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Abstract: PO1800

Comparing the Lectin and Mass Spectrometry-Based Approaches to Quantify Galactose-Deficient IgA1 in IgA Nephropathy

Session Information

Category: Glomerular Diseases

  • 1202 Glomerular Diseases: Immunology and Inflammation

Authors

  • Lardinois, Olivier, NIEHS, Mass Spectrometry Research and Support Group, Research Triangle Park, North Carolina, United States
  • Nachman, Patrick H., University of Minnesota, Division of Renal Diseases and Hypertension, Minneapolis, Minnesota, United States
  • Williams, Jason G., NIEHS, Mass Spectrometry Research and Support Group, Research Triangle Park, North Carolina, United States
  • Deterding, Leesa, NIEHS, Mass Spectrometry Research and Support Group, Research Triangle Park, North Carolina, United States
Background

Abnormalities in O-glycosylation of circulating IgA1 are implicated in the pathogenesis of IgAN. This was initially demonstrated by the altered binding of lectins with specificity for O-linked glycans and confirmed later by mass spectrometry. Nevertheless, combining and interpreting the results from these two orthogonal techniques is difficult, due to their different levels of complexity. We applied the two approaches to quantify galactose-deficient IgA1 (Gd-IgA1) in plasma samples of IgAN patients and matched controls. We aim to identify potential sources of discrepancy between the two analytical methods.

Methods

IgAs were affinity purified from plasma samples from 23 patients with IgAN and 36 controls. We used enzyme-linked lectin assay and the lectin from Vicia villosa (VVL) to measure defective galactosylation of O-linked oligosaccharides. Monomeric (mIgA) and polymeric (pIgA) forms of IgA1 were size-separated by gel electrophoresis. IgA1-containing bands were in-gel digested with trypsin, the released glycopeptides were analyzed by electrospray ionization liquid mass spectrometry.

Results

A significantly larger fraction of IgA1 molecules in the circulation of IgAN patients exist as high molecular mass complexes, as compared with the control group (48.8 vs 42.8%, p=4.25E-02). The reactivity of VVL lectin with unfractionated IgA1 was higher in the IgAN group compared with healthy controls (10.9 vs 9.1 A.U., p=8.60E-02). In both groups, IgA1 binding to VVL was much stronger for pIgA than mIgA. Mass spectrometry showed that the level of Gal was higher in pIgA than in mIgA (3.66 vs 3.54 Gal/Heavy Chain, p=7.63E-05). However, no significant differences in glycan composition was detected between patients and controls. In all the experiments, the inter-individual differences in glycan composition were large, which may have obscured the signals from the disease-related galactose-deficient IgA1.

Conclusion

Our results suggest that the apparent increased abundance of Gd-IgA1 in circulation of patients with IgAN is, at least in part, attributable to a greater abundance of polymeric IgA1 compared with controls. However, the glycosylation profile of each form of IgA1 appeared indistinguishable in the IgAN group when compared to the corresponding form in the control group.