Characterising IgA1 solution structures in IgA nephropathy
2014-05-21T10:59:21Z (GMT) by
IgA nephropathy (IgAN) remains the commonest pattern of primary glomerulonephritis seen worldwide and results in the development of end-stage renal failure in over 30% of affected individuals. It is characterised by IgA1 containing immune complex formation and deposition within the mesangium; the reasons for which are unknown. IgAN is associated with aberrant serum O-glycoforms, characterised by over representation of IgA1 molecules displaying undergalactosylation. While a number of studies have postulated a pathogenic role for the IgA1 hinge region O-glycans in IgAN, none have investigated the impact of altered glycosylation on its three-dimensional shape. A combination of small-angle X-ray scattering (SAXS), and analytical ultracentrifugation (AUC) experiments were used to determine whether the monomeric IgA1 (mIgA1) conformational shape was altered in IgAN. Serum IgA1 from a healthy subject and three patients with IgAN with differing IgA1galactosylation were isolated using jacalin affinity chromatography. Monomeric IgA1 species were isolated by FPLC. Relative hinge region O-galactosylation was determined using a standardised helix aspersa agglutinin (HAA) lectin assay. The Guinier X-ray radius of gyration (R[subscript G]) for the healthy control, and the IgAN mIgA1 samples with both elevated and reduced galactosylation were 6.10–6.30 nm. This agreed well with that previously reported for mIgA1 and indicated that under the experimental conditions mIgA1 did not adopt a significantly altered conformation. The AUC sedimentation coefficients for all samples were 6.2 S, in agreement with this. The distance distribution curve P(r) gave an overall length (L) of 21 nm for the healthy mIgA1 control and 22–25 nm for IgAN-associated mIgA1. Purified IgAN-associated IgA1 monomers with the reduced O-galactosylation thus displayed an increased molecular length L, indicating a more extended arrangement in comparison to the healthy mIgA1 control. Elevated O-galactosylation however did not alter the length of mIgA1. The formation of dimer species was observed by AUC for all samples. Although the initial data analyses do not suggest major conformation change, these results provide a promising insight into the potential for altered O-galactosylation in IgA1 to promote self-aggregation, and unravelling of its hinge region which may expose neoantgenic epitopes for autoantibodies and alter IgA-receptor and IgA-extracellular matrix interactions.