Transglycosylation activity of glycosynthase mutants of an endo-beta-N-acetylglucosaminidase (endo-D) from streptococcus pneumoniae
AssigneeUniversity of Maryland, Baltimore
InventorLai-Xi Wang
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Abstract
The present invention provides for recombinant Endo-D and selected mutants that exhibit reduced hydrolysis activity and increased transglycosylation activity for the synthesis of glycoproteins wherein a desired sugar chain is added to a core fucosylated or nonfucosylated GlcNAc-protein acceptor by transglycosylation. Such recombinant Endo-D and selected mutants are useful for efficient glycosylation remodeling of IgG1-Fc domain.
Claims11
A method of preparing a fucosylated or nonfucosylated glycoprotein having a predetermined oligosaccharide moiety, the method comprising:
- providing a fucosylated or nonfucosylated GlcNAc acceptor protein comprising an asparagine-linked N-acetylglucosamine (GlcNAc) residue, wherein the fucosylated acceptor protein is an alpha-1-6-fucosyl-GlcNAc-protein and the nonfucosylated acceptor protein is an alpha-1-6-GlcNAc-protein;
- and
- enzymatically reacting the fucosylated or nonfucosylated GlcNAc acceptor protein with an activated oligosaccharide donor in the presence of an Endoglycosidase-D full length or truncated mutant selected from the group consisting of SEQ ID NO:
- 4, SEQ ID NO:
- 5, SEQ ID NO:
- 6, SEQ ID NO:
- 7, SEQ ID NO:
- 8, and SEQ ID NO:
- 9, wherein the activated oligosaccharide donor comprises an oxazoline covalently linked to an oligosaccharide moiety, wherein the oligosaccharide moiety comprises a mannose type N-glycan having a predetermined number of mannose residues, wherein the enzymatically reacting covalently links the oligosaccharide moiety to the fucosylated or nonfucosylated GlcNAc acceptor protein, thereby providing the fucosylated or nonfucosylated glycoprotein having the predetermined oligosaccharide moiety.
A method of synthesizing a modified antibody or fragment thereof, the method comprising;
- providing an antibody or fragment thereof comprising a fucosylated or nonfucosylated N-acetylglucosamine (GlcNAc) moiety;
- wherein the fucosylated or nonfucosylated N-acetylglucosamine (GlcNAc) moiety is positioned on a Fc region of the antibody or fragment thereof;
- enzymatically transferring an oligosaccharide having a predetermined number of saccharides to the fucosylated or nonfucosylated N-acetylglucosamine (GlcNAc) moiety under the catalysis of an Endoglycosidase-D full length or truncated mutant selected from the group consisting of SEQ ID NO:
- 4, SEQ ID NO:
- 5, SEQ ID NO:
- 6, SEQ ID NO:
- 7, SEQ ID NO:
- 8 and SEQ ID NO:
- 9, to form the modified antibody or fragment thereof with the predetermined number of saccharides.
Dependencies are inferred from the claim text.