAFIKYVFDRWLQGHGRFPSTGILSVIFSLHICDEVDLYGFGADSKGNWHHYWENN PSAGAFRKTGVHDGDFESNVTTILASINKIRIFKGR (SEQ ID NO: 18)

[0188] TM(CD94)-Fc-pST3Gall WT (TM(CD94)-PS1)MAVFKTTLWRTGLISGTLGIICLSLMATLGILLSRTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSRPCTCTRCIEEQRVSAWF DERFNRSMQPLLTAI<NAHLEEDTYI<WWLRLQREI<QPNNLNDTIRELFQVVPGNVDPLLEKRLVSCRRCAVVGNSGNLKESYYGPQIDSHDFVLRMNKAPTEGFEADVGSKTT HHFVYPESFRELAQEVSMILVPFKTTDLEWVISATTTGRISHTYVPVPAKIKVKKEKILIYHPAFIKYVFDRWLQGHGRYPSTGILSVIFSLHICDEVDLYGFGADSKGNWHHYWE NNPSAGAFRKTGVHDGDFESNVTTILASINKIRIFKGR (SEQ ID NO: 19)

[0189] TM(CD94)-Fc-pST3Gall H243A (TM(CD94)-H243A)MAVFKTTLWRTGLISGTLGIICLSLMATLGILLSRTCPPCPAPELAGAPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSRPCTCTRCIEEQRVSAWF DERFNRSMQPLLTAI<NAHLEEDTYI<WWLRLQREI<QPNNLNDTIRELFQVVPGNVDPLLEKRLVSCRRCAVVGNSGNLKESYYGPQIDSHDFVLRMNKAPTEGFEADVGSKTT HHFVYPESFRELAQEVSMILVPFKTTDLEWVISATTTGRISHTYVPVPAKIKVKKEKILIYHPAFIKYVFDRWLQGHGRYPSTGILSVIFSLHICDEVDLYGFGADSKGNWHAYWE NNPSAGAFRKTGVHDGDFESNVTTILASINKIRIFKGR (SEQ ID NO:20)

[0190] TM(CD94)-Fc-pST3Gall Q49A / Y174A / Y210F (TM(CD94)-Dead)MAVFKTTLWRTGLISGTLGIICLSLMATLGILLSRTCPPCPAPELAGAPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSRPCTCTRCIEEQRVSAWF DERFNRSMQPLLTAI<NAHLEEDTYI<WWLRLAREI<QPNNLNDTIRELFQVVPGNVDP LLEKRLVSCRRCAVVGNSGNLKESYYGPQIDSHDFVLRMNKAPTEGFEADVGSKTT HHFVYPESFRELAQEVSMILVPFKTTDLEWVISATTTGRISHTAVPVPAKIKVKKEKIL IYHPAFIKYVFDRWLQGHGRFPSTGILSVIFSLHICDEVDLYGFGADSKGNWHHYWE NNPSAGAFRKTGVHDGDFESNVTTILASINKIRIFKGR (SEQ ID NO:21)

[0191] TM(DPP4)-Fc-pST3Gall-FLAG (TM(DPP4)-PS1-FLAG)MKTPWKTGVLLGLLGAAALVTIITVPVVLLSRTCPPCPAPELAGAPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSRPCTCTRCIEEQRVSAWFDE RFNRSMQPLLTAI<NAHLEEDTYI<WWLRLQREI<QPNNLNDTIRELFQVVPGNVDPLLEKRLVSCRRCAVVGNSGNLKESYYGPQIDSHDFVLRMNKAPTEGFEADVGSKTTHH FVYPESFRELAQEVSMILVPFKTTDLEWVISATTTGRISHTYVPVPAKIKVKKEKILIY HPAFIKYVFDRWLQGHGRYPSTGILSVIFSLHICDEVDLYGFGADSKGNWHHYWEN NPSAGAFRKTGVHDGDFESNVTTILASINKIRIFKGRGSDYKDDDDK (SEQ ID NO:22)

[0192] TM(DPP4)-Fc-hST6Gall WTMKTPWKTGVLLGLLGAAALVTIITVPVVLLSRTCPPCPAPELAGAPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSEASFQVWNKDSSSKNLIPRL QKIWKNYLSMNKYKVSYKGPGPGIKFSAEALRCHLRDHVNVSMVEVTDFPFNTSEW EGYLPKESIRTKAGPWGRC AVVS S AGSLKS SQLGREIDDHD AVLRFNGAPT ANFQQD VGTKTTIRLMNSQLVTTEKRFLKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNFFN NYKTYRKLHPNQPFYILKPQMPWELWDILQEISPEEIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDVCYYYQKFFDSACTMGAYHPLLYEKNLVKHLNQGTDEDIYLLG KATLPGFRTIHC (SEQ ID NO:23)

[0193] TM(DPP4)-Fc-hST6Gall Y354AMKTPWKTGVLLGLLGAAALVTIITVPVVLLSRTCPPCPAPELAGAPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSEASFQVWNKDSSSKNLIPRL QKIWKNYLSMNKYKVSYKGPGPGIKFSAEALRCHLRDHVNVSMVEVTDFPFNTSEW EGYLPKESIRTKAGPWGRC AVVS S AGSLKS SQLGREIDDHD AVLRFNGAPT ANFQQD VGTKTTIRLMNSQLVTTEKRFLKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNFFN NYKTYRKLHPNQPFYILKPQMPWELWDILQEISPEEIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDVCAYYQKFFDSACTMGAYHPLLYEKNLVKHLNQGTDEDIYLLG KATLPGFRTIHC (SEQ ID NO:24)

[0194] TM(DPP4)-Fc-hST3Gal4 WTMAVFKTTLWRTGLISGTLGIICLSLMATLGILLSRTCPPCPAPELAGAPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSKEPCLQGEAESKASKLFG NYSRDQPIFLRLEDYFWVKTPSAYELPYGTKGSEDLLLRVLAITSSSIPKNIQSLRCRRCVVVGNGHRLRNSSLGDAINKYDVVIRLNNAPVAGYEGDVGSKTTMRLFYPESAHF DPKVENNPDTLLVLVAFKAMDFHWIETILSDKKRVRKGFWKQPPLIWDVNPKQIRIL NPFFMEIAADKLLSLPMQQPRKIKQKPTTGLLAITLALHLCDLVHIAGFGYPDAYNKK QTIHYYEQITLKSMAGSGHNVSQEALAIKRMLEMGAIKNLTSF (SEQ ID NO:25)

[0195] TM(DPP4)-Fc-hST3Gal4 H294AMAVFKTTLWRTGLISGTLGIICLSLMATLGILLSRTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSKEPCLQGEAESKASKLFG NYSRDQPIFLRLEDYFWVKTPSAYELPYGTKGSEDLLLRVLAITSSSIPKNIQSLRCRR CVVVGNGHRLRNSSLGDAINKYDVVIRLNNAPVAGYEGDVGSKTTMRLFYPESAHFDPKVENNPDTLLVLVAFKAMDFHWIETILSDKKRVRKGFWKQPPLIWDVNPKQIRIL NPFFMEIAADKLLSLPMQQPRKIKQKPTTGLLAITLALHLCDLVHIAGFGYPDAYNKK QTIAYYEQITLKSMAGSGHNVSQEALAIKRMLEMGAIKNLTSF (SEQ ID NO:26)

[0196] TM(DPP4)-Fc-hST6GalNAc2 WTMKTPWKTGVLLGLLGAAALVTIITVPVVLLSRTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSGQACRHLLHLAIQRHPHFRG LFNLSIPVLLWGDLFTPALWDRLSQHKAPYGWRGLSHQVIASTLSLLNGSESAKLFA PPRDTPPKCIRCAVVGNGGILNGSRQGPNIDAHDYVFRLNGAVIKGFERDVGTKTSFYGFTVNTMKNSLVSYWNLGFTSVPQGQDLQYIFIPSDIRDYVMLRSAILGVPVPEGLD KGDRPHAYFGPEASASKFKLLHPDFISYLTERFLKSKLINTHFGDLYMPSTGALMLLTALHTCDQVSAYGFITSNYWKFSDHYFERKMKPLIFYANHDLSLEAALWRDLHKAGI LQLYQR (SEQ ID NO:27)

[0197] TM(DPP4)-Fc-hST6GalNAc2 H336AMKTPWKTGVLLGLLGAAALVTIITVPVVLLSRTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSGQACRHLLHLAIQRHPHFRG LFNLSIPVLLWGDLFTPALWDRLSQHKAPYGWRGLSHQVIASTLSLLNGSESAKLFA PPRDTPPKCIRCAVVGNGGILNGSRQGPNIDAHDYVFRLNGAVIKGFERDVGTKTSFY GFTVNTMKNSLVSYWNLGFTSVPQGQDLQYIFIPSDIRDYVMLRSAILGVPVPEGLD KGDRPHAYFGPEASASKFKLLHPDFISYLTERFLKSKLINTHFGDLYMPSTGALMLLT ALHTCDQVSAYGFITSNYWKFSDAYFERKMKPLIFYANHDLSLEAALWRDLHKAGI LQLYQR (SEQ ID NO:28)

[0198] TM(DPP4)-Fc-SP_ST3Gall (TM(DPP4)-SP31)MKTPWKTGVLLGLLGAAALVTIITVPVVLLSRTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSGGQRCRRLWKKGTSEWFDE KYNDSLLPVWMKENKKMSEEIGKWWLSLQRRENSDYIGALDKAFEVIPNPQRFLTR NVSRCLRCAVVGNSGNLRNSGYGTAIDKHDVVVRINQAKVKGFEKDVGQKETHRL MYPESFMDIAPETNFVLLSFKVIDLQWARSAITTGEITKTYTNVRRKIRVTPSKILFYN PALMYHIHREWIDRKGRYPSSGTLAVFFALQFCDEVSVYGMGANSKGFWDHYWEVNDGSRNSAFLKTHVHDSVHEFEVIKKLADEKIITMFQGVR (SEQ ID NO:29)

[0199] TM(DPP4)-Fc-CB ST3Gall (TM(DPP4)-CB31)MAVFKTTLWRTGLISGTLGIICLSLMATLGILLSRTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSGQCGCRQCMTELEDDPW FIERFNRSVPPLMSRKNSLLTDDTYHWWQLQIEEDPANFSEVVEKLFQVIPDDDDLY MDAGPERCRTCAVVGNSGNLKGSRYGPLIDSSDVIIRMNKAPTSGFEKDVGSRTTHH VMYPESAIDLDNTTSLLLIPFKTLDLQWITSALTTGSIEKTYIPVLVYSPTFFKYVYDT WLESHGRYPSTGFLSLLFAVHICDKVNVYGFGGDQYGNWHHYWENNDQGGAFRETGVHDADYEYNATLLLADKHKISIFKGL (SEQ ID NO:30)

[0200] TM(DPP4)-Fc-XT ST3Gall (TM(DPP4)-XT31)MKTPWKTGVLLGLLGAAALVTIITVPVVLLSRTCPPCPAPELAGAPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSRQCGCDTCVAEPEASTWFD ERFNLSVLPLLSKQNNVIPDTVYKWWLTLQGETKPKDIYEVMEELFEVIPGDLDLMD QGPYRCRTCAVVGNSGNLKSSNYGPEIDEHDFVLRMNHAPTARFEKDVGGKTTHHF VYPESVRDLQANVSMILIPFKTLDLQWLASALTHGTINRTYVQVPRKIRVSKDKVLV YSPELMKYVYDKWLLNHGRYPSTGLLAVIFALHVCDKVDLYGFGADSKGHWHHYWENNASAGAFRLTGVHDGDFEASILANLTSINKVLMFRGR (SEQ ID N0:31)

[0201] TM(DPP4)-Fc-MM ST3Gall (TM(DPP4)-MM31)MKTPWKTGVLLGLLGAAALVTIITVPVVLLSRTCPPCPAPELAGAPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSRPCTCTHCIGQRKLSVWFDE RFNQTVQPLLTAQNALLEDDTYRWWLRLQREKKPNNLNDTIKELFRVVPGNVDPML EKRSVGCRRCAVVGNSGNLRESSYGPEIDRHDFVLRMNKAPTAGFEADVGTKTTHH LVYPESFRELGDNVSMILVPFKTIDLEWVVSATTTGTISHTYVPVPAKIRVKQDKILIYHPAFIKYVFDNWLQGHGRYPSTGILSVIFSMHVCDEVDLYGFGADSKGNWHHYWE NNPSAGAFRKTGVHDADFESNVTATLASINKIRIFKGR (SEQ ID NO:32)

[0202] TM(DPP4)-Fc-hST3Gall (TM(DPP4)-h31)MKTPWKTGVLLGLLGAAALVTIITVPVVLLSRTCPPCPAPELAGAPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSRPCTCTHCIGQRKLSAWFDE RFNQTMQPLLTAQNALLEDDTYRWWLRLQREKKPNNLNDTIKELFRVVPGNVDPM LEKRSVGCRRCAVVGNSGNLRESSYGPEIDSHDFVLRMNKAPTAGFEADVGTKTTH HLVYPESFRELGDNVSMILVPFKTIDLEWVVSAITTGTISHTYIPVPAKIRVKQDKILIYHPAFIKYVFDNWLQGHGRYPSTGILSVIFSMHVCDEVDLYGFGADSKGNWHHYWE NNPSAGAFRKTGVHDADFESNVTATLASINKIRIFKGR (SEQ ID NO:33)

[0203] TM(DPP4)-Fc-SP_ST3Gall-FLAG (TM(DPP4)-SP31-FLAG)MKTPWKTGVLLGLLGAAALVTIITVPVVLLSRTCPPCPAPELAGAPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSGGQRCRRLWKKGTSEWFDE KYNDSLLPVWMKENKKMSEEIGKWWLSLQRRENSDYIGALDKAFEVIPNPQRFLTR NVSRCLRCAVVGNSGNLRNSGYGTAIDKHDVVVRINQAKVKGFEKDVGQKETHRL MYPESFMDIAPETNF VLLSFKVIDLQWARS AITTGEITKTYTNVRRKIRVTPSKILFYN PALMYHIHREWIDRKGRYPSSGTLAVFFALQFCDEVSVYGMGANSKGFWDHYWEV NDGSRNSAFLKTHVHDSVHEFEVIKKLADEKIITMFQGVRGSDYKDDDDK (SEQ ID NO:34)

Claims

What is claimed is:

1. A modified protein comprising one or more amino acid changes relative to its unmodified sequence, the modified protein comprising the sequence:RPCTCTRCIEEQRVSAWFDERFNRSMQPLLTAKNAHLEEDTYKWWLRLQREKQPNN LNDTIRELFQVVPGNVDPLLEKRLVSCRRCAVVGNSGNLKESYYGPQIDSHDFVLRM NKAPTEGFEADVGSKTTHHFVYPESFRELAQEVSMILVPFKTTDLEWVISATTTGRIS HTYVPVPAKIKVKKEKILIYHPAFIKYVFDRWLQGHGRYPSTGILSVIFSLHICDEVDL YGFGADSKGNWHHYWENNPSAGAFRKTGVHDGDFESNVTTILASINKIRIFKGR (SEQ ID NO: 1); said protein comprising a change of H243; or a change of amino acid A253; or a change of amino acid F254; or a combination of said amino acid changes.

2. The modified protein of claim 1, wherein the amino acid change is H243A, A253I, F254S, or a combination thereof.

3. The modified protein of claim 2, comprising all of the H243A, A253I, and F254S amino acid changes.

4. The modified protein of claim 3, further comprising an immunoglobulin (Ig) segment, wherein said Ig segment optionally comprises the sequence:TCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<ALPAPIEI<TIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID N0:3).

5. The modified protein of claim 4, further comprising a linking sequence , wherein said linking sequence is located between the sequence of SEQ ID NO:1 and SEQ ID NO:2, said linker sequence optionally comprising the sequence GSGSGSGSGS (SEQ ID NO:4).

6. The modified protein of any one of claims 5, further comprising a secretion signal sequence, wherein said secretion signal sequence optionally comprises the sequence MIPARFAGVLLALALILPGTLC (SEQ ID NO:5).

7. The modified protein of claim 6, said protein optionally comprising a purification tag sequence, wherein the purification tag sequence optionally comprises the sequence HHHHHH (SEQ ID NO: 6).

8. The modified protein of claim 1, comprising the sequence:MIPARFAGVLLALALILPGTLCHHHHHHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSRPCTCTRCIEEQRVSAWFDERFNRSMQPLLTAI<NAHLEEDTYI<WWLRLQREI<QPNNLNDTIRELFQVVPGNVDPLLEI<RL VSCRRCAVVGNSGNLKESYYGPQIDSHDFVLRMNKAPTEGFEADVGSKTTHHFVYP ESFRELAQEVSMILVPFKTTDLEWVISATTTGRISHTYVPVPAKIKVKKEKILIYHPAFI KYVFDRWLQGHGRYPSTGILSVIFSLHICDEVDLYGFGADSKGNWHAYWENNPSAG ISRKTGVHDGDFESNVTTILASINKIRIFKGR (SEQ ID NO:7).

9. The modified protein of any one of claims 1-8, wherein the modified protein is attached to a substrate.

10. The modified protein of any one of claims 1-8, wherein the modified protein is displayed on the surface of a cell.

11. The modified protein of any one of claims 1-8, wherein the modified protein is detectably labeled.

12. A polynucleotide encoding the modified protein of any one of claims 1-8.

13. The polynucleotide of claim 12, wherein the polynucleotide comprises an expression vector.

14. A modified protein comprising or consisting of a sequence described herein as Fc- pST3Gall WT (‘PSI’), Fc-pST3Gall H243A (‘H243A’), TM(DPP4)-Fc-pST3Gall WT (‘TM-PS1’), TM(DPP4)-Fc-pST3Gall H243A (‘TM-H243A’), Fc-pST3Gall H243A / A253I / F254S (‘sCore2’), TM(CD94)-Fc-pST3Gall WT (TM(CD94)-PS1), TM(CD94)-Fc-pST3Gall H243A (TM(CD94)-H243A), TM(DPP4)-Fc-pST3Gall-FLAG (TM(DPP4)-PS1-FLAG), TM(DPP4)-Fc-hST6Gall WT, TM(DPP4)-Fc-hST6Gall Y354A TM(DPP4)-Fc-hST3Gal4 WT, TM(DPP4)-Fc-hST3Gal4 H294A, TM(DPP4)-Fc- hST6GalNAc2 WT, TM(DPP4)-Fc-hST6GalNAc2 H336A, TM(DPP4)-Fc-SP_ST3Gall (TM(DPP4)-SP31), TM(DPP4)-Fc-CB_ST3Gall (TM(DPP4)-CB31), TM(DPP4)-Fc- XT_ST3Gall (TM(DPP4)-XT31), TM(DPP4)-Fc-MM_ST3Gall (TM(DPP4)-MM31), TM(DPP4)-Fc-hST3Gall (TM(DPP4)-h31), TM(DPP4)-Fc-SP_ST3 Gall -FLAG (TM(DPP4)-SP31-FLAG).

15. A modified protein comprising SEQ ID NO: 1 with a K256V mutation, and wherein the modified protein has improved enzymatic activity relative to a protein comprising SEQ ID NO: 1 that does not contain the K256V mutation.

16. A method comprising screening a library of mutated proteins displayed on a plurality of mammalian cells for improved target binding or improved enzymatic activity or diminished enzymatic activity, relative to a control value.

17. The method of claim 16, comprising screening for the improved enzymatic activity.