2025 Theses Doctoral

Characterizing Protein Glycosylation with Single-Molecule FRET Imaging

Rubin, Joseph Ronald

IgA1 nephropathy (IGAN) is the most common form of primary glomerular nephritus1. The disease is triggered by damage to glomeruli from deposition of complexes formed between polyclonal IgA1 antibody proteins that are “galactose-deficient” (GD-IgA1) and antibodies directed to this GD-IgAI. Currently, galactose deficiencies are detected through mass spectrometry, liquid chromatography, and other ensemble methods. Ensemble measurements, however, only measure average glycan-IgA1 ratios, but cannot resolve heterogeneity of O-glycosylation between different IgA1 populations.

To resolve heterogeneity in O-glycosylated IgA1, we are creating an assay to detect GD-IgA1 using single molecule fluorescence measurements. Using single molecule fluorescence methods may allow us to detect small populations of glycosylated IgA1 or subtle differences between the glycans that are not detectable in ensemble measurements.This work utilizes Fluorescence Resonance Energy Transfer (FRET) to observe the binding of protein probes to surface adhered molecules and obtain their kinetic fingerprints. Specifically, we measured the kinetic rates of jacalin (a lectin binding to O-linked glycans) binding at individual IgA1 molecules on a surface. Adding galactose decreased binding, which demonstrated that the jacalin probe binds specifically to O-linked glycans on the hinge region of IgA1.

This result is a first step towards kinetically fingerprinting O-glycans on IgA1. Subsequently, we compared kinetic fingerprinting results for IgA1 from healthy and Myeloma plasma samples. This work using Myeloma plasma samples will serve as a proof of concept for resolving multiple IgA1 populations in IgA Nephropathy patient samples, to ultimately find biomarkers or cure the disease.