Transglycosidase‐like activity of Mucor hiemalis endoglycosidase mutants enabling the synthesis of glycoconjugates using a natural glycan donor

Abstract

Glycan conversion of glycoprotein via the transglycosylation activity of endo-β-N-acetylglucosaminidase is a promising chemoenzymatic technology for the production of glycoproteins including bio-medicines with a homogeneous glycoform. Although Endo-M is a key enzyme in this process, its product undergoes rehydrolysis, which leads to a lower yield, and limits the practical application of this enzyme. We developed several Endo-M mutant enzymes including N175Q with glycosynthase-like activity and/or transglycosidase-like activity. We found that the Endo-M N175H mutant showed glycosynthase-like activity comparable to N175Q as well as transglycosidase-like activity superior to N175Q. Using a natural sialylglycopeptide as a donor substrate, N175H readily transferred the sialo-glycan onto an N-acetylglucosamine residue attached to bovine ribonuclease B (RNase B), yielding a nonnative sialoglycosylated RNase B. These results demonstrate that use of Endo-M N175H is an alternative glycoengineering technique, which provides a relatively high yield of transglycosylation product and avoids the laborious synthesis of a sugar oxazoline as a donor substrate.

Characterization of Antibody Products Obtained through Enzymatic and Nonenzymatic Glycosylation Reactions with a Glycan Oxazoline and Preparation of a Homogeneous Antibody–Drug Conjugate via Fc N-Glycan

“…Besides monosaccharyl transferases, endo-β- N -acetylglucosaminidase (ENGase) mutants are also useful for site-specific conjugation of oligosaccharides . Various ENGase mutants, such as Endo M N175Q, Endo CC N180H, EndoS D233Q, Endo S2 D184M, and Endo S2 D184Q, have been developed. Combinations of ENGase mutants with reduced hydrolytic activity and the transition-state mimic sugar oxazoline have been used for the transfer of oligosaccharides to functional groups in acceptor molecules. ,, However, a side reaction between oxazoline and the amino group was recently reported. ,, …”

Generation of a Mutant Mucor hiemalis Endoglycosidase That Acts on Core-fucosylated N-Glycans

“…Thus, regulation of glycoforms in glycoprotein-based therapeutics (biomedicines), including antibodies and cytokines, have drawn attention in the pharmaceutical industry. This enzyme has been explored extensively to expand its utility, by protein engineering using site-directed mutagenesis, resulting in the isolation of efficient glycosynthase-like mutant enzymes, such as N175Q (17,33,34). These mutant enzymes exhibit both effective synthetic rates using sugar oxazoline as the glycosyl donor substrate and lack of hydrolytic activity, resulting in minimum degradation of the reaction products (33,35,36).…”

Synthesis of Glycosides by Glycosynthases

“…The group also described the successful synthesis of sialo-complex-type glycoforms of the bioactive peptides PAMP12 and Substance P in yields of 95 and 98%, respectively. The glycosylation reaction was also shown for the protein RNaseB bearing a single GlcNAc moiety therefore allowing glycan modification of proteins/enzymes by this method [ 66 ]. Amin et al also described the production of monoglycoforms of RNaseB by the glycosynthase Endo-A N171A ( A. protophormiae ) in yields up to 80% with chemically synthesized oxazoline glycans [ 67 ].…”