Generation of efficient mutants of endoglycosidase from Streptococcus pyogenes and their application in a novel one-pot transglycosylation reaction for antibody modification

PloS One
Mitsuhiro IwamotoJun Hasegawa

Abstract

The fine structures of Fc N-glycan modulate the biological functions and physicochemical properties of antibodies. By remodeling N-glycan to obtain a homogeneous glycoform or chemically modified glycan, antibody characteristics can be controlled or modified. Such remodeling can be achieved by transglycosylation reactions using a mutant of endoglycosidase from Streptococcus pyogenes (Endo-S) and glycan oxazoline. In this study, we generated improved mutants of Endo-S by introducing additional mutations to the D233Q mutant. Notably, Endo-S D233Q/Q303L, D233Q/E350Q, and several other mutations resulted in transglycosylation efficiencies exceeding 90%, with a single-digit donor-to-substrate ratio of five, and D233Q/Y402F/D405A and several other mutations resulted in slightly reduced transglycosylation efficiencies accompanied by no detectable hydrolysis activity for 48 h. We further demonstrated that the combined use of mutants of Endo-S with Endo-M or Endo-CC, endoglycosidases from Mucor hiemalis and Coprinopsis cinerea, enables one-pot transglycosylation from sialoglycopeptide to antibodies. This novel reaction enables glycosylation remodeling of antibodies, without the chemical synthesis of oxazoline in advance or in situ.

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Citations

Apr 3, 2020·Organic & Biomolecular Chemistry·Mingqun WangXin-Shan Ye
Mar 28, 2019·Annual Review of Biochemistry·Lai-Xi WangTiezheng Li
Dec 9, 2020·Chemical Society Reviews·Stephen J WalshDavid R Spring
Apr 23, 2021·Chemical Record : an Official Publication of the Chemical Society of Japan ... [et Al.]·Shino Manabe, Yoshiki Yamaguchi
Aug 25, 2018·Chemical Reviews·Chao Li, Lai-Xi Wang
Feb 5, 2019·Journal of the American Chemical Society·Larissa Krasnova, Chi-Huey Wong

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Methods Mentioned

BETA
glycosylation
transglycosylation
Electrophoresis

Software Mentioned

Experion

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