Fucosidase mutants and the use thereof

Abstract

To provide a novel mutant form of α-fucosidase (α-L-fucosidase), which exhibits enhanced α-(1,6) fucosidase activity.SOLUTION: The present disclosure relates to compositions comprising the novel mutant form of α-fucosidase, and methods of using the novel mutant form of α-fucosidase to cleave α-(1,6)-linked fucoses in the glycoconjugates. In one embodiment, the present disclosure provides a mutant form of α-L-fucosidase for the improved enzymatic hydrolysis of fucose in vitro.SELECTED DRAWING: None

Description

[Technical field]

[0001] Technical Field The present disclosure relates to novel mutant forms of α-fucosidase (α-L-fucosidase) that exhibit enhanced α-(1,6) fucosidase activity. The present disclosure also relates to compositions comprising the novel mutant forms of α-fucosidase and methods of using the novel mutant forms of α-fucosidase to cleave α-(1,6)-linked fucose in glycoconjugates. [Background technology]

[0002] background N-glycosylation is one of the most common post-translational modifications observed in mammalian proteins and plays an important role in regulating the intrinsic characteristics and biological functions of these proteins. For example, the attachment of N-glycans can significantly affect protein folding, stability, antigenicity, and immunogenicity. In addition, N-glycans can be directly involved in a wide range of biological recognition processes, such as cell adhesion, host-pathogen interactions, cancer metastasis, and immune responses. Although mammalian N-glycans share a basic oligosaccharide core structure, the introduction of additional modifications to the core structure, such as sialylation and fucosylation, contributes to an increased level of structural diversity that affects biological functions.

[0003] The physiological activity of therapeutic antibodies is mediated by two distinct mechanisms. The first mechanism involves target antigen neutralization or apoptosis, which contributes to therapeutic efficacy. The second mechanism involves antibody effector functions, antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which are activated by the formation of immune complexes. The importance of ADCC in the clinical efficacy of therapeutic antibodies, especially in anticancer antibodies, has been elucidated through genetic analysis of leukocyte receptor (FcγR) polymorphisms in patients. Therefore, the bioindustry has devoted considerable efforts to developing ADCC enhancement technologies, including modification of N-glycans attached to the constant region (Fc) of antibodies. It is known that the amount of α-(1,6)-linked fucose (core fucose) attached to the innermost GlcNAc of N-glycans on the Fc region of antibodies affects ADCC activity. Removal of core fucose from the innermost GlcNAc of N-glycans on the Fc region significantly enhances the ADCC activity of IgG due to increased binding affinity of nonfucosylated antibodies to the FcγRIIIα receptor.

[0004] Numerous approaches have been developed to enhance FcγRIIIα binding and ADCC by reducing IgG fucosylation. Some strategies involve the development of production cell lines that ablate or reduce the expression of α-(1,6)fucosyltransferase. An alternative strategy to reduce fucosylation includes silencing the α-(1,6)fucosyltransferase gene by using RNAi. However, enzymatic defucosylation of N-glycans in vitro remains challenging because the N-glycans are embedded between two Fc domains. The presence of the Fc domain limits the access of α-fucosidase to the fucose residues, resulting in steric hindrance and reducing the efficiency of enzymatic defucosylation.

[0005] Only a few α-L-fucosidases capable of removing core fucose from the innermost GlcNAc residue after removing the outer N-glycan of the Fc region have been disclosed, such as α-L-fucosidase from bovine kidney, α-L-fucosidase from human (FucA1), α-L-fucosidase from Bacteroides fragilis (BfFuc), and α-L-fucosidase C (AlfC) from Lactobacillus casei. The intrinsic functions and activities of the wild-type enzymes are limited and low, so there is a demand for the development of modified enzymes that efficiently cleave core fucose from N-glycans. Summary of the Invention [Means for solving the problem]

[0006] overview In one embodiment, the present disclosure provides mutant forms of α-L-fucosidase for improved enzymatic hydrolysis of fucose in vitro. The present disclosure relates to novel mutant forms of α-fucosidase (α-L-fucosidase) that exhibit enhanced α-(1,6) fucosidase activity. The present disclosure also relates to compositions comprising the novel mutant forms of α-fucosidase and methods of using the novel mutant forms of α-fucosidase to cleave α-(1,6)-linked fucose in glycoconjugates.

[0007] In one embodiment, the disclosure provides a variant α-L-fucosidase comprising a polypeptide having at least 95% sequence identity to SEQ ID NO:1 and having a substitution mutation at amino acid position 247 (K247).

[0008] In one embodiment, the disclosure provides a variant α-L-fucosidase comprising a polypeptide having at least 97% sequence identity to SEQ ID NO:1 and having a substitution mutation at amino acid position 247 (K247).

[0009] In one embodiment, the disclosure provides a variant α-L-fucosidase comprising a polypeptide having at least 99% sequence identity to SEQ ID NO:1 and having a substitution mutation at amino acid position 247 (K247).

[0010] In one embodiment, the disclosure provides a mutant α-L-fucosidase comprising a polypeptide having the sequence of SEQ ID NO:1 and having a substitution mutation at amino acid position 247 (K247).

[0011] In some embodiments, the substitution mutation is selected from the group consisting of K247A, K247C, K247D, K247E, K247F, K247G, K247I, K247L, K247M, K247N, K247P, K247Q, K247S, K247T, K247V, K247W, and K247Y.

[0012] In some embodiments, the mutant α-L-fucosidase has the sequence of any one of SEQ ID NOs: 2-18.

[0013] In some embodiments, the variant α-L-fucosidases of the present disclosure are useful for efficiently cleaving core fucose in native glycoproteins without denaturing or functionally degrading the glycoprotein.

[0014] In some embodiments, the fucosidases described herein are capable of hydrolyzing one or more of α-(1,2), α-(1,3), α-(1,4), and α-(1,6) linked fucose. The fucose may be present in N- and / or O-linked glycans in glycoconjugates.

[0015] In some embodiments, the α-fucosidase is a recombinant Bacteroides α-fucosidase.

[0016] In some embodiments, the α-fucosidase has an optimum pH of 6.5 to 7.5.

[0017] In some embodiments, the present disclosure provides compositions comprising the mutant α-L-fucosidases described above to facilitate Fc glycoengineering of antibodies, such as therapeutic antibodies, or Fc fusion proteins.

[0018] In some embodiments, the composition further comprises at least one glycosidase.

[0019] In some embodiments, the glycosidase can be an exoglycosidase.

[0020] In some embodiments, the glycosidase can be an endoglycosidase, including, but not limited to, endo-β-N-acetylglucosaminidase (NAG), EndoA, EndoF1, EndoF2, EndoF3, EndoH, EndoM, EndoS, EndoS2, and variants thereof.

[0021] In some embodiments, the compositions are useful and efficient for performing defucosylation of glycoconjugates in vitro. In particular, the compositions described herein are useful for performing core defucosylation of glycoproteins in vitro.

[0022] In some embodiments, the core defucosylation is core α-(1,6) defucosylation.

[0023] In some embodiments, the core defucosylation is core α-(1,3) defucosylation.

[0024] In some embodiments, defucosylation can be performed without denaturation or functional degradation of the glycoprotein.

[0025] In one embodiment, the disclosure provides a method for producing defucosylated glycoconjugates in vitro, comprising sequentially or simultaneously contacting a glycoconjugate containing one or more fucoses with an endoglycosidase and a mutant α-L-fucosidase as described above.

[0026] In some embodiments, the method comprises the following sequence of steps: (a) contacting a glycoconjugate with an endoglycosidase; and (b) contacting the glycoconjugate with a mutant α-L-fucosidase as described above.

[0027] In some embodiments, the method further comprises step (c) terminating the reaction.

[0028] In some embodiments, step (c) is carried out at about 65° C. for 15 to 25 minutes.

[0029] In some embodiments, step (a) and / or step (b) are carried out at about 37° C. for 0.5 to 2 hours.

[0030] In some embodiments, the method comprises the following sequence of steps: (a) contacting a glycoconjugate with an endoglycosidase, including but not limited to endo-β-N-acetylglucosaminidase (NAG), EndoA, EndoF1, EndoF2, EndoF3, EndoH, EndoM, EndoS, EndoS2, and variants thereof; (b) contacting the glycoconjugate with a mutant α-fucosidase of the invention as described above. The glycoconjugate comprises one or more fucose selected from α-(1,2), α-(1,3), α-(1,4), and α-(1,6) linked fucose. The fucose may be present in N- and / or O-linked glycans in the glycoconjugate.

[0031] In some embodiments, the method comprises contacting a composition comprising the above-mentioned mutant α-L-fucosidase with at least one glycosidase. Endoglycosidases include, but are not limited to, endo-β-N-acetylglucosaminidase (NAG), EndoA, EndoF1, EndoF2, EndoF3, EndoH, EndoM, EndoS, EndoS2, and variants thereof. The glycoconjugate comprises one or more fucose selected from α-(1,2), α-(1,3), α-(1,4), and α-(1,6) linked fucose. The fucose may be present in the N- and / or O-linked glycans in the glycoconjugate. In an embodiment of the present invention, for example, the following items are provided: (Item 1) A mutant α-L-fucosidase comprising a polypeptide having at least 95% sequence identity to SEQ ID NO:1 and having a substitution mutation at amino acid position 247 (K247). (Item 2) 2. The mutant α-L-fucosidase according to item 1, wherein the substitution mutation is selected from the group consisting of K247A, K247C, K247D, K247E, K247F, K247G, K247I, K247L, K247M, K247N, K247P, K247Q, K247S, K247T, K247V, K247W, and K247Y. (Item 3) 3. The mutant α-L-fucosidase according to item 1 or 2, having any one of the sequences set forth in SEQ ID NOs: 2 to 18. (Item 4) A composition comprising the mutant α-L-fucosidase according to any one of items 1 to 3 and at least one glycosidase. (Item 5) A method for producing defucosylated glycoconjugates in vitro, comprising the steps of sequentially or simultaneously contacting a glycoconjugate containing one or more fucoses with an endoglycosidase and a mutant α-L-fucosidase according to any one of items 1 to 3. (Item 6) (a) contacting said glycoconjugate with said endoglycosidase; and (b) contacting the glycoconjugate with a mutant α-L-fucosidase according to any one of items 1 to 3. 6. The method according to item 5, comprising the successive steps of: (Item 7) The method according to item 5 or 6, further comprising the step of (c) terminating the reaction. (Item 8) 8. The method according to claim 7, wherein the step (c) is carried out at about 65° C. for 15 to 25 minutes. (Item 9) 9. The method according to any one of items 6 to 8, wherein the step (a) and / or step (b) is carried out at about 37° C. for 0.5 to 2 hours. (Item 10) The method according to any one of items 6 to 9, wherein the endoglycosidase is endo-β-N-acetylglucosaminidase (NAG), EndoA, EndoF1, EndoF2, EndoF3, EndoH, EndoM, EndoS, EndoS2, and variants thereof. (Item 11) 11. The method of claim 10, wherein EndoS2 comprises a substitution mutation selected from the group consisting of T138D, T138E, T138F, T138H, T138K, T138L, T138M, T138N, T138Q, T138R, T138V, T138W, D182Q, D226Q, T227Q, and T228Q. (Item 12) 12. The method according to any one of items 5 to 11, wherein the fucose is selected from the group consisting of α-(1,2), α-(1,3), α-(1,4) and α-(1,6) linked fucose. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Detailed explanation For convenience, certain terms used in the context of this disclosure are collected here. Unless otherwise defined, 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 disclosure belongs.

[0033] The singular forms "a," "an," and "the" are used herein to include plural references unless the context clearly indicates otherwise.

[0034] The term "substantial identity" or "substantially identical" when referring to a polypeptide refers to a polypeptide sequence identity of at least 95%, more preferably at least 96%, 97%, 98%, or 99% of the amino acid residues to the entire sequence of the reference polypeptide sequence when the appropriate polypeptide insertion or deletion is optimally aligned with another amino acid residue, as measured by any well-known algorithm of sequence identity, such as BLAST, ALIGN, or Megalign (DNASTAR) software. A polypeptide sequence having substantial identity to a reference polypeptide sequence may exhibit the same function.

[0035] As used herein, ranges are expressed as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, an embodiment includes the range from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations by using the word "about," it is understood that the particular value forms another embodiment. Moreover, it is understood that each endpoint of a range is significant both in relation to the other endpoint and independently of the other endpoint. As used herein, the term "about" refers to ±20%, preferably ±10%, more preferably ±5%.

[0036] As used herein, the term "glycan" refers to a polysaccharide or oligosaccharide. Glycans can be homopolymers or heteropolymers of monosaccharide residues and can be linear or branched. Glycans can also be used to refer to the carbohydrate moiety of a glycoconjugate such as a glycoprotein, glycolipid, or proteoglycan.

[0037] As used herein, the term "glycosidase" refers to an enzyme that catalyzes the hydrolysis of a glycosidic linkage or bond.

[0038] As used herein, the term "glycoconjugate" refers to any molecule in which at least one sugar moiety is covalently attached to at least one other moiety, including, but not limited to, N-linked glycoproteins, O-linked glycoproteins, glycolipids, proteoglycans, and the like.

[0039] As used herein, the term "N-linked glycoprotein" refers to a glycoprotein having a glycan attached to the nitrogen of a typically asparagine or arginine side chain.

[0040] As used herein, the term "O-linked glycoprotein" refers to a glycoprotein in which a glycan is attached to a hydroxy oxygen, usually of a serine, threonine, tyrosine, hydroxylysine, or hydroxyproline side chain, or to an oxygen on a lipid, such as ceramide.

[0041] A first aspect of the present invention relates to a polypeptide fragment comprising a variant form of α-L-fucosidase from Lactobacillus casei having substantial sequence identity to polypeptide SEQ ID NO:1 and having a substitution mutation at position 247.

[0042] SEQ ID NO:1 refers to the sequence of an α-fucosidase from Lactobacillus casei, with GeneBank accession number CAQ67984.1. [ka]

[0043] In some embodiments, the substitution mutation is selected from the group consisting of K247A, K247C, K247D, K247E, K247F, K247G, K247I, K247L, K247M, K247N, K247P, K247Q, K247S, K247T, K247V, K247W, and K247Y, preferably K247D and K247E.

[0044] In some embodiments, the substitution mutation is K247D and the mutant α-L fucosidase has [ka] The sequence is:

[0045] In some embodiments, the substitution mutation is K247E and the mutant α-L fucosidase has [ka] The sequence is:

[0046] In some embodiments, the substitution mutation is K247F and the mutant α-L fucosidase has [ka] The sequence is:

[0047] In some embodiments, the substitution mutation is K247A and the mutant α-L fucosidase has [ka] The sequence is:

[0048] In some embodiments, the substitution mutation is K247C and the mutant α-L fucosidase has [ka] The sequence is:

[0049] In some embodiments, the substitution mutation is K247G and the mutant α-L fucosidase has [ka] The sequence is:

[0050] In some embodiments, the substitution mutation is K247I and the mutant α-L fucosidase has [ka] The sequence is:

[0051] In some embodiments, the substitution mutation is K247L and the mutant α-L fucosidase has [ka] The sequence is:

[0052] In some embodiments, the substitution mutation is K247M and the mutant α-L fucosidase has [ka] The sequence is:

[0053] In some embodiments, the substitution mutation is K247N and the mutant α-L fucosidase has [ka] The sequence is:

[0054] In some embodiments, the substitution mutation is K247P and the mutant α-L fucosidase has [ka] The sequence is:

[0055] In some embodiments, the substitution mutation is K247Q and the mutant α-L fucosidase has [ka] The sequence is:

[0056] In some embodiments, the substitution mutation is K247S and the mutant α-L fucosidase has [ka] The sequence is:

[0057] In some embodiments, the substitution mutation is K247T and the mutant α-L fucosidase has [ka] The sequence is:

[0058] In some embodiments, the substitution mutation is K247V and the mutant α-L fucosidase has [ka] The sequence is:

[0059] In some embodiments, the substitution mutation is K247W and the mutant alpha-L fucosidase has the sequence [ka]

[0060] In some embodiments, the substitution mutation is K247Y and the mutant α-L fucosidase has [ka] The sequence is:

[0061] The variant α-fucosidase is capable of hydrolyzing one or more of α-(1,2), α-(1,3), α-(1,4), and α-(1,6)-linked fucose. The fucose may be present in N- and / or O-linked glycans in glycoconjugates. The fucose may be core α-(1,3) fucose or core α-(1,6) fucose.

[0062] A second aspect of the present invention relates to a composition comprising the above-described mutant α-fucosidase and at least one glycosidase.

[0063] In some embodiments, the glycosidase is an exoglycosidase.

[0064] In some embodiments, the glycosidase is an endoglycosidase, including, but not limited to, endo-β-N-acetylglucosaminidase (NAG), EndoA, EndoF1, EndoF2, EndoF3, EndoH, EndoM, EndoS, EndoS2, and variants thereof.

[0065] In some embodiments, the endoglycosidase is EndoS that includes a mutation at position D233.

[0066] In some embodiments, the endoglycosidase is EndoS2 that includes mutations at positions T138, D226, T227, and / or T228.

[0067] In some embodiments, the endoglycosidase is EndoS2 comprising a mutation selected from the group consisting of T138D, T138E, T138F, T138H, T138K, T138L, T138M, T138N, T138Q, T138R, T138V, T138W, D182Q, D226Q, T227Q, and T228Q.

[0068] In some embodiments, the compositions are useful and efficient for defucosylation of glycoconjugates in vitro. In particular, the compositions described herein are useful for core defucosylation of glycoproteins in vitro. Defucosylation can be performed without denaturation or loss of functionality of the glycoprotein.

[0069] In some embodiments, the compositions of the present invention relate to efficient hydrolysis of N-glycans on the Fc region of an antibody. As used herein, the term "N-glycan" refers to N-linked oligosaccharides bound by N-acetylglucosamine (GlcNAc) linked to the amide nitrogen of an asparagine residue of the Fc region of an antibody or fragment thereof.

[0070] In some embodiments, the N-glycans are high mannose, hybrid or complex, with or without core fucose.

[0071] A third aspect of the invention relates to a method for producing defucosylated glycoconjugates in vitro. The glycoconjugates can be treated sequentially or simultaneously with an endoglycosidase and a mutant α-fucosidase as described above.

[0072] In some embodiments, the method comprises the steps of: (a) contacting a glycoconjugate with an endoglycosidase, including but not limited to endo-β-N-acetylglucosaminidase (NAG), EndoA, EndoF1, EndoF2, EndoF3, EndoH, EndoM, EndoS, EndoS2, and variants thereof; (b) contacting the glycoconjugate with a mutant α-fucosidase of the invention as described above. The glycoconjugate comprises one or more fucose selected from α-(1,2), α-(1,3), α-(1,4), and α-(1,6) linked fucose. The fucose may be present in N- and / or O-linked glycans in the glycoconjugate.

[0073] In some embodiments, the method comprises contacting at least one glycosidase with a composition comprising the mutant α-L-fucosidase described above. Endoglycosidases include, but are not limited to, Endo-β-N-acetylglucosaminidase (NAG), EndoA, EndoF1, EndoF2, EndoF3, EndoH, EndoM, EndoS, EndoS2, and variants thereof. The glycoconjugate comprises one or more fucose selected from α-(1,2), α-(1,3), α-(1,4), and α-(1,6) linked fucose. The fucose may be present in the N- and / or O-linked glycans in the glycoconjugate.

[0074] In some embodiments, the endoglycosidase is Streptococcus pyogenes endoglycosidase S2, Streptococcus pyogenes endoglycosidase S having a mutation at amino acid position D233, Streptococcus pyogenes endoglycosidase S2 having a mutation at amino acid position T138, Streptococcus pyogenes endoglycosidase S2 having a mutation at amino acid position D182, Streptococcus pyogenes endoglycosidase S2 having a mutation at amino acid position D184, Streptococcus pyogenes endoglycosidase S2 having a mutation at amino acid position D186, Streptococcus pyogenes endoglycosidase S2 having a mutation at amino acid position D226, or Streptococcus pyogenes endoglycosidase S2 having a mutation at amino acid position T227.

[0075] In some embodiments, the endoglycosidase is EndoS that includes a mutation at position D233.

[0076] In some embodiments, the endoglycosidase is EndoS2 that includes mutations at positions T138, D226, T227, and / or T228.

[0077] In some embodiments, the endoglycosidase is EndoS2 comprising a mutation selected from the group consisting of T138D, T138E, T138F, T138H, T138K, T138L, T138M, T138N, T138Q, T138R, T138V, T138W, D182Q, D226Q, T227Q, and T228Q.

[0078] In some embodiments, the duration of the reaction mixture is set to at least 20, 30, 40, 50, 60, 70, 80, 90 or 100 minutes, preferably less than 60 minutes. The reaction temperature is preferably performed at room temperature, more preferably at about 20° C., 25° C., 30° C., 35° C., 40° C. or 45° C., most preferably at about 37° C. EXAMPLES

[0079] Example 1. Protein Expression Constructs

[0080] Wild-type α-fucosidase C (AlfC) was amplified by polymerase chain reaction (PCR) from Lacticaseibacillus casei BL23 genomic DNA (ATCC 393) and cloned into pET47N with an N-terminal His-tag fusion. Substitutions of the wild-type lysine at position 247 with each of the other 19 natural amino acids were generated by saturation mutagenesis using the following forward primers and the plasmid-encoded wild-type AlfC as template: [Table 1]

[0081] All substitutions were made using the following as the reverse primer: 5'-CGG GAT CTC GTT ATC GCC CAG-3' (SEQ ID NO: 38). The PCR program used for saturation mutagenesis was as follows: 95°C for 3 min, 16 cycles of 95°C for 45 s, 58°C for 50 s, 68°C for 8 min, with a final step of 72°C for 10 min. Mutations were confirmed by DNA sequencing. Other enzymes used in the study, such as EndoS or EndoS2, were cloned into pET28a with N-terminal His-tag fusions.

[0082] Example 2. Protein expression and purification Protein expression constructs were transformed into BL21(DE3) (EMD Biosciences, San Diego, CA). Proteins were overexpressed for 20 h at 16°C by 0.1 mM isopropyl β-D-thiogalactopyranoside (IPTG) induction. Cells were disrupted with a high-pressure homogenizer, and the supernatant was collected after centrifugation. The collected supernatant was loaded onto a Ni-NTA agarose column and washed with 10 column volumes of washing buffer (Tris-HCl, pH 7.4, 200 mM sodium chloride, and 40 mM imidazole). Elution was performed with 2 column volumes of elution buffer (Tris-HCl, pH 7.4, 200 mM sodium chloride, and 300 mM imidazole). Protein purity was checked by SDS-PAGE and exchanged into reaction buffer (50 mM Tris-HCl, 50 mM mannitol, 50 mM sorbitol, pH 7.0) using Amicon® Ultra-0.5 Centrifugal Filter Devices (UFC5010BK, 10 kDa cutoff).

[0083] Example 3. Core Defucosylation of Glycoproteins The core defucosylation activity of the mutant fucosidase was performed by analyzing the core fucose percentage after enzyme treatment. Trastuzumab (Herceptin®) (24 mg) was treated with EndoS2 (15.2 μg) at 37° C. for 1 h, followed by reaction with AlfC fucosidase mutant (2.8 μg) at 37° C. for another 1 h. The reaction was suspended by heat inactivation at 65° C. for 20 min and washed three times with ddH2O in an Amicon® Ultra filter device. The supernatant was collected for mass spectrometry (Table 2). The percentage of core fucose on N-glycans in the blank group (without enzyme) was about 87%. The defucosylation activity of wild-type AlfC enzyme was about 19%. Mutation of the K247 residue to a negatively charged amino acid residue significantly improved the defucosylation activity to about 80%, while other mutants of K247, except for positively charged residues, also showed improved defucosylation activity. The defucosylation percentage was calculated as follows:

number

[0084] While the present disclosure has been described in conjunction with the specific embodiments above, many alternatives, modifications, and variations thereof will be apparent to those skilled in the art, and all such alternatives, modifications, and variations are deemed to be within the scope of the present disclosure.

[0085] References

[0086] US11193155B2

[0087] Klontz EH, Li C, Kihn K, Fields JK, Beckett D, Snyder GA, Wintrode PL, Deredge D, Wang LX, Sundberg EJ. Structure and dynamics of an α-fucosidase reveal a mechanism for highly efficient IgG transfucosylation. Nat Commun. 2020 Dec 4;11(1):6204

[0088] Li C, Zhu S, Ma C, Wang LX. Designer α1,6-Fucosidase Mutants Enable Direct Core Fucosylation of Intact N-Glycopeptides and N-Glycoproteins. J Am Chem Soc. 2017 Oct 25;139(42):15074-15087.

[0089] Osanjo G, Dion M, Drone J, Solleux C, Tran V, Rabiller C, Tellier C. Directed evolution of the alpha-L-fucosidase from Thermotoga maritima into an alpha-L-transfucosidase. Biochemistry. 2007 Jan 30;46(4):1022-33.

[0090] US10415021

Claims

1. A mutant α-L-fucosidase comprising a polypeptide having at least 95% sequence identity to SEQ ID NO:1 and having a substitution mutation at amino acid position 247 (K247).

2. 2. The mutant α-L-fucosidase of claim 1, wherein the substitution mutation is selected from the group consisting of K247A, K247C, K247D, K247E, K247F, K247G, K247I, K247L, K247M, K247N, K247P, K247Q, K247S, K247T, K247V, K247W, and K247Y.

3. The mutant α-L-fucosidase according to claim 1 or 2, having any one of the sequences set forth in SEQ ID NOs: 2 to 18.

4. A composition comprising the mutant α-L-fucosidase of any one of claims 1 to 3 and at least one glycosidase.

5. 10. A method for producing defucosylated glycoconjugates in vitro, comprising sequentially or simultaneously contacting a glycoconjugate containing one or more fucoses with an endoglycosidase and a mutant α-L-fucosidase according to any one of claims 1 to 3.

6. (a) contacting said glycoconjugate with said endoglycosidase; and (b) contacting said glycoconjugate with the mutant α-L-fucosidase of any one of claims 1 to 3. The method of claim 5, comprising the successive steps of:

7. 7. The method of claim 5 or 6, further comprising the step of: (c) terminating the reaction.

8. 8. The method of claim 7, wherein step (c) is carried out at about 65° C. for 15 to 25 minutes.

9. The method of any one of claims 6 to 8, wherein step (a) and / or step (b) is carried out at about 37°C for 0.5 to 2 hours.

10. The method of any one of claims 6 to 9, wherein the endoglycosidase is Endo-β-N-acetylglucosaminidase (NAG), EndoA, EndoF1, EndoF2, EndoF3, EndoH, EndoM, EndoS, EndoS2, and variants thereof.

11. 11. The method of claim 10, wherein EndoS2 comprises a substitution mutation selected from the group consisting of T138D, T138E, T138F, T138H, T138K, T138L, T138M, T138N, T138Q, T138R, T138V, T138W, D182Q, D226Q, T227Q, and T228Q.

12. 12. The method of any one of claims 5 to 11, wherein the fucose is selected from the group consisting of α-(1,2), α-(1,3), α-(1,4) and α-(1,6) linked fucose.

Images