title: Fragments of bacterial endoglycosidase s and immunoglobulin g reveal subdomains of each that contribute to deglycosylation.
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.
authors.affiliation: From the Oxford Glycobiology Institute, Department of Biochemistry and emma.dixon@msdtc.ox.ac.uk.
authors.forename: Emma V
authors.initials: EV
authors.lastname: Dixon
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Rd., Oxford OX1 3QU, United Kingdom, and.
authors.forename: Jolyon K
authors.initials: JK
authors.lastname: Claridge
authors.affiliation: From the Oxford Glycobiology Institute, Department of Biochemistry and School of Life Sciences, University of Warwick, Gibbet Hill Campus, Coventry, CV4 7AL, United Kingdom.
authors.forename: David J
authors.initials: DJ
authors.lastname: Harvey
authors.affiliation: From the Oxford Glycobiology Institute, Department of Biochemistry and.
authors.forename: Kavitha
authors.initials: K
authors.lastname: Baruah
authors.affiliation: From the Oxford Glycobiology Institute, Department of Biochemistry and.
authors.forename: Xiaojie
authors.initials: X
authors.lastname: Yu
authors.affiliation: From the Oxford Glycobiology Institute, Department of Biochemistry and.
authors.forename: Snezana
authors.initials: S
authors.lastname: Vesiljevic
authors.affiliation: From the Oxford Glycobiology Institute, Department of Biochemistry and.
authors.forename: Susan
authors.initials: S
authors.lastname: Mattick
authors.affiliation: From the Oxford Glycobiology Institute, Department of Biochemistry and.
authors.forename: Laura K
authors.initials: LK
authors.lastname: Pritchard
authors.affiliation: From the Oxford Glycobiology Institute, Department of Biochemistry and.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Krishna
authors.affiliation: From the Oxford Glycobiology Institute, Department of Biochemistry and.
authors.forename: Christopher N
authors.initials: CN
authors.lastname: Scanlan
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Rd., Oxford OX1 3QU, United Kingdom, and.
authors.forename: Jason R
authors.initials: JR
authors.lastname: Schnell
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Rd., Oxford OX1 3QU, United Kingdom, and.
authors.forename: Matthew K
authors.initials: MK
authors.lastname: Higgins
authors.affiliation: From the Oxford Glycobiology Institute, Department of Biochemistry and.
authors.forename: Nicole
authors.initials: N
authors.lastname: Zitzmann
authors.affiliation: From the Oxford Glycobiology Institute, Department of Biochemistry and max.crispin@bioch.ox.ac.uk.
authors.forename: Max
authors.initials: M
authors.lastname: Crispin
mesh_terms: D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000378:metabolism*; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D011134:Polysaccharides / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D013297:Streptococcus pyogenes / Q000201:enzymology*
chemical_list: D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D011134:Polysaccharides; D006026:Glycoside Hydrolases
keywords: Antibody; Antibody engineering; Enzyme Mechanism; Glycoprotein; Glycosylation