Designer α 6-fucosidase mutants enable direct core fucosylation of intact N-glycopeptides and N-glycoproteins
AssigneeUniversity of Maryland, College Park
InventorsLai-Xi Wang, Chao Li, Shilei Zhu
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Abstract
The present invention provides for novel fucosidase mutants that server as fuco-ligases for core fucosylation of a range of biological glycopeptides and glycoproteins including intact therapeutic antibodies. Several mutants with mutation at the general acid/base residue E274 of the Lactobacillus casei α1,6-fucosidase, including E274A, E274S, and E274G, were able to efficiently fucosylate a wide variety of complex N-glycopeptides and intact glycoproteins. The site specific mutants enable the transfer of fucose to a core GlcNAc-Asn residue and useful for drug delivery and vaccine development.
Claims11
A mutated α1-6-fucosidase enzyme selected from the group consisting of E274A (SEQ ID NO:
- 1), E274S (SEQ ID NO:
- 2), and E274G (SEQ ID NO:
- 3).
A kit comprising a least one Lactobacillus casei α-fucosidase mutant selected from the group consisting of E274A (SEQ ID NO:
- 1), E274S (SEQ ID NO:
- 2), and E274G (SEQ ID NO:
- 3).
A catalytic method for fucosylating a N-glycopeptide or N-glycoprotein, the method comprising:
- providing a GlcNAc acceptor substrate comprising a GlcNAc containing peptide or protein, or an oligosaccharide containing a terminal GlcNAc moiety;
- providing an activated glycosyl donor for access to a sugar fucose;
- and
- providing an α1,6-fucosidase mutant enzyme for enzymatically transfucosylating the GlcNAc acceptor substrate with the sugar fucose,
- wherein the α1,6-fucosidase mutant enzyme is a Lactobacillus casei α1,6-fucosidase mutant, thereby synthesizing a fucosylated glycopeptide or glycoprotein, wherein α1,6-fucosidase mutant enzyme is selected from a group consisting of E274A (SEQ ID NO:
- 1), E274S (SEQ ID NO:
- 2), and E274G (SEQ ID NO:3) derived from Lactobacillus casei α-fucosidase (SEQ ID NO:
- 4).
A chemoenzymatic method for the preparation of homogeneous fucosylated glycopeptides or glycoproteins, comprising:
- providing a nonfucosylated glycopeptide or glycoprotein acceptor;
- and
- reacting the nonfucosylated glycopeptide or glycoprotein acceptor with a donor substrate including an activated fucose containing moiety, in the presence of a Lactobacillus casei al-6-fucosidase E274 mutant protein or fragment thereof comprising a catalytic domain having a E274 mutation and exhibits increased transfucosylation and reduced hydrolytic activity relative to the wild type α1-6-fucosidase E274 enzyme (SEQ ID NO:
- 4) to transfer the activated fucose containing moiety to the nonfucosylated glycopeptide or glycoprotein acceptor and yielding the homogeneous fucosylated glycopeptides or glycoproteins, wherein α1,6-fucosidase mutant enzyme is selected from a group consisting of E274A (SEQ ID NO:
- 1), E274S (SEQ ID NO:
- 2), and E274G (SEQ ID NO:3).
Dependencies are inferred from the claim text.