Fragments of Bacterial Endoglycosidase S and Immunoglobulin G Reveal Subdomains of Each That Contribute to Deglycosylation

Abstract

Background: Endoglycosidase S (EndoS) is an immune evasion factor from Streptococcus pyogenes that impedes IgG effector functions by deglycosylation.Results: Analysis of fragments of enzyme and substrate identify components of each that contribute to catalysis.Conclusion: Regions outside of the catalytic domain of EndoS contribute to IgG deglycosylation, and catalysis does not require all antibody subunits.Significance: Engineering of EndoS specificity requires consideration of both catalytic and non-catalytic regions.

EndoSd: An IgG Glycan Hydrolyzing Enzyme in Streptococcus Dysgalactiae Subspecies Dysgalactiae

“…For comparison, glycan profiles of both human and bovine IgGs generated by PNGase F were used. The UHPLC analysis of released glycan from IgG confirmed that the cleavage site of EndoSd is identical to that of EndoS (Figure 6) [18]. EndoSd was found to hydrolyze all major glycoforms of both human and bovine IgG.…”

“…Analysis of truncated EndoS mutants further showed that the C-terminus is not required for activity. However, C-terminally truncated EndoS mutants exhibited a reduced enzymatic activity, especially toward glycoforms modified with a bisecting GlcNAc [18]. As EndoS, EndoS2 and EndoSd show somewhat different glycan substrate specificities and vary the most in the C-terminal domains; it is, therefore, tempting to speculate that the C-terminus with the carbohydrate binding module mediates glycoform specificity in EndoS-like proteins.…”

“…With one notable exception, in contrast to EndoS [16], EndoSd only released minute amount of glycans modified with a bisecting GlcNAc (Figure 6). Previous studies have shown that EndoS lacking its carbohydrate binding domain was considerably slower in releasing structures with bisecting GlcNAc as compared with other glycoforms, indicating that these structures might be more resistant to endoglycosidases [18]. As bovine IgG is almost completely devoid of glycans with a bisecting GlcNAc (Figure 6) [35], this limitation in substrate specificity is unlikely to hinder EndoSd efficacy in its natural host.…”

“…EndoS is a multidomain protein containing an N-terminal GH18 chitinase domain, a leucine-rich repeat domain, a hybrid Ig domain, a carbohydrate binding module and a three-helix bundle [10]. Systematic analysis of truncated human IgG and EndoS revealed that EndoS is active on isolated CH2 domains of IgG, and that C-terminally truncated EndoS is still enzymatically active against IgG glycans but at a slower rate [18].…”

Redirecting adenoviruses to tumour cells using therapeutic antibodies: Generation of a versatile human bispecific adaptor

“…EndoS was cloned and purified as previously described (Dixon et al, 2014) using a plasmid containing an N-terminally glutathione S-transferase (GST) tagged construct of the full-length codonoptimized EndoS (Goodfellow et al, 2012). For release of N-linked glycans by EndoS, reactions were performed in PBS at a molar ratio of 1:10 EndoS to substrate.…”