Designer α1,6-Fucosidase Mutants Enable Direct Core Fucosylation of Intact N-Glycopeptides and N-Glycoproteins

Abstract

Core fucosylation of N-glycoproteins plays a crucial role in modulating the biological functions of glycoproteins. Yet, the synthesis of structurally well-defined, core-fucosylated glycoproteins remains a challenging task due to the complexity in multi-step chemical synthesis or the inability of the biosynthetic α1,6-fucosyltransferase (FUT8) to directly fucosylate full-size mature N-glycans in a chemoenzymatic approach. We report in this paper the design and generation of potential α1,6-fucosynthase and fucoligase for direct core-fucosylation of intact N-glycoproteins. We found that mutation at the nucleophilic residue (D200) did not provide a typical glycosynthase from this bacterial enzyme, but several mutants with mutation at the general acid/base residue E274 of the Lactobacillus casei α1,6-fucosidase, including E274A, E274S, and E274G, acted as efficient glycoligases that could fucosylate a wide variety of complex N-glycopeptides and intact glycoproteins by using α-fucosyl fluoride as a simple donor substrate. Studies on the substrate specificity revealed that the α1,6-fucosidase mutants could introduce an α1,6-fucose moiety specifically at the Asn-linked GlcNAc moiety not only to GlcNAc-peptide, but also to high-mannose and complex type N-glycans in the context of N-glycopeptides, N-glycoproteins, and intact antibodies. This discovery opens a new avenue to a wide variety of homogeneous, core-fucosylated N-glycopeptides and N-glycoproteins that are hitherto difficult to obtain for structural and functional studies.

A Single Route to Mammalian N‐Glycans Substituted with Core Fucose and Bisecting GlcNAc

“…Chemoenzymatic approaches using recombinant α1,6‐fucosyltransferases or endoglycosidases have simplified the introduction of core fucose only for appropriate substrates. Recently, a mutated fucosidase was developed capable of adding a core fucose onto glycopeptides and glycoproteins . Herein, we show that chemical α1,6‐fucosylation of complex N‐glycans can reliably be carried out with nearly exclusive α‐selectivity using suitably protected fucosyl donors.…”

A Single Route to Mammalian N‐Glycans Substituted with Core Fucose and Bisecting GlcNAc

“…[3] Furthermore core fucose and bisecting GlcNAc are frequently associated with cancer progression and serve as atumor marker. [10] Herein, we show that chemical a1,6-fucosylation of complex N-glycans can reliably be carried out with nearly exclusive a-selectivity using suitably protected fucosyl donors.T he new approach is compatible with the modular one-pot introduction of abisecting GlcNAc moiety and can furnish anomeric azides of all mammalian complex-type core-fucosylated N-glycans with an optional bisecting GlcNAc. [7] Chemoenzymatic approaches using recombinant a1,6-fucosyltransferases [8] or endoglycosidases [9] have simplified the introduction of core fucose only for appropriate substrates.R ecently,amutated fucosidase was developed capable of adding ac ore fucose onto glycopeptides and glycoproteins.…”

“…[7] Chemoenzymatic approaches using recombinant a1,6-fucosyltransferases [8] or endoglycosidases [9] have simplified the introduction of core fucose only for appropriate substrates.R ecently,amutated fucosidase was developed capable of adding ac ore fucose onto glycopeptides and glycoproteins. [10] Herein, we show that chemical a1,6-fucosylation of complex N-glycans can reliably be carried out with nearly exclusive a-selectivity using suitably protected fucosyl donors.T he new approach is compatible with the modular one-pot introduction of abisecting GlcNAc moiety and can furnish anomeric azides of all mammalian complex-type core-fucosylated N-glycans with an optional bisecting GlcNAc.…”

Employment of fucosidases for the synthesis of fucosylated oligosaccharides with biological potential

“…Other types of mutant enzymes are fucoligases, which are created by mutation of a general acid–base residue, thereby disabling hydrolysis by the enzyme. In this case, the enzyme requires a donor substrate with the same anomeric configuration for transglycosylation . Usually the donor substrate that is employed in reactions catalyzed by fucosynthases or fucoligases is Fuc‐F.…”

“…This compound has been employed as a substrate for the 1,2/3/6‐α‐ l ‐fucosynthases that were engineered from the α‐fucosidases from B. bifidum . An improvement in this strategy has been the use of β‐fucosyl azide as an alternative donor substrate that was successfully used with the α‐ l ‐fucosynthase from Sulfolobus solfataricus . Li et al.…”