Protein targeting editor and application thereof

Abstract

The invention discloses a protein targeting editor and application thereof, and particularly relates to a protein editor for targeting a target N-glycosylated protein in a receptor biological cell and application of the protein editor. The constructed protein editor comprises three elements: a protein targeting peptide, a linker and a protein editing enzyme. The construction of the deglycosylated protein sequence editor is beneficial to removal of an N-sugar chain of a target protein, and deamidation of an asparagine residue at the site is carried out to convert the asparagine residue into an aspartic acid residue. This deglycosylation editing results in a significant reduction in the stability of the target protein and accelerates the rate of degradation thereof by proteasomes. Therefore, the invention provides a tool capable of performing deglycosylation protein sequence editing on an N-glycosylation modification site of a specific target protein in a cell, which is expected to reveal the functions of N-glycosylation modification and protein sequence editing of asparagine deamidation into aspartic acid in living organisms. And the method has the potential of serving as an alternative method for developing a protein degradation technology.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a protein targeting editor and its application, and in particular to a protein editor that targets disease-related N-glycosylated proteins in recipient biological cells and its application. Background Art

[0002] To date, editing technologies in the biological field have primarily focused on the nucleic acid level. For example, since 2012, the development of CRISPR / Cas9 technology has revolutionized gene editing. This technology utilizes a specifically designed guide RNA (gRNA) that binds to the Cas9 protein to form a complex that precisely homes in on a specific location in the genome and cuts the DNA strand. This process triggers the involvement of DNA repair mechanisms, enabling the editing and alteration of the target gene. Subsequently, novel technologies for single-base DNA editing, RNA editing, and gene transcription activation and repression have emerged based on CRISPR / Cas9. Currently, research in the field is focusing on single-base DNA editing, which aims to directly modify a single base within a gene without requiring the entire gene to be spliced ​​or replaced. This technology enables the precise repair of single-base mutations, which has important implications for the treatment of some genetic diseases and the study of gene function.

[0003] At the protein level, current research hotspots mainly revolve around protein degradation technology, such as the widely-watched protein-targeted degradation chimera technology (PROTAC). PROTAC (Proteolysis-Targeting Chimeras) technology is a new molecular tool for degrading specific proteins. PROTAC mainly consists of two parts, one is a small molecule that binds to the target protein, and the other is a small molecule that binds to the ubiquitin ligase system. The two parts are connected by a linker. This carefully designed and screened bifunctional small molecule binds to the target protein to form a fusion complex. This fusion complex guides the target protein to the ubiquitin ligase system in the cell, causing the target protein to be ubiquitinated. The ubiquitinated protein will be degraded by the ubiquitin-proteasome, thereby achieving the degradation of the target protein.

[0004] While these technologies represent emerging therapeutic strategies, offering new possibilities for treating currently intractable diseases and profoundly impacting life science research and medicine, they nonetheless face challenges that remain unresolved. The rapid development of CRISPR / Cas9-related technologies has been accompanied by ethical and safety concerns. For example, gene editing can cause permanent changes and carry significant off-target toxicity. Studies have shown that the cleavage process initiated by CRISPR / Cas9 on target DNA can lead to chromosome breakage, necessitating careful consideration of the risks involved in its application. Similarly, while PROTAC technology has generated widespread interest in the field of protein degradation, it still faces challenges. For example, the design and synthesis of PROTAC molecules is a complex undertaking, requiring consideration of multiple factors, including structure, affinity, and pharmacokinetics. This makes the development of PROTAC drugs relatively difficult and time-consuming. Furthermore, different proteins may require unique PROTAC small molecules for degradation, resulting in a lack of versatility and high costs. Furthermore, PROTAC technology itself presents challenges such as nonspecific effects and toxicity.

[0005] Inspired by the rise of various DNA / RNA editing technologies and protein targeted degradation technologies, we believe that a protein targeted editing technology can be developed that can directly modify or edit the target protein of interest at the protein level. In fact, similar to the natural occurrence of DNA / RNA single-base editing in organisms, Gary Ruvkun et al. have discovered that protein sequence editing also occurs spontaneously in organisms and plays an important role in the function of eukaryotic proteins (Lehrbach, NJ, PC Breen, and G. Ruvkun, Protein Sequence Editing of SKN-1A / Nrf1 by Peptide: N-Glycanase Controls Proteasome Gene Expression. Cell, 2019.177(3): p.737-750e15). For example, it has been confirmed that in Caenorhabditis elegans and humans, PNG1 / NGLY1-mediated deglycosylation and deamidation "sequence editing" dependent on N-glycosylation modification sites leads to the nuclear localization of SKN-1A / Nrf1 and the transcriptional activation of proteasome subunit genes. Studies have shown that this amino acid sequence change (from asparagine to aspartic acid) activates the transcription factor activity of the SKN-1A protein, allowing it to translocate from the cytoplasm to the nucleus and upregulate the expression of proteasome subunit genes. This protein deglycosylation sequence editing phenomenon is crucial for the survival of C. elegans cells under conditions of impaired proteasome function. Protein glycosylation is one of the most important post-translational modifications in organisms, occurring on 50%-80% of proteins in cells. It is well known that N-glycosylation is the attachment of polysaccharides to the free amino groups of asparagine in the peptide chain of a protein, followed by the assembly of various types of sugar chains onto the protein. In eukaryotic cells, most glycoproteins on the cell membrane undergo extensive N-glycosylation. This post-translational modification plays a crucial role in various biological functions, such as protein folding and stability, intercellular communication, membrane protein transport, pathogen invasion, and immune response. To date, most research on deglycosylation has focused on inhibiting N-glycosylation biosynthesis. This is achieved through various techniques, including gene mutation technology (such as site-directed mutagenesis of DNA sites corresponding to asparagine (Asn), knockout or deletion of genes related to N-glycan generation and transfer (such as MGAT1 and STT3A genes), treatment with chemical inhibitors (such as streptavidin, NGI-1), metabolic inhibitors (such as dideoxyglucose), and in vitro enzymatic treatment methods, such as the use of peptide N-glycosidase (PNGase F) or endo-glucosidase H (Endo H).However, there is no reported technology that can directly perform deglycosylation sequence editing at the protein level in cells, which involves removing N-glycans and converting the amino acid residues to which they are connected from asparagine to aspartic acid in living cells. In 2021, Christina M.Woo's team developed a nanobody-fused split O-glucose-N-acetylglucosaminase (SplitOGA) tool that can selectively target target proteins from living cells and remove O-linked N-acetylglucosamine (O-GlcNAc), but this does not cause changes in amino acids in the protein sequence (Ge, Y., et al., Target protein deglycosylation in living cells by a nanobody-fusedsplit O-GlcNAcase. Nat Chem Biol, 2021.17(5): p.593-600).

[0006] Previous studies have shown that programmed cell death protein-1 (PD-1) and its ligand, programmed cell death-1 ligand (PD-L1), each possess four N-glycosylation sites. Accumulating evidence suggests that N-glycosylation of PD-1 and PD-L1 is crucial for maintaining their protein synthesis, stability, and interaction. Blocking any of these glycosylation sites (N49, N58, N74, and N116 on PD-1) reduces PD-1 stability and expression, suggesting a close positive correlation between PD-1 glycosylation and its stability and expression. Glycosylation at N192, N200, and N219 (but not N35) on PD-L1 may exert a steric effect, inhibiting PD-L1 degradation via the GSK3β-mediated 26S proteasome mechanism, thereby playing a role in stabilizing PD-L1. Furthermore, inhibiting PD-1 / PD-L1 N-glycosylation reduces their interactions, significantly enhancing anti-tumor efficacy.

[0007] In addition to human proteins, many membrane-bound proteins derived from human pathogenic viruses, such as the SARS-CoV-2 spike protein, are also highly N-glycosylated, with 22 confirmed N-glycosylation sites. These N-glycosylation modifications contribute to the correct folding and structural stability of proteins, promote the transport of expressed proteins within infected host cells and their secretion from cells, and influence the interaction of proteins with host cell receptors, which is crucial for viral entry into host cells and subsequent infection. Acting as a "glycosylated shield," N-glycans can conceal vulnerable epitopes on these proteins, making them more difficult for the immune system to identify and attack the virus.

[0008] Consequently, scientists and researchers have conducted extensive research across a wide range of fields, encompassing biology, transmission pathways, immunology, and SARS-CoV-2 vaccine development, in an effort to understand, prevent, and ultimately treat COVID-19. Numerous studies have highlighted the critical role of N-glycosylation modifications on the SARS-CoV-2 Spike protein in viral invasion of host cells. However, most research has focused on using various techniques to inhibit the biosynthesis of N-glycosylation on the Spike protein to explore the role of N-glycans on the Spike protein. However, these methods are unable to produce the naturally occurring deglycosylated form of the protein within living cells, sparking our interest in further research into this unexplored area.

[0009] The novel coronavirus, SARS-CoV-2, belongs to the genus Betacoronavirus and is an enveloped virus containing positive-stranded single-stranded RNA. The SARS-CoV-2 virus is primarily composed of the spike protein (S protein), nucleocapsid protein (N protein), membrane protein (M protein), envelope protein (E protein), and RNA. The spike protein, located on the viral surface, plays a key role in viral invasion, mediating recognition and membrane fusion between the virus and host cells. The spike protein, typically found in the viral envelope, is a trimer, with each monomer consisting of an S1 subunit and an S2 subunit, totaling 1273 amino acids. The S1 subunit contains a signal peptide (SP) and a receptor-binding domain (RBD), responsible for binding to the host cell receptor angiotensin-converting enzyme 2 (ACE2). The S2 subunit comprises two heptad repeats (HR1 / 2), a central helix, and a transmembrane region, mediating fusion between the viral envelope and the cell membrane. As a transmembrane glycoprotein, the Spike protein is covered with numerous sugar chains, including 22 N-glycosylation sites and 17 O-glycosylation sites. The presence of these sugar chains not only affects its folding and structural stability but is also crucial for its invasion, recognition, and immune evasion, preventing viral recognition by the host immune system and thus escaping immune control. Most studies have shown that inhibiting Spike protein N-glycosylation modification by using N-glycosylation inhibitors (such as tunicamycin and kifunicine), knocking out or knocking down relevant glycan-producing genes (such as MGAT1 and STT3A), or site-directed mutagenesis (N→Q) of N-glycosylation sites effectively reduces the ability of the spike protein to package and infect the host, indicating that glycosylation modification of the spike protein is essential for viral packaging, maturation, and host cell infection. Previous studies have shown that treating mature pseudoviral particles with peptide N-glycosidase (PNGaseF) in vitro can effectively prevent pseudoviral infection of host cells. However, no studies have yet investigated the impact of targeted deglycosylation editing of the SARS-CoV-2 spike protein on its ability to infect cells.

[0010] N-glycoprotein deglycosylase (PNGase or PNG1), also known as peptide N-glycosidase, is widely distributed in prokaryotes and eukaryotes. It specifically targets asparagine (Asn) residues within N-glycosylation motifs (Asn-X-Ser / Thr, where X can be any amino acid except proline), releasing the sugar chain from the protein backbone and converting Asn to Asp. PNGases from different species vary in gene length, amino acid sequence, and domain structure, resulting in differences in biological function. For example, PNGase F (abbreviated as PNGF) from Pseudomonas miricola is a commercial enzyme that exhibits high activity against a variety of native and denatured glycoproteins and glycopeptides, and has broad substrate specificity. It can completely hydrolyze and release all types of N-glycans (including high mannose, hybrid, and complex) and deamidate asparagine (Asn) to aspartic acid residues (Asp). This enzyme is widely used to study and analyze protein glycosylation patterns and functions, and has promoted glycoproteomics research. However, PNG1 from eukaryotic organisms can usually only recognize and hydrolyze misfolded or denatured glycoproteins, and is particularly prone to hydrolyzing high mannose-rich N-glycoprotein substrates. However, studies have shown that deleting the N-terminal H1 helix from yeast PNG1 (Png1p-ΔH1, referred to as ScPNG1ΔH1 in this invention) enhances its enzymatic activity towards denatured N-glycoproteins. It also exhibits efficient deglycosylation activity towards native N-glycoproteins, with peak activity at 30°C and almost no activity above 37°C. However, this has not yet been tested in mammalian cells. Therefore, in this invention, two peptide N-glycosidases, derived from prokaryotes and eukaryotes, were used for testing and application as protein editing enzymes. Summary of the Invention

[0011] In view of the above background, the present invention provides a protein targeted editing technology, which is specifically related to combining a high-affinity small peptide targeting a target protein with two PNGase enzymes from prokaryotes and eukaryotic organisms, respectively, through a linker, in order to directly target N-glycoproteins for deglycosylation, and edit the amino acid residues at the target glycoprotein N-glycosylation modification site to deamidate asparagine into aspartic acid. The present invention has the potential to promote the study of specific N-glycosylation modification-related functions and protein sequence editing. In addition, the present invention greatly destroys protein stability and accelerates the protein degradation process through deglycosylation editing, and also provides a novel perspective and alternative method for the field of protein degradation. The main purpose of the present invention is to develop a deglycosylated protein sequence editing technology that can be used for targeting disease-related N-glycoproteins in receptor biological cells.

[0012] The present invention provides a protein targeting editor, which comprises three elements: a protein targeting peptide, a linker and a protein editing enzyme. Preferably, the protein editing enzyme is peptide N-glycosidase (abbreviated as PNGase).

[0013] Among them, peptide N-glycosidases are used to remove N-linked sugar chains on target proteins and catalyze the deamidation of asparagine to aspartic acid, such as PNGase F (abbreviated as PNGF) from Pseudomonas michelbikova, or PNG1 (abbreviated as ScPNG1ΔH1) from Saccharomyces cerevisiae that has been truncated and modified to be active.

[0014] Protein targeting peptides are used to specifically target target proteins, such as, but not limited to, PD-1, PD-L1, and the SARS-CoV-2 Spike protein. For specific target proteins, targeted literature and patent searches can be conducted to identify experimentally validated peptides with high affinity for the target glycoprotein. Alternatively, such peptides can be designed and screened to serve as protein targeting peptides.

[0015] The linker, including a flexible or rigid linker, is used to connect the protein targeting peptide and the protein editing enzyme.

[0016] Specifically, the arrangement and combination of the three elements include but are not limited to the protein targeting peptide fused to the amino terminus (N terminus) of peptide N-glycosidase via a flexible linker, the protein targeting peptide fused to the carboxyl terminus (C terminus) of peptide N-glycosidase via a flexible linker, the protein targeting peptide fused to the N terminus of peptide N-glycosidase via a rigid linker, or the protein targeting peptide fused to the C terminus of peptide N-glycosidase via a rigid linker.

[0017] The flexible joints include but are not limited to GGGGS, (2) (GGGGS) 3; the rigid joints include but are not limited to (1) (EAAAK) 2, (2) (EAAAK) 3, (3) A (EAAAK) 2A.

[0018] The present invention provides nucleic acids for editing the above-mentioned protein editors, preferably nucleic acids that have been optimized for human codons.

[0019] The present invention provides a nucleic acid expression element containing the above nucleic acid.

[0020] The present invention further provides a recombinant expression vector comprising the aforementioned nucleic acid or its expression element, preferably capable of expressing the protein editor. Preferably, a shuttle vector is employed, for example, capable of replication and amplification in Escherichia coli as well as amplification and expression in mammalian cells. A specific starting vector is pcDNA3.1.

[0021] After introduction into the recipient biological cells, the vector can achieve varying degrees of removal of the target protein's N-glycans within the cells and successfully deamidate asparagine (Asn) residues at N-glycosylation sites on the target protein to aspartic acid (Asp). The protein editor provided by the present invention is successfully expressed in the cells, significantly reducing the stability of the target protein and accelerating its degradation in the ubiquitin-proteasome.

[0022] The present invention also provides a recombinant host cell containing the above-mentioned nucleic acid expression element or the above-mentioned recombinant expression vector, preferably, the host cell is an animal cell, such as a mammalian cell.

[0023] The present invention provides the use of a protein editor, or a nucleic acid encoding the same, or a recombinant vector thereof in target protein editing. Preferably, the use of a protein editor in selectively targeting the deglycosylation editing of the novel coronavirus spike protein, for example, the use of the deglycosylation editing thereof in significantly inhibiting cell-cell fusion and significantly reducing the infection efficiency of the novel coronavirus pseudovirus.

[0024] The protein sequence editor provided by the present invention is used for selectively editing target proteins in cells, for example, it can effectively target PD-1, PD-L1 and SARS-CoV-2 Spike protein, resulting in the removal of the target protein N-glycan and the deamidation of the asparagine residue at the site into an aspartic acid residue, thereby reducing the protein stability and accelerating its degradation process in the ubiquitin-proteasome.

[0025] Experimental studies have shown that the editor for targeting the SARS-CoV-2 Spike protein provided in the present invention can significantly reduce the formation of syncytia, inhibit the maturation and packaging of pseudoviruses, and reduce the efficiency of virus infection of host cells (HEK293T-hACE2, Calu-3, PC-9). Among them, the best editor LCB1-PNGF is characterized in that the targeting peptide LCB1 is connected to the N-terminus of PNGF through a flexible linker (GGGGS) containing five amino acid residues. The LCB1-PNGF editor reduces the cell fusion efficiency by about 80% and the pseudovirus infection efficiency by about 80%. The LCB1-PNGF editor provided in the present invention was detected by liquid chromatography-mass spectrometry to be able to edit 15 N-glycosylation sites of the SARS-CoV-2 Spike protein.

[0026] Therefore, the editor constructed and optimized in the present invention, for example, by editing the N-glycosylation modification site of the SARS-CoV-2 Spike protein, results in the production of a deglycosylated form of the Spike protein, significantly reducing the expression level of the Spike protein, effectively inhibiting the formation of syncytia, and ultimately significantly reducing the pseudovirus packaging and infection efficiency. Of course, the present invention can also be applied to target other target proteins, including target proteins of other receptor biological cells, to achieve corresponding different purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the protein editor constructed by the present invention and related element information. Figure 1 Figure A in the middle is a simplified schematic diagram of the constructed protein deglycosylation sequence editing tool; Figure 1 Middle B is the peptide N-glycosidase PNGF and ScPNG1 from two different species; Figure 1 In the C, a variety of protein targeting editors with different permutations and combinations were constructed, including (1) protein targeting peptides; (2) peptide N-glycosidases from two different species (PNGF with the signal peptide sequence removed and ScPNG1 with H1 removed, i.e., ScPNG1ΔH1) were used in the present invention); (3) a linker was used for connection, and a 3×Flag tag was connected to the C-terminus of the protein editor to facilitate the detection of subsequent protein expression levels; Figure 1 D in the middle represents a high-affinity small peptide used for PD-1, PD-L1, and SARS-CoV-2 Spike protein, as well as different forms of flexible or rigid linkers used in this example.

[0028] Figure 2 This is a schematic diagram of the protein domains and N-glycosylation modification sites of the disease-related target glycoproteins PD-1, PD-L1, and SARS-CoV-2 Spike.

[0029] Figure 3 The effect of the protein-targeting editor was verified by immunoblotting experiments. Figure 3 Figure 5 AC shows the catalytic effects of two peptide N-glycosidases from different species on the target proteins PD-1, PD-L1, and SARS-CoV-2 Spike protein. As shown in the figure, PNGF exhibits high deglycosylation activity on PD-1, PD-L1, and SARS-CoV-2 Spike protein, while ScPNG1ΔH1 has moderate or low deglycosylation activity on the N-glycans of these glycoproteins. This is due to the high enzymatic activity of PNGF and the relatively low enzymatic activity of ScPNG1ΔH1. Figure 3 Middle D shows that compared with the two protein-targeted editing chimeras (bPD-1-PNGF and PNGF-bPD-1), PNGF alone achieved better PD-1 deglycosylation editing effects; Figure 3 Middle E shows that compared with ScPNG1ΔH1 itself, the fusion of bPD-1 peptide and ScPNG1ΔH1 (N-terminus or C-terminus) results in the production of a lower molecular weight deglycosylated form of PD-1 protein, indicating that the fusion of the targeting peptide bPD-1 and ScPNG1ΔH1 with lower enzymatic activity has obvious targeting activity. Figure 3 Figure F shows that, similar to the results of PD-1 editing, the deglycosylation editing effect of PD-L1 was best achieved using PNGF alone compared with bPD-L1-PNGF and PNGF-bPD-L1. Figure G shows that compared with using ScPNG1ΔH1 alone, bPD-L1-ScPNG1ΔH1 and ScPNG1ΔH1-bPD-L1 produced lower molecular weight deglycosylated PD-L1 protein bands.

[0030] Figure 4 The deglycosylation editing effect of the protein editor on the Spike protein was verified, and the specific editing sites and the interaction between the protein editor and the Spike protein were detected. Figure 4 Figures A and B show that compared with PNGF, the LCB1-PNGF pair fused with targeting peptides has a higher affinity for wild-type spike protein (S-WT) and mutant spike protein (S-Mutant, 682 'RRAR' 685 The mutation of the site to 'GGSG') resulted in a more extensive deglycosylation editing effect, and its expression level was lower than that of PNGF; Figure 4 Middle C shows that compared with ScPNG1ΔH1 alone, the LCB1-ScPNG1ΔH1 fusion protein exhibits better deglycosylation editing effect on the full-length Spike protein; Figure 4Middle D shows that the catalytic activity of LCB1-PNGF towards Spike protein is significantly higher than that of LCB1-ScPNG1ΔH1. Figure 4 The EF team confirmed the existence of a strong protein-protein interaction between the constructed editors (PNGF and LCB1-PNGF) and the target Spike protein through co-immunoprecipitation experiments (co-IP). It also proved that LCB1-PNGF, which has a lower expression level, has a more significant deglycosylation editing effect on the mutant spike protein than PNGF. Figure 4 Liquid chromatography-mass spectrometry (LC-MS) analysis was used to detect the deglycosylation-dependent editing sites (asparagine deamidation to aspartic acid) caused by PNGF or LCB1-PNGF editor in the Spike protein mutant (S-Mutant) sample, and the results were statistically analyzed through four biological replicates. Consistent with the immunoblotting results, more deamidation sites (15) were found in the LCB1-PNGF sample compared with the PNGF sample (12 deamidation sites were counted).

[0031] Figure 5 The editor's de-N-glycosylation editing of the SARS-CoV-2 Spike protein disrupts cell-cell fusion and inhibits the efficiency of pseudovirus infection of host cells. Figure 5 In Figure A, immunoblotting experiments were performed to verify the deglycosylation effects of the constructed deglycosylation protein editors on the SARS-CoV-2 Spike protein, as well as the protein expression levels of the editors in living cells. Figure 5 Figure 3B accurately quantified the effects of various editors on cell-cell fusion efficiency using a luciferase-based quantitative analysis method, where editing of LCB1-PNGF resulted in a significant reduction in fusion efficiency of approximately 80%. Figure 5 Figure C clearly shows that pretreatment of Spike protein with LCB1-PNGF significantly inhibited cell fusion through fluorescence microscopy. Figure 5 Figure D is a schematic diagram of SARS-CoV-2 pseudovirus generation and entry into target cells. Figure 5 Figure E detected the Spike protein expression on the surface of the pseudovirus treated with the LCB1-PNGF editor by immunoblotting, showing deglycosylation and a lower protein level than that of the empty vector pcDNA3.1. Figure 5 Through pseudovirus infection experiments, FH found that the pseudovirus processed by the LCB-PNGF editor lost more than 80% of its viral infection ability when infecting three commonly used host cells, including HEK293T, Calu-3 and PC-9.

[0032] Figure 6The designed and optimized protein editors destroyed the protein stability and accelerated the degradation of the target glycoprotein. Figure 6 The cycloheximide treatment experiment confirmed that compared with PNGF alone, the three protein targeting editors bPD-1-PNGF, bPD-L1-PNGF and LCB1-PNGF significantly accelerated the degradation of PD-1, PD-L1 and new coronavirus Spike proteins. Figure 6 Figure E showed that flow cytometry analysis showed that LCB1-PNGF caused a decrease of approximately 50% in the level of SARS-CoV-2 Spike protein compared with the empty vector pcDNA3.1, while PNGF alone resulted in less than 20% decrease. Figure 6 Zhong FG used MG132 treatment experiments to prove that the deglycosylated form of the S1 subunit of the new coronavirus Spike protein induced by different editors mainly undergoes the proteasome-mediated endoplasmic reticulum-associated degradation pathway rather than the autophagy pathway. Figure 6 Zhong H used the CellTiter-Glo kit to perform cell activity analysis and found that the appropriate use of various constructed editors including LCB1-PNGF in six-well plates (the dose of editor used in the six-well plate in this example did not exceed 2 μg) had no obvious toxicity to the cells. DETAILED DESCRIPTION

[0033] To facilitate understanding of the present invention, the present invention is further described in detail with reference to the following specific embodiments and accompanying drawings. The processes, conditions, experimental methods, etc. for implementing the present invention, except for those specifically mentioned below, are common knowledge and common common sense in the art and are not particularly limited in the present invention.

[0034] Example 1: Construction of a plasmid for expressing a de-N-glycosylated protein sequence editor in mammalian cells

[0035] First, the amino acid sequences and corresponding gene sequences of PNGF (UniProt ID: P21163) from Pseudomonas elizabethii and ScPNG1 (UniProt ID: Q02890) from Saccharomyces cerevisiae were obtained from the UniProt and KEGG websites. The signal peptide sequence (1 to 40 amino acids) at the beginning of PNGF and the H1 sequence (1 to 32 amino acids) at the beginning of ScPNG1 were deleted. Human codon optimization was performed by GenWeiZhi (Suzhou, China). PNGF and ScPNG1ΔH1 fragments were synthesized, and 3×Flag tag sequences were fused to the carboxyl termini for subsequent protein expression testing. These gene fragments were cloned into the mammalian expression vector pcDNA3.1 (with dual restriction enzyme sites XhoI and EcoRI) using Gibson seamless cloning to generate the pcDNA3.1-PNGF-3×Flag and pcDNA3.1-ScPNG1ΔH1-3×Flag initial editors, respectively. Then, the inactive mutants of pcDNA3.1-PNGF(D60N)-3×Flag and pcDNA3.1-ScPNG1ΔH1(D204A)-3×Flag were obtained by site-directed mutagenesis using polymerase chain reaction (PCR).

[0036] The amino acid sequences of small peptides with high affinity for PD-1, PD-L1, and SARS-CoV-2 Spike proteins were obtained through literature search. Human codon optimization was performed by GENEWIZ (Suzhou, China), and different forms of linkers (flexible or rigid) were added to their N-termini or C-termini to synthesize the corresponding gene fragments. The fragments were cloned into corresponding plasmids such as pcDNA3.1-PNGF-3×Flag and pcDNA3.1-ScPNG1ΔH1-3×Flag by Gibson seamless cloning technology. Figure 1 As shown, through various permutations and combinations, different protein targeting peptides and two types of peptide N-glycosidases were connected with flexible or rigid linkers to obtain various types of protein editor plasmids in Table 1.

[0037] The amino acid sequence of the PD-1 protein targeting peptide PD-MP1 (named bPD-1 in this example) is as follows (SEQ ID NO: 1): CLC WCARTKPFHRRYGKYLYGTRLQCKKWLSECAQQNPGARVNIQC.

[0038] The corresponding nucleotide sequence synthesized in this example is as follows (SEQ ID NO: 2): TGCCTGTGCTGGTGCGCTAGAACCAA GCCCTTCCACAGAAGATACGGCAAGTACCTGTACGGCACAAGACTGCAGTGCAAGAAGTGGCTGAG CGAGTGCGCTCAGCAGAACCCCGGCGCTAGAGTGAACATTCAGTGC.

[0039] The amino acid sequence of the protein targeting peptide MOPD-1 (named bPD-L1 in this example) of PD-L1 is as follows (SEQ ID NO: 3): IQIREY KRCGQDEERVRRECKERGERQNCHYVIHKEGNCYVCGIICL.

[0040] The corresponding nucleotide sequence synthesized in this example is as follows (SEQ ID NO: 4): ATTCAGATCAGAGAGTACAAGAGATG CGGCCAAGACGAGGAGAGAGTGAGAAGAGAGTGCAAGGAGAGAGGCGAGAGACAGAACTGCCACT ACGTGATCCACAAGGAGGGCAACTGCTACGTGTGCGGCATCATCTGCCTG.

[0041] The amino acid sequence of the protein targeting peptide LCB1 of SARS-CoV-2Spike is as follows (SEQ ID NO: 5): DKEWILQKIYEIMRLL DELGHAEASMRVSDLIYEFMKKGDERLLEEAERLLEEVER.

[0042] The corresponding nucleotide sequence synthesized in this example is as follows (SEQ ID NO: 6): GACAAGGAGTGGATCCTGCAGAAGA TCTACGAGATCATGAGACTGCTGGACGAACTGGGCCACGCCGAGGCTAGCATGAGAGTGAGCGACCT GATCTACGAGTTCATGAAGAAGGGCGACGAAAGACTGCTGGAAGAAGCTGAGCGGCTGCTGGAAGA GGTGGAAAGA.

[0043] The amino acid sequence of the protein targeting peptide 23mer (A2N) of SARS-CoV-2Spike is as follows (SEQ ID NO: 7): QNKTFLDKFNHE AEDLFYQSSLA.

[0044] The corresponding nucleotide sequence synthesized in this example is as follows (SEQ ID NO: 8): CAGAACAAGACATTTTTGGACAAGTTT AACCACGAAGCCGAAGACCTGTTCTATCAAAGTTCACTTGCT.

[0045] The PNGF nucleotide sequence optimized with human codons synthesized in this embodiment is as follows (SEQ ID NO: 9): ATGGCCCCCGCCGACAACACCGTGAACATCAAGACCTTCGACAAGGTGAAGAACGCCTTCGGCGACGGCCTGAGCCAGAGCGCCGAGGGCACCTTCACCTTCCCCGCCGACGTGACCACCGTGAAGACCATCAAGATGTTCATCAAGAACGACTGCCCCAACAAGACCTGCGACGAGTGGGACCGCTACGCCAACGTGTACGTGAAGAACAAGACCACCGGCGAGTGGTACGAGATCGGCCGCTTCATCACCCCCTACTGGGTGGGCACCGAGAAGCTGCCCCGCGGCCTGGAGATCGACGTGACCGACTTCAAGAGCCTGCTGAGCGGCAACACCGAGCTGAAGATCTACACCGAGACCTGGCTGGCCAAGGGCCGCGAGTACAGCGTGGACTTCGACATCGTGTACGGCACCCCCGACTACAAGTACAGCGCCGTGGTGCCCGTGATCCAGTACAACAAGAGCAGCATCGACGGCGTGCCCTACGGCAAGGCCCACACCCTGGGCCTGAAGAAGAACATCCAGCTGCCCACCAACACCGAGAAGGCCTACCTGCGCACCACCATCAGCGGCTGGGGCCACGCCAAGCCCTACGACGCCGGCAGCCGCGGCTGCGCCGAGTGGTGCTTCCGCACCCACACCATCGCCATCAACAACGCCAACACCTTCCAGCACCAGCTGGGCGCCCTGGGCTGCAGCGCCAACCCCATCAACAACCAGAGCCCCGGCAACTGGGCCCCCGACCGCGCCGGCTGGTGCCCCGGCATGGCCGTGCCCACCCGCATCGACGTGCTGAACAACAGCCTGACCGGCAGCACCTTCAGCTACGAGTACAAGTTCCAGAACTGGACCAACAACGGCACCAACGGCGACGCCTTCTACGCCATCAGCAGCTTCGTGATCGCCAAGAGCAACACCCCCATCAGCGCCCCCGTGGTGACCAAC。

[0046]

[0047] Table 1: Constructed and optimized protein deglycosylation sequence editors

[0048]

[0049] Table 1 lists the de-N-glycosylation protein sequence editing tool plasmids targeting three disease-related glycoproteins, PD-1, PD-L1 and SARS-CoV-2 Spike, constructed and optimized in the present invention, which also includes three negative control plasmids (empty pcDNA3.1, inactivated mutant PNGF (D60N) and inactivated mutant ScPNG1ΔH1 (D204A)).

[0050] Example 2: Construction of target protein PD-1, PD-L1 and SARS-CoV-2 Spike expression plasmids

[0051] like Figure 2 As shown, the present invention selected three disease-related membrane proteins with N-glycosylation modifications as target proteins, namely PD-1, PD-L1 and SARS-CoV-2 Spike protein. The nucleotide sequences encoding PD-1 (NCBI-GeneID: 5133) and PD-L1 (NCBI-GeneID: 29126) were obtained from the KEGG website. The full-length gene fragments encoding PD-1 and PD-L1 were synthesized by GenWeiZhi (Suzhou, China). Then, they were constructed into the mammalian expression vector pcDNA3.1 (double restriction sites XhoI and EcoRI) by Gibson seamless cloning technology to obtain pcDNA3.1-PD-1 and pcDNA3.1-PD-L1 expression plasmids, respectively.

[0052] The SARS-CoV-2 Spike protein nucleotide sequence was derived from the pcDNA3.1.2S (GenBank: MT613044.1) plasmid reported in the literature. The corresponding full-length gene fragment encoding the Spike protein was synthesized by Jin Weizhi (Suzhou, China). Together with the EGFP gene, the Spike and EGFP gene sequences were integrated into the lentiviral expression vector pCDH vector by Gibson seamless cloning technology to obtain the pCDH-S-WT-EGFP fusion expression plasmid. At the same time, the pCDH-S-WT-T2A-EGFP expression plasmid was constructed with the self-cleaving polypeptide T2A in the middle. In order to obtain the complete full-length Spike protein that is not cleaved by host proteases in cells, the S1 / S2 cleavage site was modified by polymerase chain reaction (PCR) site-directed mutagenesis. 682 RRAR 685 Mutation 682 GGSG 685, integrated into the pCDH plasmid together with EGFP, and connected in the middle with the self-cleavage polypeptide T2A to obtain the pCDH-S-Mutant-T2A-EGFP plasmid. The S1 subunit gene fragment of the Spike protein was obtained by polymerase chain reaction (PCR) amplification technology, and integrated into the pCDH vector together with the EGFP gene, and connected in the middle with the self-cleavage polypeptide T2A to obtain the pCDH-S1-T2A-EGFP lentiviral expression plasmid. The literature reports that deleting the nucleotide sequence encoding the last 19 amino acids at the end of the Spike gene can obtain a pseudovirus with a higher viral titer. Therefore, in this example, the S-WT-Δ19 gene fragment was amplified by PCR amplification technology, and then integrated into the pcDNA3.1 expression vector by Gibson seamless cloning technology to obtain the pcDNA3.1-S-WT-Δ19 expression plasmid.

[0053] Example 3: Construction of HEK293T-S-WT, HEK293T-S-Mutant and HEK293T-S1 stable cell lines

[0054] According to the needs of the present invention, we successfully constructed three stably expressing S1, S full length and S-Mutant mutant (S1 / S2 cleavage site 682 RRAR 685 Mutation 682 GGSG 685 ) HEK293T stable cell lines (named HEK293T-S1 stable strain, HEK293T-S-WT stable strain and HEK293T-S-Mutant stable strain, respectively).

[0055] The specific experimental process of constructing a stable cell line by the lentiviral packaging method is as follows: 18-24 hours before transfection, passage an appropriate amount of HEK293T cells to a 10-cm cell culture dish (approximately 3-5 million cells), and transfect when the cell density reaches 70-80% on the next day. Lentiviral transfection system (10 μg each of psPAX2, pVSVG, pCDH-S-WT / pCDH-S-Mutant / pCDH-S1) was added to 750 μL DMEM and allowed to stand for 5 minutes. Then, 50 μL Lipo8000 (Cat.C0533, Biyuntian) was added and allowed to stand at room temperature for 15 minutes. The cell culture medium was replaced with DMEM culture medium containing 2% FBS + 1% penicillin / streptomycin. The culture medium could be supplemented appropriately during the process. After culturing for about 72 hours, the lentiviral titer in the supernatant was quickly detected using Clotech Lenti-X GoStix Plus lentiviral titer detection test paper. After reaching the usable titer, the lentiviral supernatant was collected into a 50 mL centrifuge tube and centrifuged at 5000 rpm at 4°C for 30 minutes. The precipitate was discarded and filtered with a 0.45 μm filter membrane (Millipore). Then, the supernatant was filtered using an ultrafiltration tube (Amicon Ultra 100kDa) at 4°C, 2000rpm and concentrated to about 500μL, which can be directly used for lentiviral infection. The remaining lentiviral particles can be frozen in a -80°C refrigerator.

[0056] 18-24 hours before infection, passage the 293T cells to be infected to 6-well or 12-well plates; the next day, when the cell density reaches 70-80%, use an appropriate amount of virus (to establish a virus gradient) to infect the cells, and reduce the volume of the infection medium by half (for infection of six-well plates, virus + serum-free medium = 1 mL), add 10 μg / mL infection reagent Polybrene (C0351-1 ml, Biyuntian), mix gently, then add to the culture plate, and culture in a constant temperature incubator at 37°C, 5% CO2 for 6-8 hours. After that, add fresh complete medium containing 10% FBS + 1% penicillin / streptomycin to the normal culture volume.

[0057] 72 hours after lentiviral infection, the culture medium was replaced with DMEM (DMEM + 10% FBS + 1% penicillin / streptomycin) containing 2 μg / mL puromycin for resistance screening. After 4-7 days of antibiotic selection, GFP-positive cells were isolated by flow cytometry, expanded, and cryopreserved in liquid nitrogen. The resulting stable cell lines, HEK293T-S-WT, HEK293T-S-Mutant, and HEK293T-S1, were used for subsequent experimental procedures.

[0058] Example 4: Verification of the deglycosylation editing effect of the constructed and optimized protein editor on the target protein in cells

[0059] Basic process: well-grown HEK293T cells or corresponding stable cell lines (HEK293T-S-WT, HEK293T-S-Mutant) were passaged into six-well plates, 2.5 mL of fresh complete medium (DMEM + 10% FBS + 1% penicillin / streptomycin) was added, and cultured in a 37°C, 5% CO2 constant temperature incubator for 18-24 hours. When the cell density reached 70-80%, an appropriate amount of constructed plasmid was added through Lipo8000. TM Transfection reagent (Cat.C0533, Biyuntian) was transiently transfected into cells cultured in six-well plates. 48 hours after transfection, cells were collected and washed once with 1× PBS buffer, and then lysed with 200μL of RIPA high-efficiency lysis buffer (Cat.R0010, Solebo) per well at 4°C for 30 minutes. Centrifuge at 4°C, 15,000 rpm for 15 minutes, and the supernatant was collected. Protein concentration was quantified using the Pierce BCA protein quantification kit (Cat.23225, Thermo Fisher Scientific). 30μg of protein was mixed with 1× SDS-PAGE loading buffer (Aibotech) and heated at 100°C for 5-10 minutes. The proteins were then separated by SurePAGE, Bis-Tris 4-12% gel (Cat.M00654, GenScript) (constant voltage 170V, 50 minutes), and subsequently transferred to a 0.45μm PVDF membrane (Cat.88518, Thermo Fisher Scientific) using the eBlotTM L1 system (GenScript). After transfer, the PVDF membrane was removed, washed in TBST buffer for 1-2 minutes, and then blocked with 20mL of blocking solution (5% skim milk powder dissolved in TBST buffer) at 4°C overnight or at room temperature for 2 hours to prevent nonspecific binding of the antibody. After blocking, the blocking solution was removed and the protein expression levels of the various editors constructed in the present invention were detected using an appropriately diluted primary antibody (e.g., anti-Flag antibody (Cat.AE092, Abotek)). The deglycosylation effect of the target protein was verified using an antibody against the corresponding target protein. Incubation with the primary antibody was performed at room temperature for 2 hours, followed by a 5-minute TBST wash, and repeated three times. The cells were incubated with secondary antibody HRP Goat Anti-Rabbit IgG (H+L) (1:10,000) (Cat. AS014, Abotec) diluted in blocking buffer at room temperature for 40 minutes, washed again with TBST for 5 minutes, and repeated three times. Finally, luminescence imaging detection (Tanon 5200, Shanghai Tianneng) was performed using an ultrasensitive ECL chemiluminescence detection kit (Cat. S6009M, Bioscan Biotechnology).

[0060] The specific implementation process is as follows:

[0061] (1) Verification of the deglycosylation effect of PD-1 protein: The well-grown HEK293T cells were passaged into six-well plates and cultured for 18-24 hours. When the cell density reached 70-80%, 1 μg of PD-1 expression plasmid (pcDNA3.1-PD-1) and 1 or 2 μg of targeted PD-1 protein editor (4 μL of Lipo8000 per well) were transfected at the same time. After 48 hours of transfection, cell lysis, protein concentration determination and immunoblotting (Western blotting) experiments were performed according to the above experimental procedures. The protein expression levels of various PD-1-targeting editors constructed in the present invention were detected by anti-Flag antibody (1:3000), and the deglycosylation effect of the target protein PD-1 was detected by PD-1 antibody (1:2000) (Cat.A5584, Abotek).

[0062] (2) Verification of the deglycosylation effect of PD-L1 protein: The well-grown HEK293T cells were passaged into six-well plates and cultured for 18-24 hours. When the cell density reached 70-80%, 1 μg of PD-L1 expression plasmid (pcDNA3.1-PD-L1) and 1 or 2 μg of targeted PD-L1 protein editor (4 μL of Lipo8000 per well) were transfected at the same time. After 48 hours of transfection, cell lysis, protein concentration determination and immunoblotting (Western blotting) experiments were performed according to the above experimental procedures. The protein expression levels of various PD-L1-targeting editors constructed in the present invention were detected by anti-Flag antibody (1:3000), and the deglycosylation effect of the target protein PD-L1 was detected by PD-L1 antibody (1:2000) (Cat.A1645, Abotek).

[0063] (3) Verification of the deglycosylation effect of the new crown Spike protein: The well-grown HEK293T-S-WT or HEK293T-S-Mutant stable cell line cells were passaged into six-well plates and cultured for 18-24 hours. When the cell density reached 70-80%, 2 μg of targeted spike protein editor was transfected (the dosage of Lipo8000 per well was 4 μL). After 48 hours of transfection, cell lysis, protein concentration determination and immunoblotting (Western blotting) experiments were performed according to the above experimental procedures. The protein expression levels of various spike-targeting editors constructed in the present invention were detected by anti-Flag antibody (1:3000), and the deglycosylation effect of the target protein new crown spike protein was detected by anti-RBD antibody (1:2000) (Cat.40592-T62, Yiqiao Shenzhou).

[0064] like Figure 3As shown in Figures AC, first, due to the high enzymatic activity of PNGF itself and its broad substrate selectivity, PNGF expressed in HEK293T cells successfully caused obvious deglycosylation editing of PD-1, PD-L1 and Spike proteins; secondly, the truncated ScPNG1ΔH1 also had a certain degree of deglycosylation editing effect on PD-L1 and Spike proteins. Figure 3 Figure D shows that compared with the fusion targeting peptide bPD-1 at the N-terminus or C-terminus of its PNGF, the expression of PNGF alone showed a superior deglycosylation editing effect on PD-1. This may be due to the high enzymatic activity of PNGF itself, resulting in the effect of the targeting peptide not yet being exerted. Therefore, the enzyme engineering of PNGF to reduce its enzymatic activity, as well as the selection of targeting peptides and appropriate fusion methods need to be further optimized to improve the targeting activity; similar to targeting PD-1, compared with the fusion targeting peptide bPD-L1, the expression of PNGF alone also showed a better deglycosylation editing effect on PD-L1 ( Figure 3 However, when the targeting peptide bPD-1 was fused to the N-terminus or C-terminus of ScPNG1ΔH1, a lower molecular weight deglycosylated protein band was observed compared to when ScPNG1ΔH1 was expressed alone ( Figure 3 Similarly, fusion of the targeting peptide bPD-L1 to the N-terminus or C-terminus of ScPNG1ΔH1 showed a better degree of deglycosylation of the target protein PD-L1 than expressing ScPNG1ΔH1 alone ( Figure 3 (G), indicating that the fusion of targeting peptides bPD-1 and bPD-L1 with ScPNG1ΔH1 with lower enzymatic activity effectively promoted the targeting activity of deglycosylation editing of target proteins PD-1 and PD-L1.

[0065] like Figure 4 As shown in Figures AD, compared with expressing only PNGF or ScPNG1ΔH1 itself, the protein-targeted editing chimeras of PNGF (i.e., LCB1-PNGF) or ScPNG1ΔH1 (i.e., LCB1-ScPNG1ΔH1) fused with the targeting peptide LCB1 have better deglycosylation editing effects on Spike proteins stably expressed in cells; at the same time, LCB1-PNGF exhibits superior deglycosylation editing effects than LCB1-ScPNG1ΔH1.

[0066] Example 5: Verification of the mutual binding of PNGF and LCB1-PNGF editors to the novel coronavirus Spike protein and detection of the deamidation site of the Spike protein after de-N-glycosylation

[0067] Since the LCB1-PNGF editor has a significant effect on the deglycosylation editing of the new crown Spike protein, PNGF and LCB1-PNGF were used as examples to detect the mutual binding ability of the two editors with the Spike protein by co-immunoprecipitation technology (co-IP), and then the specific deglycosylation sites of the Spike protein were detected and counted by liquid chromatography-mass spectrometry (LC-MS). Since the Spike protein is easily cleaved into S1 and S2 subunits by furin protease in the host cell, the HEK293T-S-Mutant stable cell line constructed in the present invention was used. After the S-Mutant was mutated ( 682 RRAR 685 Mutation 682 GGSG 685 ) inactivates the S1 / S2 cleavage site without affecting the protein structure, enabling the complete full-length S protein to be obtained.

[0068] Co-immunoprecipitation (co-IP) experiment: 18-24 hours before transfection, well-grown HEK293T-S-Mutant stable cell lines were passaged into 10-cm dishes (approximately 3-5 million cells / dish). When the cells were confluent to 50-70% density, an appropriate amount of the editor plasmid (750 μL DMEM, 10 μg PNGF or LCB1-PNGF, 25 μL Lipo8000) was transiently transfected into each dish using the Lipo8000 liposome method. After 48 hours, the cells were collected and lysed using NP-40 lysis buffer at 4°C for 5 minutes. The cells were centrifuged at 15,000 rpm at 4°C for 5 minutes, and the supernatant was collected and quantified using the Pierce BCA protein quantification kit (Cat. 23225, Thermo Fisher Scientific, Inc., New York, NY). Scientific) for quantification, a portion of the protein supernatant was used as input, and the remaining protein samples were divided into two equal groups. One group of protein samples was immunoprecipitated with the Flag tag protein immunoprecipitation kit (magnetic bead method) (Cat. P2181S, Biyuntian) to obtain Flag-tagged PNGF or LCB1-PNGF, as well as all proteins bound thereto. The expression of PNGF or LCB1-PNGF and the deglycosylation of the S-Mutant protein bound thereto were detected by Western blotting. At the same time, another group of protein samples was immunoprecipitated with the Anti-SARS-CoV-2 SpikeS1-NTD Magnetic Beads Immunoprecipitation (IP) Kit (Cat. MB40591-R235, Sino Biological), to obtain the S-Mutant protein and all proteins bound thereto. The degree of deglycosylation of the S-Mutant and the expression level of the PNGF or LCB1-PNGF bound thereto were then detected by Western blotting.

[0069] Liquid chromatography-mass spectrometry (LC-MS) experiment: 18-24 hours before transfection, well-grown HEK293T-S-Mutant stable cell lines were passaged into 10-cm dishes (approximately 3-5 million cells / dish). When the cells reached 50-70% confluence, an appropriate amount of the editor plasmid (750 μL DMEM, 10 μg PNGF or LCB1-PNGF, 25 μL Lipo8000) was transiently transfected into each dish using the Lipo8000 liposome method. After 48 hours, the cells were harvested and lysed using NP-40 lysis buffer at 4°C for 5 minutes. The cells were centrifuged at 15,000 rpm at 4°C for 5 minutes, and the supernatant was collected and quantified using the Pierce BCA protein quantification kit (Cat. 23225, Thermo Fisher Scientific, Inc., New York, NY). Equal amounts of protein samples were quantified using a Flag Tag Protein Immunoprecipitation Kit (Magnetic Bead Method) (Cat. P2181S, Beyotime) to obtain Flag-tagged PNGF or LCB1-PNGF, as well as all proteins associated with it (including S-mutant proteins). SDS-PAGE gel electrophoresis was performed to obtain S-mutant protein strips of the corresponding molecular weight. After sample processing, 1 μL of each sample was separated using a nanoElute nanoliter liquid phase system and then coupled to a mass spectrometer equipped with a nanoliter ion source (Bruker timsTOF Pro 2) for data acquisition. The .d files acquired by mass spectrometry were converted to MGF files using Data Analysis (vversion, Bruker MGF). Proteome Discoverer (v 2.5.0.400, Thermo) software was used for database comparison and analysis, ultimately identifying the deamidation site of the Spike protein's N-glycosylation site.

[0070] like Figure 4 As shown in E and F, immunoprecipitation experiments have demonstrated that both PNGF and LCB1-PNGF editors have significant protein-protein interactions with S-Mutant proteins, indicating that both editors can bind to the target protein S-Mutant and perform deglycosylation editing on it. The results of immunoblotting experiments showed that compared with PNGF, LCB1-PNGF can cause a deeper deglycosylation editing effect on the new crown Spike protein, indicating that the fusion of LCB1 peptide and PNGF effectively improves the targeting activity. Consistent with this result, the results of LC-MS experiments showed that ( Figure 4(G) Compared to expressing PNGF alone, LCB1-PNGF significantly increased the number of deglycosylation editing sites on the S-Mutant protein. Statistics from four sets of biological replicates revealed that LCB1-PNGF caused deglycosylation and deamidation editing of 15 N-glycosylation sites on the Spike protein, while PNGF caused deglycosylation and deamidation editing of 12 N-glycosylation sites. These results suggest that the 56-amino acid residue-long LCB1 peptide effectively targets the Spike protein and guides its fusion protein, PNGF, to cause deeper deglycosylation editing of the Spike protein.

[0071] Example 6: LCB1-PNGF editor significantly inhibits cell fusion efficiency

[0072] Since N-glycosylation modification plays an important role in the interaction between the new coronavirus Spike protein and the ACE2 protein expressed on the surface of host cells and mediating subsequent cell fusion, the present invention focuses on studying the deglycosylation editing effects of various types of protein editors including LCB1-PNGF (see Table 1) on the Spike protein and its impact on cell fusion efficiency.

[0073] To verify the effect of Spike protein deglycosylation editing, 18-24 hours before transfection, passage the same number of well-grown HEK293T-S-WT stable cell lines into six-well plates (approximately 600,000 cells per well). Add 2 mL of fresh complete medium (DMEM + 10% FBS + 1% penicillin / streptomycin) to each well and incubate in a 37°C incubator (5% CO2). When the cell density reaches 70-80%, replace with fresh complete medium. Add the prepared transfection complex (125 μL DMEM, 2 μg of the different editor-related plasmids, and 4 μL Lipo8000) to each well, gently shake to mix, and continue to incubate in a 37°C incubator (5% CO2). 48 hours after transfection, cells were washed once with 1× PBS buffer. Then, 200 μL of RIPA high-performance lysis buffer was added to each well and lysed at 4°C for 30 minutes. The cells were centrifuged at 15,000 rpm for 15 minutes at 4°C. The supernatant was collected and protein concentration was quantified using the Pierce BCA Protein Assay Kit (Cat. 23225, Thermo Fisher Scientific). 30 μg of protein was mixed with 5× SDS-PAGE loading buffer (Cat. RM00001, Abotek) and heated at 100°C for 5–10 minutes. Proteins were then separated by SurePAGE using a Bis-Tris 4–12% gel (Cat. M00654, GenScript) at a constant voltage of 170 V for 50 minutes and transferred to a 0.45 μm PVDF membrane (Cat. 88518, Thermo Fisher Scientific) using the eBlot™ L1 system (GenScript). After transfer, remove the PVDF membrane and rinse in TBST buffer for 1-2 minutes. Then, block with 20 mL of blocking buffer (5% nonfat dry milk dissolved in TBST buffer) at 4°C overnight or at room temperature for 2 hours to prevent nonspecific antibody binding. After blocking, remove the blocking buffer and detect protein expression levels of various Spike-targeting editors using anti-Flag antibodies (1:3000). Deglycosylation of the target protein, the SARS-CoV-2 spike protein, was detected using anti-RBD antibodies (1:2000) (Cat. 40592-T62, Sino Biological). Incubate with the primary antibody at room temperature for 2 hours, wash with TBST for 5 minutes, and repeat three times. Incubate with the secondary antibody HRP Goat Anti-Rabbit IgG (H+L) (1:10,000) (Cat. AS014, Abiotech) diluted in blocking buffer for 40 minutes at room temperature. Wash again with TBST for 5 minutes, and repeat three times. Finally, an ultrasensitive ECL chemiluminescence detection kit (Cat.S6009M, Bioscan Biotechnology) was used for luminescence imaging (Tanon 5200, Shanghai Tianneng) detection.

[0074] Luciferase assay for precise quantification of cell fusion efficiency: 18-24 hours before transfection, passage the same number of HEK293T-S-WT stable cell lines into six-well plates (approximately 600,000 cells per well) and add 2 mL of fresh complete medium (DMEM + 10% FBS + 1% penicillin / streptomycin) to each well for culture. Passage the same number of HEK293T-ACE2 stable cell lines into 10-cm dishes (3-5 million cells), add 10 mL of fresh complete medium (DMEM + 10% FBS + 1% penicillin / streptomycin), and culture in a 37°C incubator (5% CO2). When the cell density reaches 70-80%, replace with fresh complete medium. To each well of a six-well plate containing HEK293T-S-WT cells, add the prepared transfection complex (125 μL DMEM, 1.5 μg pFR-luc plasmid, 2 μg of the various editor-related plasmids, and 5 μL Lipo8000). Gently shake to mix, and incubate in a 37°C incubator (5% CO2). To a 10-cm dish containing HEK293T-ACE2 cells, add the prepared transfection complex (750 μL DMEM, 10 μg pBD-NF-κB plasmid, and 25 μL Lipo8000). Gently shake to mix, and continue incubating in a 37°C incubator (5% CO2). 24 hours after transfection, passage an appropriate number of HEK293T-ACE2 cells into a 24-well plate (approximately 150,000 cells per well). 48 hours after transfection, the HEK293T-S-WT cells in the six-well plate were resuspended with an appropriate amount of 0.25% trypsin, the cell number was counted, and then evenly plated on the HEK293T-ACE2 cells in the 24-well plate at a 1:1 ratio. Continue to culture for 16-24 hours, aspirate the cell culture medium, and add 200μL of reporter gene cell lysate to each well of the 24-well plate after cell fusion. After sufficient lysis at 4°C, centrifuge at 15000g for 10 minutes, take 20μL of supernatant and add it to the white opaque 96-well plate. Add 100μL of firefly luciferase detection reagent (Cat.RG051S, Beyotime) that has been equilibrated to room temperature, incubate at room temperature for 5 minutes, and use the SpectraMax iD5 multi-function microplate reader to measure the luminescence value and calculate the cell fusion efficiency.

[0075] Fluorescence microscopy observation of cell fusion: To visualize the inhibitory effect of deglycosylation editing of the Spike protein on cell fusion, appropriate amounts of pcDNA3.1-PNGF plasmids and pcDNA3.1-LCB1-PNGF plasmids, as well as empty vector and inactive mutant PNGF (D60N) as negative controls, were transiently transfected into six-well plates of HEK293T-S-WT-EGFP stable cell lines at a cell density of 70-80%. Forty-eight hours after transfection, the cells were digested with 0.25% trypsin and plated at a ratio of approximately 1:1 on the surface of HEK293T-hACE2 cells at a cell density of 70-80%. Culture was continued for approximately 24 hours, and images of syncytia formation were captured using an OLYMPUS IX73 inverted microscope and cellSens Entry software.

[0076] First, if Figure 5 As shown in Figure A, among all the editors constructed and optimized to target the novel coronavirus Spike protein, except for the negative control group (including blank vectors and inactivated mutants), all other editors were able to have varying degrees of deglycosylation editing effects on the Spike protein. Overall, compared with the ScPNG1ΔH1-related editors, the PNGF-related editors (fused with LCB1 peptide and 23mer (A2N) peptide, respectively) caused more deglycosylation editing of the Spike protein.

[0077] In order to accurately quantify the effects of deglycosylation editing on Spike protein and its effects on cell fusion, we used a modified luciferase quantification method to accurately quantify the cell fusion efficiency. The principle is: HEK293T-S-WT stable cells are transfected with a pFR-Luc plasmid, which contains a synthetic promoter and a luciferase gene. The synthetic promoter consists of five tandem repeats of a regulatory yeast GAL4 binding site to regulate the expression of the luciferase gene. HEK293T-ACE2 stable cells are transfected with a pBD-NF-κB plasmid, which contains a fusion protein encoding a GAL4 DNA binding domain and a NF-κB transcriptional activation domain. When cell fusion occurs between S-WT and ACE2 cells, when the GAL4-NF-κB fusion protein binds to the GAL4 binding site in the promoter region of the pFR-Luc plasmid, luciferase gene expression is activated, and the expression of the luciferase gene is detected using BrightLumi TM The luciferase activity was measured by a luciferase reporter gene assay kit (Cat.RG051S, Beyotime) to accurately quantify the syncytium formation efficiency.

[0078] Although immunoblotting experiments showed that the fusion editors of LCB1 and PNGF (with different intermediate linkers) had little difference in deglycosylation editing effects, Figure 5As shown in Figure B, LCB1-PNGF editor had the most significant effect in inhibiting cell fusion (cell fusion efficiency was reduced by about 80%), followed by LCB1-(EAAAK)2-PNGF and LCB1-(GGGGS)3-PNGF. Since LCB1-PNGF was the most effective in inhibiting cell fusion, in order to visualize cell fusion, syncytium formation was observed using a fluorescence microscope ( Figure 5 Middle C). Compared to an empty vector and an inactive mutant, the LCB1-PNGF editor significantly reduced syncytia formation. Overall, the LCB1-PNGF editor effectively targets the Spike protein for deglycosylation, significantly inhibiting cell fusion.

[0079] Example 7: LCB1-PNGF editor significantly reduces pseudovirus packaging and infection efficiency

[0080] It is known that the LCB1-PNGF editor can effectively reduce the efficiency of cell fusion, so the present invention further studied its effect on pseudovirus packaging and host cell infection efficiency. The third-generation lentiviral system was used for pseudovirus packaging, and the S-WT-Δ19 protein sequence with 19 amino acids truncated at the carboxyl terminus was used to replace the VSV-G envelope protein sequence to produce a high viral titer SARS-CoV-2 Spike pseudovirus ( Figure 5 (D) Schematic diagram of pseudovirus packaging and infection.

[0081] Immunoblotting was used to detect the expression and deglycosylation of Spike protein in the packaged pseudovirus: 18-24 hours before transfection, an appropriate number of HEK293T cells in good growth condition were passaged into 150-mm dishes. The next day, when cells reached 70–80% confluence, fresh medium was replaced 1 h before transfection. Lipo8000 was used to transfect the relevant plasmids (10 μg pLVX-IRES-ZsGreen1-Luc, 10 μg psPAX2, 20 μg S-WT-Δ19, 10 μg pcDNA3.1, or LCB1-PNGF). Seventy-two hours after transfection, the cell supernatant was collected and centrifuged at 5000 rpm for 30 min at 4°C. The pellet was discarded and filtered through a 0.45 μm filter (Millipore). Pseudoviruses were concentrated using a universal virus concentration kit (Cat. C2901L, Beyotime), and pseudovirus titers were determined using a lentivirus titer ELISA kit (Cat. BF06203, Biolong). Western blotting of concentrated SARS-CoV-2 Spike pseudovirus particles was performed, using P24 protein as an internal control protein to detect Spike protein expression levels and the effect of deglycosylation editing on the pseudoviruses.

[0082] Pseudovirus packaging and infection efficiency: 18-24 hours before transfection, passage an appropriate number of HEK293T cells in good growth condition into 150-mm dishes. The next day, when the cells reached 70-80% confluence, fresh medium was replaced 1 hour before transfection. Lipo8000 was used to transfect the relevant plasmids (10 μg pLVX-IRES-ZsGreen1-Luc, 10 μg psPAX2, 20 μg S-WT-Δ19, 10 μg pcDNA3.1 or PNGF(D60N) or LCB1-PNGF(D60N) or PNGF or LCB1-PNGF). 72 hours after transfection, the cell supernatant was collected and centrifuged at 5000 rpm at 4°C for 30 minutes. The pellet was discarded and filtered through a 0.45 μm filter (Millipore). Pseudovirus was concentrated using a universal virus concentration kit (Cat. C2901L, Bio-Tech), and pseudovirus titer was determined using a lentivirus titer ELISA kit (Cat. BF06203, Bio-Tech). The concentrated pseudovirus sample can be directly used for infection experiments. 18-24 hours before infection, appropriate amounts of HEK293T-ACE2 cells, Calu-3, and PC-9 cells were passaged into 96-well plates, with approximately 30,000 cells per well. After approximately 24 hours of passaging, when the cells were confluent in the well plate, the original culture medium was discarded, the cells were washed once with 1× PBS, and different concentrated pseudovirus samples of the same virus titer were added to establish an appropriate virus titer gradient. 48 hours after pseudovirus infection, the pseudovirus infection was detected by luciferase fluorescence activity assay: the original virus mixture in the 96-well plate was discarded, the plate was washed twice with PBS, and 100 μL of Firefly luciferase reporter gene assay cell lysis buffer (Cat.RG126M, Beyotime) was added to each well. The cells were lysed at room temperature for 10 minutes and centrifuged at 12,000 rpm at 4°C for 10 minutes to obtain the supernatant. 20 μL of the supernatant was taken from each well and added to a white opaque 96-well plate. 100 μL of Firefly luciferase detection reagent (Cat.RG051S, Beyotime) equilibrated to room temperature was added to each well. The plates were incubated at room temperature for 5 minutes. The luminescence value was then measured using a BioTek Synergy Neo2 multi-function microplate reader to calculate the pseudovirus infection efficiency.

[0083] like Figure 5 As shown in E, with an equal amount of P24 protein as the internal reference protein, the Spike protein on the concentrated pseudovirus particles was pretreated with the LCB1-PNGF editor. Compared with the blank vector pcDNA3.1, both the full-length Spike protein and the S1 subunit protein showed obvious deglycosylated bands, and their expression levels were significantly reduced, indicating that after the pseudovirus particles were pretreated with the LCB1-PNGF editor, the protein expression level of the Spike protein packaged thereon was significantly reduced after deglycosylation editing. Figure 5Figures FH in Figure 3 showed that SARS-CoV-2 Spike pseudovirus pretreated with LCB1-PNGF significantly reduced its efficiency in infecting host cells (including HEK293T-ACE2, Calu-3 and PC-9) (reduced by about 80%).

[0084] Example 8: Deglycosylation editing causes target protein instability and accelerates degradation

[0085] Cycloheximide (CHX) blocking method: By inhibiting protein synthesis with cycloheximide (CHX) and monitoring changes in protein levels at different time intervals, the half-life of a specific protein can be determined. Specific experimental steps: (1) Well-grown HEK293T cells were passaged into six-well plates. After culturing for about 18-24 hours, when the cell density reached 70-80%, appropriate amounts of PD-1 expression plasmid and targeted PD-1 editor plasmid (PNGF, bPD-1-PNGF) or blank control plasmid pcDNA3.1 were simultaneously transfected using Lipo8000 high-efficiency transfection reagent; 30 hours after transfection, cycloheximide (CHX) (Cat. C112766, Aladdin) at a final concentration of 100 μM was added to each well, and the cells were cultured for another 12 hours. Cell samples were collected every 4 hours, lysed using RIPA high-efficiency lysis buffer at 4°C for 30 minutes, centrifuged at 15,000 rpm at 4°C for 15 minutes, and the supernatant was collected. The protein concentration was quantified using the Pierce BCA protein quantification kit (Cat. 23225, Thermo Fisher Scientific). 30 μg of protein was mixed with 5× SDS-PAGE loading buffer and heated at 100°C for 5-10 minutes. PD-1 protein degradation was then detected by Western blotting. (2) PD-L1 expression plasmid and targeted PD-L1 editor plasmid (PNGF, bPD-L1-PNGF) or blank control plasmid pcDNA3.1 were simultaneously transfected into HEK293T cells. The remaining steps were the same as above. (3) Appropriate amounts of targeted Spike protein-related editor plasmids (including PNGF, LCB1-PNGF, ScPNG1ΔH1, LCB1-ScPNG1ΔH1) were transiently transfected into HEK293T-S-WT stable cells. The remaining steps were the same as above.

[0086] like Figure 6 As shown in Figures A and B, although the deglycosylation effect of the PNGF editor fused with the targeting peptide bPD-1 or bPD-L1 is not as good as that of expressing PNGF itself, the stability of its target proteins PD-1 and PD-L1 is greatly weakened. Figure 6Middle C shows that the deglycosylation editing of Spike protein by LCB1-PNGF editor makes Spike protein extremely unstable, especially its S1 subunit protein is almost undetectable. In comparison, although LCB1-ScPNG1ΔH1 can also cause significant deglycosylation editing of Spike protein, its effect on its stability is not obvious ( Figure 6 Middle D).

[0087] Fluorescence flow cytometry: The well-grown HEK293T cells were passaged into six-well plates and cultured for about 18-24 hours. When the cell density reached 70-80%, the pCDH-S-WT-EGFP plasmid and the related editor plasmid (PNGF or LCB1-PNGF) were transiently transfected simultaneously using Lipo8000 high-efficiency transfection reagent. The empty vector pcDNA3.1 and the inactivated mutant PNGF (D60N) were used as negative controls. After 48 hours of transfection, the cells were collected and the overall expression level of the Spike protein was detected by the average fluorescence intensity of eGFP in flow cytometry (BD LSR Fortessa X-20). Figure 6 As shown in E, targeted editing of LCB1-PNGF resulted in a decrease of approximately 50% in the overall expression of Spike protein in cells.

[0088] MG-132 method to identify protein degradation pathways: The corresponding editor plasmids (including PNGF, LCB1-PNGF, ScPNG1ΔH1, ScPNG1ΔH1 and negative control) were transiently transfected into HEK293T-S1 stable cell lines. After 48 hours of transfection, 10 μM proteasome inhibitor MG-132 (Cat.HY-13259, MedChemExpress) or 50 nM lysosomal inhibitor NH4Cl (Cat.HY-Y1269, MedChemExpress) were added, and the cells were collected after incubation for 12 hours. The protein degradation pathway was identified by western blotting. As shown in Figures F and G, after the addition of the proteasome inhibitor MG-132, the protein expression level of the S1 subunit of the new crown Spike protein can be significantly restored, while the addition of the lysosomal inhibitor NH4Cl has no obvious effect, indicating that the deglycosylated S1 subunit protein is mainly degraded through the proteasome pathway rather than the lysosomal pathway.

[0089] Example 9: Cytotoxicity Assessment of Protein Editors

[0090] Well-grown HEK293TS-WT cells were passaged into six-well plates and cultured for about 18-24 hours. When the cell density reached 70-80%, 2 μg of different editor plasmids were transiently transfected using Lipo8000 high-efficiency transfection reagent. 48 hours after transfection, the cells were collected and the cell number was quantified using a LUNAIITM automatic cell counter. 40,000 cells were taken for each sample in an opaque 96-well plate. 100 μl of CellTiter-Glo reagent (Promega) was added to each well and incubated at room temperature for 10 minutes. The luminescence intensity was then measured using a BioTek Synergy Neo2 multi-mode microplate reader to assess cell viability, thereby evaluating the cytotoxicity of the protein editor. Figure 6 As shown in Figure H, the protein editor plasmids did not significantly affect cell viability, indicating that the cytotoxicity they caused was not high and the cell tolerance was acceptable.

Claims

1. A protein editor comprising three elements: a protein targeting peptide, a linker and a protein editing enzyme, preferably the protein editing enzyme is a peptide N-glycosidase.

2. The protein editor according to claim 1, wherein The peptide N-glycosidase is used to remove N-linked sugar chains on target proteins and catalyze the deamidation of asparagine to aspartic acid, for example, from Pseudomonas mikkelsenii ( Elizabethkingiamiricola ) or PNGase F from Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) PNG1 that has been truncated and transformed to be active (ScPNG1ΔH1); Protein targeting peptides are used to specifically target target proteins, such as but not limited to PD-1, PD-L1, and SARS-CoV-2 Spike protein; The linker, including a flexible or rigid linker, is used to connect the protein targeting peptide and the protein editing enzyme.

3. The protein editor according to claim 1, wherein The arrangement and combination of the three elements include but are not limited to the protein targeting peptide fused to the N-terminus of the peptide N-glycosidase via a flexible linker, the protein targeting peptide fused to the C-terminus of the peptide N-glycosidase via a flexible linker, the protein targeting peptide fused to the N-terminus of the peptide N-glycosidase via a rigid linker, or the protein targeting peptide fused to the C-terminus of the peptide N-glycosidase via a rigid linker.

4. The protein editor according to claim 1, wherein The flexible joints include but are not limited to GGGGS, (2) (GGGGS) 3; the rigid joints include but are not limited to (1) (EAAAK) 2, (2) (EAAAK) 3, (3) A (EAAAK) 2A.

5. A nucleic acid encoding the protein editor according to any one of claims 1 to 4, preferably a nucleic acid that has been optimized for human codons.

6. An expression element comprising the nucleic acid according to claim 5.

7. A recombinant expression vector containing the expression element as described in claim 6, preferably the vector is capable of expressing the protein editor; preferably a shuttle vector is used, for example, the starting vector is pcDNA3.

1.

8. A recombinant host cell containing the nucleic acid expression element according to claim 6 or the recombinant expression vector according to claim 7, preferably an animal cell, such as a mammalian cell.

9. Use of the protein editor according to any one of claims 1 to 4, or its encoding nucleic acid, or its recombinant vector in target protein editing.

10. The use according to claim 9, characterized in that Application of deglycosylation editing in selectively targeting disease-associated glycoproteins PD-1, PD-L1, and SARS-CoV-2 Spike protein.

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