The pathogenomonic feature in multiple myeloma (MM) is the abnormal monoclonal immunoglobulin, of which immunoglobulin G (IgG) paraprotein is the most common. The paraproteinemic state is also associated with neuropathy due to immunoglobulins targeting peripheral nerve antigen such as GM1 ganglioside, belonging to the glycosphingolipid families. All subclasses of immunoglobulins are post-translationally modified by the addition of N-glycans, influencing structure, stability, and biological function.
Objectives
1. To investigate and characterise the N-glycosylation profiles in polyclonal IgG and paraprotein across the myeloma disease spectrum.
2. To explore the involvement of N-glycans on the dynamic binding relationship between myeloma paraprotein and FcγRIIb receptor and its effect on plasma cell apoptosis.
3. To explore the use of neutral to sialylated oligosaccharide ratio (N:S) in monitoring for disease progression and overall survival.
Methods
Polyclonal IgG is extracted and quantified from sera of patients across the plasma cell
disorder spectrum and age-matched control, using Protein G affinity chromatography.
Cation exchange chromatography is employed to purify out the monoclonal
immunoglobulin (paraprotein). N-glycans are enzymatically liberated from both
polyclonal and monoclonal IgG, fluorescently labelled, and profiled using hydrophilic
interaction ultra-performance liquid chromatography. Exoglycosidase digestions and
mass spectrometry are used to elucidate the glycan structures and positional specificity.
Localisation analyses are performed to determine the distribution of N-glycans in the Fc
and Fab regions using a combination of enzymatic digestions and chemical reduction
followed by SDS-PAGE separation of the resulting protein fragments and subsequent ingel
digestion of the N-glycans. Non-supervised principal component analysis (PCA) is
employed to detect distinguishable chromatographic features among the studied groups.
Membrane lipid array is used to identify lipid antigens that interact with IgG paraprotein.
In addition, IgG paraprotein-FcγR binding interactions are explored using surface
plasmon resonance (SPR)-based biosensor system (BIAcore), and apoptosis assay.
