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J Biol Chem. 2014 May 16;289(20):13876-89. doi: 10.1074/jbc.M113.532812. Epub 2014 Mar 25.

Fragments of bacterial endoglycosidase s and immunoglobulin g reveal subdomains of each that contribute to deglycosylation.

Author information

1
From the Oxford Glycobiology Institute, Department of Biochemistry and emma.dixon@msdtc.ox.ac.uk.
2
Department of Biochemistry, University of Oxford, South Parks Rd., Oxford OX1 3QU, United Kingdom, and.
3
From the Oxford Glycobiology Institute, Department of Biochemistry and School of Life Sciences, University of Warwick, Gibbet Hill Campus, Coventry, CV4 7AL, United Kingdom.
4
From the Oxford Glycobiology Institute, Department of Biochemistry and.
5
From the Oxford Glycobiology Institute, Department of Biochemistry and max.crispin@bioch.ox.ac.uk.

Abstract

Endoglycosidase S (EndoS) is a glycoside-hydrolase secreted by the bacterium Streptococcus pyogenes. EndoS preferentially hydrolyzes the N-linked glycans from the Fc region of IgG during infection. This hydrolysis impedes Fc functionality and contributes to the immune evasion strategy of S. pyogenes. Here, we investigate the mechanism of human serum IgG deactivation by EndoS. We expressed fragments of IgG1 and demonstrated that EndoS was catalytically active against all of them including the isolated CH2 domain of the Fc domain. Similarly, we sought to investigate which domains within EndoS could contribute to activity. Bioinformatics analysis of the domain organization of EndoS confirmed the previous predictions of a chitinase domain and leucine-rich repeat but also revealed a putative carbohydrate binding module (CBM) followed by a C-terminal region. Using expressed fragments of EndoS, circular dichroism of the isolated CBM, and a CBM-C-terminal region fusion revealed folded domains dominated by β sheet and α helical structure, respectively. Nuclear magnetic resonance analysis of the CBM with monosaccharides was suggestive of carbohydrate binding functionality. Functional analysis of truncations of EndoS revealed that, whereas the C-terminal of EndoS is dispensable for activity, its deletion impedes the hydrolysis of IgG glycans.

KEYWORDS:

Antibody; Antibody engineering; Enzyme Mechanism; Glycoprotein; Glycosylation

PMID:
24668806
PMCID:
PMC4022860
DOI:
10.1074/jbc.M113.532812
[Indexed for MEDLINE]
Free PMC Article

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