Underglycosylation of IgAl hinge plays a certain role for its glomerular deposition in IgA nephropathy

Abstract

This study was performed to isolate and investigate the IgAl that could accumulate in glomeruli (glomerulophilic IgAl). IgAl was fractionated by the electric charge and the reactivity to Jacalin. Serum IgAl of IgA nephropathy patients was separated and fractionated using a Jacalin column and subsequent ion-exchange chromatography. The fractions were divided into three groups of relatively cationic (C), neutral (N), and anionic (A). IgAl was also divided into Jacalin low (L), intermediate (I), and high (H) affinity fractions by serial elution using 25, 100, and 800 mM galactose. The left kidneys of Wistar rats were perfused with 2, 5, or 10 mg of each group of IgAl. The rats were sacrificed 15 min, 30 min, 3 h, or 24 h after the perfusion. The accumulation of each IgAl in the glomeruli was then observed by immunofluorescence. The IgAl of the fractions N and H separated by the two methods was definitely accumulated in the rat glomeruli with a similar pattern. The electrophoresis revealed that the macromolecular IgAl was increased in fraction H compared with other fractions. Therefore, Jacalin high-affinity IgAl (fraction H) was applied on a diethylaminoethyl column and divided into electrically cationic (HC), neutral (HN), and anionic (HA). Only the asialo-Galβ1,3GalNAc chain was identified in the fraction HN IgAl by gas-phase hydrazinolysis. Furthermore, the IgAl fraction was strongly recognized by peanut agglutinin, Vicia Villosa lectins, and antisynthetic hinge peptide antibody. These results indicated that the IgAl molecules having the underglycosylated hinge glycopeptide played a certain role in the glomerular accumulation of IgAl in IgA nephropathy.