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

Israel Silman,Mitsuhiro Iwamoto,Jun Hasegawa,Yukiko Sekiguchi,Kensuke Nakamura,Yoshirou Kawaguchi,Takeshi Honda

doi:10.1371/journal.pone.0193534

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.

Highlights

Antibodies are major components of humoral immunity that protect the host from infection by binding to pathogens
We describe improved mutants of endoglycosidase from Streptococcus pyogenes (Endo-S) that enable efficient transglycosylation using a reduced concentration of oxazoline; the mutants were obtained by site-directed mutagenesis based on structural information and methods adapted from studies of various ENGases [17,22,23,24,25,26]
The Endo-S D233Q/E350Q mutant in place of the D233Q mutant could improve the maximum efficiency to 97%, while wild-type Endo-S severely diminished the transglycosylation activity (Fig 4A)

Summary

Introduction

Antibodies are major components of humoral immunity that protect the host from infection by binding to pathogens. IgG, the most common antibody found in the circulation, contributes to host defense via several mechanisms, including complement-dependent cytotoxicity [1,2,3,4,5] and antibody-dependent cell-mediated cytotoxicity [6,7,8,9], and the extent of these contributions are significantly influenced by the glycoform of the conserved N-linked glycan in the Fc region. In an effort to improve the efficacy and safety of recombinant. The application of efficient Endo-S mutants in a novel one-pot transglycosylation for antibody modification these authors are articulated in the ‘author contributions’ section

Methods

Results

Discussion

Conclusion