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This study was performed to isolate and investigate the IgA1 that could accumulate in glomeruli (glomerulophilic IgA1). IgA1 was fractionated by the electric charge and the reactivity to Jacalin. Serum IgA1 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). IgA1 was also divided into Jacalin low (L), intermediate (I), and high (H) affinity fractions by serial elu- tion 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 IgA1. The rats were sacrificed 15 min, 30 min, 3 h, or 24 h after the perfusion. The accumulation of each IgA1 in the glomeruli was then observed by immunofluorescence. The IgA1 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 IgA1 was increased in fraction H compared with other frac- tions. Therefore, Jacalin high-affinity IgA1(fraction H) was applied on a diethylaminoethyl column and divided into elec- trically cationic (HC), neutral (HN), and anionic (HA). Only the asialo-Galb1,3GalNAc chain was identified in the fraction HN IgA1 by gas-phase hydrazinolysis. Furthermore, the IgA1 fraction was strongly recognized by peanut agglutinin, Vicia Villosa lectins, and antisynthetic hinge peptide antibody. These results indicated that the IgA1 molecules having the undergly- cosylated hinge glycopeptide played a certain role in the glo- merular accumulation of IgA1 in IgA nephropathy.

The aim of this study was to investigate the role of carbohydrate moieties attached to IgA 1 hinge region in IgA 1 self-aggregation and adhesion to extracellular matrix (ECM) proteins previously reported in IgA nephropathy. Serum IgAI samples isolated from healthy individuals were digested with neuraminidase (NA), NA + f3-galactosidase, and NA + 13-ga- lactosidase + a-N-acetylgalactosaminidase to remove the car- bohydrates from the hinge region and were named asialo, agalacto, and naked IgA I , respectively. First, polyacrylamide gel electrophoresis was performed under the native condition, and consequently, a broad band indicating IgA I self-aggrega- tion was clearly observed in asiabo, agalacto, and naked IgA I, but not in native IgA I. However, the broad band disappeared in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under the nonreducing condition. Second, it was shown that IgAb adhesion activities to type IV collagen, fibronectin, and laminin were significantly higher in asiabo, agalacto, and naked IgAl than in native IgA1, using enzyme-linked immunosor- bent assay (asiabo, agalacto, and naked versus native: P < 0.0 1). In addition, agalacto IgA 1 had the highest affinity for all of the ECM proteins among the deglycosybated IgA I (agalacto versus asiabo and naked, P < 0.05). These results indicated that the removal of carbohydrates from the IgA I molecule resulted in noncovalent self-aggregation and a significant increase in adhesion to the ECM proteins. It was therefore suggested that the IgAl glycans played a protective role against aggregation and adhesion and that the underglycosylation of the IgA I molecule found in IgA nephropathy could be involved in the nonimmunobogic gbomerular accumulation of IgA I.