Unusual Transglycosylation Activity of Flavobacterium meningosepticum Endoglycosidases Enables Convergent Chemoenzymatic Synthesis of Core Fucosylated Complex N‐Glycopeptides

Abstract

Structurally well-defined, homogeneous glycopeptides and glycoproteins are indispensable tools for functional glycomics studies. By screening of various endo-β-N-acetylglucosaminidases using an appropriate synthetic donor and acceptor substrates, we have found that the Flavobacterium meningosepticum endo-β-N-acetylglucosaminidases glycosidases (GH family 18), including Endo-F2 and Endo-F3, were able to glycosylate α-1,6-fucosylated GlcNAc derivative to provide natural, core-fucosylated complex type N-glycopeptides. The Endo-F2 and Endo-F3 were efficient for transferring both sialylated and asialylated glycans and were highly specific for an α-1,6-fucosyllated GlcNAc-peptide as acceptor for transglycosylation, as they showed only marginal activity on non-fucosylated GlcNAc-peptide. In contrast, we found that the commonly used endoglycosidases such as Endo-A and Endo-M, which belong to GH family 85, were unable to take α-1,6-fucosyl-GlcNAc derivative as acceptor for transglycosylation. The novel activity of Endo-F2 and Endo-F3 was successfully applied for a highly convergent chemoenzymatic synthesis of a full-size CD52 glycopeptide antigen carrying both terminal sialic acid and core fucose. This is the first report on endoglycosidases that are able to glycosylate α-1,6-fucosylated GlcNAc derivatives to form natural core-fucosylated glycopeptides.

Endo-F3 Glycosynthase Mutants Enable Chemoenzymatic Synthesis of Core-fucosylated Triantennary Complex Type Glycopeptides and Glycoproteins

“…We found that Endo-F3 D165A and D165Q both showed apparent transglycosylation activity with the core-fucosylated GlcNAc-Asn acceptor (2) to give the transglycosylation product (4) (Fig. 3B), which was identical to the authentic compound characterized previously by ESI-MS (56). Interestingly, no transglycosylation was detected when the non-fucosylated GlcNAc-Asn (3) was used as the substrate (Fig.…”

“…Materials-Fmoc-Asn(Fuc␣1,6GlcNAc)-OH and core-fucosylated CD52 antigen were synthesized following our previously reported procedures (56). Monoclonal antibody rituximab (rituxan, Genetech Inc., South San Francisco, CA) was purchased through Premium Health Services Inc. (Columbia, MD).…”

“…We have previously discovered a transglycosylation activity of wild type Endo-F3 using SCTox (1) as the donor substrate and Fmoc-Asn(Fuc␣1,6GlcNAc)-OH (core-fucosylated) (2) or Fmoc-Asn(GlcNAc)-OH (3) as the acceptor substrates (56). In this study, the same reactions were applied to test the transglycosylation activity of the Endo-F3 mutants (Fig.…”

“…We have recently reported that several endoglycosidases from bacterium Elizabethkingia meningoseptica of the GH18 family, formerly named as Flavobacterium meningosepticum or Chryseobacterium meningosepticum (53)(54)(55), possess significant transglycosylation activity at neutral pH (partly due to the decreased hydrolytic activity at the less acidic pH) (56). Of these endoglycosidases, Endo-F3 has shown transglycosylation activity on core-fucosylated GlcNAc-peptides.…”

A common glycan structure on immunoglobulin G for enhancement of effector functions

“…Compared with the other endoglycosidases of the Glycoside Hydrolase Family 18 (GH18), like endo H, endo F1, F2, and F3, endoS showed the best specific hydrolytic activity for the asparagine-linked biantennary glycan of human IgG to give mono-GlcNAc antibody. In addition, some endoglycosidases of GH18 can also be used as glycosynthases with glycan oxazolines as substrates to form the chitobiose linkage, a strategy first demonstrated by Shoda and coworkers using chitinases (28,29) and further elaborated by Wang with endoglycosidase mutants developed using Wither's approach, including endoS D233Q and endoM N175Q (26,30,31).…”