N-Glycan Analysis of Polyclonal IgG from Patients with Multiple Myeloma Enables Classification of Stage Specific Pathologies

Background

Multiple myeloma (MM) is an incurable plasma cell malignancy, with eventual disease refractory and relapse. Its benign precursor, monoclonal gammopathy of undetermined significance (MGUS), has an annual transformation rate of 1%, while that of the asymptomatic smouldering myeloma (SMM) is 10%. The pathognomonic feature is the presence abnormal monoclonal immunoglobulin, of which immunoglobulin G (IgG) paraprotein is the most common. All subclasses of Igs are post-translationally modified by the addition of N-glycans, reportedly influencing structure, stability, and biological function. Previous studies in MM IgG had suggested an increase in the glycosylation of the antigen-binding fragment (Fab), and an overall elevation of sialylation. Using glycomic platforms, we aimed to investigate and characterise the IgG N-glycosylation profiles across the myeloma disease spectrum.

Methods

Polyclonal IgG was extracted from sera of patients with MM, SMM, MGUS, and age-matched control, using Protein G affinity chromatography. The quantity of extracted IgG was determined using the Bradford assay. N-glycans were enzymatically liberated from a normalised quantity of purified IgG, fluorescently labelled and profiled using hydrophilic interaction ultra-performance liquid chromatography. A combination of exoglycosidase digestions and mass spectrometry were used to elucidate the glycan structures with full linkage and positional specificity. Localisation analyses were performed to determine the distribution of N-glycans at asparagine 297 in the Fc region of the antibody and those present at any additional glycosylation sites present in the Fab region using a combination of enzymatic digestion using the commercially available IdeS enzyme and chemical reduction followed by SDS-PAGE separation of the resulting protein fragments and subsequent in-gel digestion of the N-glycans. Non-supervised principal component analysis (PCA) was employed to detect distinguishable chromatographic features among the studied groups. Longitudinal analyses of samples from individual patients collected during multiple clinical assessments were also performed.

Results

N-glycan analysis of polyclonal IgG showed unique N-glycan peaks with statistically significant chromatogram variation across the 4 studied groups. PCA identified specific patterns of glycosylation present in the glycan profiles, thus demonstrating the ability to distinguish between MGUS, SMM, MM and control. Sialylated biantennary N-glycans and N-glycans containing bisecting GlcNAc residues contributed most to the PCA separation. Further characterisation of the glycans using sialic acid linkage specific derivatisation and LC-MS analysis confirmed that sialic acids were present in an α2-6 linked configuration.

Localisation analysis revealed N-glycans present in the Fc region of the extracted polyclonal antibodies consisted of the standard biantennary type glycans with core fucosylation, variable degrees of galactosylation and low levels of sialylation. Such oligosaccharide structures suggest that the Fc regions of polyclonal IgG present in patients with varying stages of plasma cell disorder maintain a correct orientation to facilitate interaction with Fcγ receptors. Sialylated biantennary N-glycans, identified during global polyclonal IgG glycosylation profiling, were found to be located predominantly in the Fab region of the antibody. The formation of these larger highly sialylated N-glycan structures is likely due to the removal of steric hindrance resulting in more facilitated access by the associated glycosyltransferases required for oligosaccharide biosynthesis. The presence of these charged oligosaccharide structures, with their inherent structural dynamics, are likely to affect the ability of the antibody to recognise antigen and form an immune complex.

Conclusions

Glycan analysis of polyclonal IgG extracted from the sera of patients with varying stages of myeloma progression is reported. Differential glycosylation on polyclonal IgG was observed between the patients with MGUS, SMM, and MM, with alterations in the levels of sialylated biantennary structures and glycans bearing bisecting GlcNAc residues capable of distinguishing between the patient groups in the spectrum of plasma cell disorders.