Understanding The 3d Shape Of Dimeric IgA1 in IgA Nephropathy

Background: IgA nephropathy (IgAN) is characterised by mesangial deposition of J-chain containing dimeric IgA1 (dIgA1). Three-dimensional (3D) conformational protein structure has been shown to determine the antigenic epitopes in other autoimmune kidney diseases, but a role for changes in the 3D structure of dIgA1 and development of IgAN remains unknown. Given that deposited IgA1 in IgAN is almost exclusively J-chain containing dIgA1, the study of solution structures of dIgA1 in precisely phenotyped IgAN patients may provide further insights into the pathogenesis of the disease. Method: dIgA1 from three IgAN patients and a healthy control was isolated via jacalin affinity chromatography and fast protein liquid chromatography and detailed analysis of the 3D structure of dIgA1 using small angle X-ray (SAXS) & neutron scattering (SANS), and analytical ultracentrifugation (AUC) were performed. We then correlate 3D conformational changes in dIgA1 structure with serum IgA-IgG immune complex levels, activated human mesangial cells (hMC) IL-6 production and the pattern of IgA1 N- and O-glycosylation (determined via Helix aspersa (HA) lectin assay and mass spectrometry). Results: The degree of dIgA1 hinge region O-glycosylation, as measured by HA lectin binding, varied between the four subjects and was lower in IgAN subjects with active renal biopsy Oxford score and clinically progressive IgAN. Decreased O-glycosylation of hinge region of dIgA1 (high lectin binding) in IgAN associates with smaller radii of gyration (RG0) in dIgA1; RG0 on SAXS for subjects with normal O-glycosylation (Healthy Control, 8.75±0.05 nm and Patient A, 8.90±0.06 nm) were significantly higher than subjects with low O-glycosylation (Patients B, 8.62±0.05 nm and C, 8.62±0.07 nm). The 3D changes associate with increased levels of serum IgA-IgG immune complexes and increased in vitro activated hMC IL-6 production. However, there are no major differences in the proportion of the different hinge region O- and N-glycoforms distribution in dIgA1 between subjects on mass spectrometry, suggesting changes in the position, but not the number, of sugar moieties may be important. Conclusion: dIgA1 hinge region O-glycosylation correlates with conformational structural changes, which in turn relates to propensity for mesangial IgA deposition and ability to generate renal injury both in vitro and in vivo.