DESIGNER alpha 6-FUCOSIDASE MUTANTS ENABLE DIRECT CORE FUCOSYLATION OF INTACT N-GLYCOPEPTIDES AND N-GLYCOPROTEINS
AssigneeUniversity of Maryland, Baltimore County
InventorsLAI-XI WANG, CHAO LI, SHILEI ZHU
Life of this patent
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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.
Claims19
A catalytic method for fucosylating a N-glycopeptide or N-glycoprotein, the method comprising:
- providing a GlcNAc containing peptide, protein, or other glycans as an GlcNAc acceptor (acceptor substrate);
- 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 with the sugar fucose, wherein the α1,6-fucosidase mutant enzyme is a Lactobacillus casei α-fucosidase mutant, thereby synthesizing a fucosylated glycopeptide or glycoprotein.
A chemoenzymatic method for the preparation of homogeneous fucosylated glycopeptides or glycoproteins, comprising:
- providing an 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 α1-6-fucosidase E274 mutant protein or fragment thereof that includes the catalytic domain and exhibits increased transfucosylation and reduced hydrolytic activity relative to the wild type α1-6-fucosidase E274 enzyme to transfer the activated fucose containing moiety to the nonfucosylated glycopeptide or glycoprotein acceptor and yielding the homogeneous fucosylated glycopeptides or glycoproteins.
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).
(canceled)
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).
(canceled)
Dependencies are inferred from the claim text.