Endoglycosidase S enables a highly simplified clinical chemistry assay procedure for direct assessment of serum IgG undergalactosylation in chronic inflammatory disease

Abstract

Over the past 30 years, it has been firmly established that a wide spectrum of (autoimmune) diseases such as rheumatoid arthritis, Crohn’s and lupus, but also other pathologies like alcoholic and non-alcoholic steatohepatitis (ASH and NASH) are driven by chronic inflammation and are hallmarked by a reduced level of serum IgG galactosylation. IgG (under)galactosylation is a promising biomarker to assess disease severity, and monitor and adjust therapy. However, this biomarker has not been implemented in routine clinical chemistry due to a complex analytical procedure that necessitates IgG purification, which is difficult to perform and validate at high throughput.We addressed this issue by using endo-β-N-acetylglucosaminidase from Streptococcus pyogenes (endoS) to specifically release Fc N-glycans in whole serum. The entire assay can be completed in a few hours and only entails adding endoS and labeling the glycans with APTS. Glycans are then readily analyzed through capillary electrophoresis. We demonstrate in two independent patient cohorts that IgG undergalactosylation levels obtained with this assay correlate very well with scores calculated from PNGaseF-released glycans of purified antibodies. Our new assay allows to directly and specifically measure the degree of IgG galactosylation in serum through a fast and completely liquid phase protocol, without the requirement for antibody purification. This should help advancing this biomarker towards clinical implementation.

Protein Glycosylation as a Diagnostic and Prognostic Marker of Chronic Inflammatory Gastrointestinal and Liver Diseases

“…113 Liver disease is an attractive field for the study of the serum glycome because most N-glycans found in whole serum are attached to serum proteins produced in the liver 21 and to a lesser extent to Igs, 20,70,114 which are produced by B cells. Alterations in serum glycan structures have been reported in patients with liver diseases 20,22,90,92,114,115,116 (Figure 4). Some glycomic-based markers have potential for clinical use (Figures 3, 5, and 6).…”

Molecular Basis of Broad Spectrum N-Glycan Specificity and Processing of Therapeutic IgG Monoclonal Antibodies by Endoglycosidase S2

“…2,4 EndoS has been investigated in numerous applications: as a therapy for antibody-mediated autoimmune diseases, such as systemic lupus erythematosus, epidermolysis bullosa acquisita, and idiopathic thrombocytopenia purpua; 5−8 as an enhancer of monoclonal antibody therapy; 9 as a tool for the chemoenzymatic synthesis of antibodies with homogeneous glycosylation or drug conjugates; 10−12 and as a method to screen for chronic inflammatory disease states. 13 Another Streptococcus pyogenes antibody-disrupting enzyme, IdeS, cleaves antibodies in their hinge region, and has shown promise in clinical trials for reducing antibody-mediated organ transplant rejection, revealing yet another possible application for endoglycosidases. 14 It was recently discovered that Streptococcus pyogenes serogroup M49 secretes a unique endoglycosidase, EndoS2, which may offer several potential therapeutic and biotechnological advantages compared to EndoS.…”