A novel glycosidase plate-based assay for the quantification of galactosylation and sialylation on human IgG

Osmond D Rebello,Paulina A Urbanowicz,David Falck,Richard A Gardner,Daniel I R Spencer,Lucy I Crouch,David N Bolam

doi:10.1007/s10719-020-09953-9

Abstract

Changes in human IgG galactosylation and sialylation have been associated with several inflammatory diseases which are a major burden on the health care system. A large body of work on well-established glycomic and glycopeptidomic assays has repeatedly demonstrated inflammation-induced changes in IgG glycosylation. However, these assays are usually based on specialized analytical instrumentation which could be considered a technical barrier for uptake by some laboratories. Hence there is a growing demand for simple biochemical assays for analyzing these glycosylation changes. We have addressed this need by introducing a novel glycosidase plate-based assay for the absolute quantification of galactosylation and sialylation on IgG. IgG glycoproteins are treated with specific exoglycosidases to release the galactose and/or sialic acid residues. The released galactose monosaccharides are subsequently used in an enzymatic redox reaction that produces a fluorescence signal that is quantitative for the amount of galactosylation and, in-turn, sialylation on IgG. The glycosidase plate-based assay has the potential to be a simple, initial screening assay or an alternative assay to the usage of high-end analytical platforms such as HILIC-FLD-MSn when considering the analysis of galactosylation and sialylation on IgG. We have demonstrated this by comparing our assay to an industrial established HILIC-FLD-MSn glycomic analysis of 15 patient samples and obtained a Pearson’s r correlation coefficient of 0.8208 between the two methods.

Highlights

Changes in IgG glycosylation have been associated with certain inflammatory diseases such as rheumatoid arthritis [1, 2], systemic lupus erythematosus [3], type 2 diabetes [4] and Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Center, Leiden, Netherlands 3 Biosciences Institute, Faculty of Medical Sciences, NewcastleUniversity, Newcastle upon Tyne, UK 4 Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK inflammatory bowel disease (IBD) [5]
Building on the need to achieve a simple and adoptable assay for glycan epitope quantification, we have developed a novel glycosidase plate-based assay for the quantification of galactosylation and sialylation on IgG glycoproteins
This fluorescence signal is used for calculating the molar amounts of terminal galactosylation and total galactosylation, and their difference is equivalent to the sialylation on the N-glycans (Fig. 1b)

Summary

Introduction

A loss of systemic and/or cellular homeostasis may trigger a change in the glycosylation of IgG molecules [6, 7]. These changes have been shown to alter the IgG glycoprotein interactions to FcγR receptors among other proteins, and tuning the immunoregulatory responses [8,9,10]. Increased agalactosylation of these glycans counteracts this immune suppressive ability by directly reducing the abundance of sialylated epitopes These agalactosylated glycans further induce a pro-inflammatory response via activation of the complement system through interaction with the mannose binding proteins [16]. We focus here on the quantification of galactosylation and sialylation on IgG as they are indicative of the proinflammatory and anti-inflammatory states of the patient, respectively

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Conclusion