Quantitative analysis of immunoglobulin subclasses and subclass specific glycosylation by LC–MS–MRM in liver disease

Highlights

• We quantified plasma immunoglobulins and site specific glycoforms of IgG 1-4 subclasses in liver disease progression to HCC.

• Increased galactose-deficient core fucosylated glycoforms were consistently observed in CIR and HCC patients.

• Increased degree of fucosylation was detected in the IgG1 and IgG3 glycoforms.

• Both quantities and glycoforms of immunoglobulins change significantly in liver disease progression to HCC.

Abstract

Aberrant glycosylation of IgGs has been linked to human diseases, including liver disease. In this study, we have quantified plasma immunoglobulins in cirrhosis (CIR) and hepatocellular carcinoma (HCC) and employed a novel LC–MS–MRM assay to quantify glycoforms of IgG subclasses 1–4. Glycan oxonium ions and peptide-GlcNAc fragment ions were utilized to quantify the IgG glycoforms purified by affinity chromatography with normalization to the unique peptide for each IgG subclass. Our results indicate that HCC patients have increased circulating IgG1, IgG3, IgA1, and IgM compared to healthy controls; comparison of HCC and CIR patients shows that HCC patients have significantly higher concentration of IgG1 and IgM but lower concentration of IgG2. An increase in galactose-deficient core fucosylated glycoforms was consistently observed in CIR and HCC patients. The FA2G0 and FA2BG0 glycoforms increase approximately 2-fold in all IgG subclasses accompanied by a decrease in the FA2G2 glycoform. Fucosylation changes are less pronounced but we have detected increased degree of fucosylation in the IgG1 and IgG3 glycoforms. In conclusion, we have optimized a sensitive and selective LC–MS–MRM method for the quantification of immunoglobulin subclasses and their site specific glycoforms, demonstrating that both quantities and glycoforms of immunoglobulins change significantly in liver disease progression to HCC.

Biological significance

We have demonstrated that both quantities and glycoforms of immunoglobulin subclasses change significantly in liver disease progression to HCC through quantitative study of immunoglobulin subclasses and their site specific glycoforms using a sensitive and selective LC–MS–MRM method. Redistribution of the glycoforms of specific immunoglobulin subclasses could have important implications for receptor mediated responses affecting the progression of liver disease.

Introduction

In the United States, hepatitis C viral (HCV) infection is the leading cause of chronic liver disease including cirrhosis and hepatocellular carcinoma (HCC), the most serious complication of the viral infection [1]. HCC is the third leading cause of cancer death in the world and a cancer with continuously increasing incidence in the United States [2], [3]. Approximately 80% of HCC is associated with chronic viral infections world-wide [4] and, in the US, 50–60% of HCC patient are HCV infected [1].

Stimulation of immune response by HCV antigens leads to an increase in specific subclasses of immunoglobulins dominated by the IgG1 and IgG3 subclasses [5]. The disease-associated shift in immunoglobulin distribution has been well documented [6], [7], [8]. Broadly neutralizing antibodies targeting the E1/E2 glycoprotein have been isolated but are not common due to the high variability and extensive glycosylation of the viral envelope [9], [10], [11]. Immune response is typically considered part of the pathogenesis of liver damage in chronic HCV infection but the mechanism remains undefined [12]. Nonetheless, antibody dependent cellular cytotoxicity (ADCC) was associated with antibodies to E2 envelope glycoprotein at all stages of HCV infection [13]. In addition to the HCV directed antibodies, liver disease leads to general increase in antibody titers in association with leakage of intestinal antigens [14], [15], [16]. A significant increase in serum IgA and IgG was reported at the stage of hepatic fibrosis [6], [17]. And a progressive increase of circulating serpin squamous cell carcinoma antigen-IgM complexes has been found to be associated with liver tumor development [8].

In addition to quantitative changes of specific immunoglobulin subclasses, N-glycosylation of immunoglobulins provides critical regulation of functional responses mediated by Ig-receptors and other interacting partners [18]. Glycosylation is a frequent and heterogeneous translational modification which regulates many biological processes including protein folding, stability, and host–pathogen interactions [19], [20], [21]. Each immunoglobulin has conserved glycosylation sites on their heavy chain (HC) while the glycosylation of the light chains is variable. We and others have shown that glycosylation of immunoglobulins changes in liver disease [19], [22], [23], [24], [25]. Immunoglobulins A and G have been found to be the major glycoproteins contributing to the observed changes in composition of total serum N-glycome in cirrhotic patients [22]. GlycoFibroTest stages fibrosis based on the log ratio of a-galactosylated biantennary glycan derived from immunoglobulins to triantennary complex glycan derived from liver secreted proteins [17]. And decreased galactosylation of anti-Gal IgG was associated with the progression of fibrosis to cirrhosis of hepatitis C viral etiology [15]. In all the above studies, glycosylation was monitored at the level of total IgG, primarily by analysis of the enzymatically detached glycans; the distribution of the glycosylation changes between subclasses of IgG in liver disease remains unknown. Because of the association of immunoglobulins with liver disease progression and because of the importance of glycosylation in regulation of IgG responses, we decided to quantify changes in the site specific glycoforms of IgG1–4 in liver disease. For this purpose, we have optimized LC–MS–MRM assays for simultaneous quantification of immunoglobulins and site specific glycoforms of IgG1–4 subclasses and report application of these assays to a pilot examination of liver disease progression from CIR to HCC.