MAPPING RAT MEGALIN - THE 2ND CLUSTER OF LIGAND-BINDING REPEATS CONTAINS A 46-AMINO ACID PATHOGENIC EPITOPE INVOLVED IN THE FORMATION OF IMMUNE DEPOSITS IN HEYMANN NEPHRITIS

Megalin (gp330), an epithelial endocytic receptor, is a major target a ntigen of Heymann nephritis (HN), an autoimmune disease in rats. To el ucidate the mechanisms of HN, we have mapped a pathogenic epitope in m egalin that binds anti-megalin antibodies. We focused our attention on four clusters of cysteine-rich, low density lipoprotein receptor (LDL R) ligand binding repeats in the extracellular domain of megalin becau se they represent putative ligand binding regions and therefore would be expected to be exposed in vivo and to be able to bind circulating a ntibodies. Rat megalin cDNA fragments I through IV encoding the first through fourth clusters of ligand-binding repeats, respectively, were expressed in a baculovirus system. All four expression products were d etected by immunoblotting with two antisera capable of inducing passiv e HN (pHN). When antibodies eluted from glomeruli of rats with pHN wer e used for immunoblotting, only the expression product encoded by frag ment II was detected. This indicates that the second cluster of LDLR l igand binding repeats is directly involved in binding anti-megalin ant ibodies and in the induction of pHN. To narrow the major epitope in th is domain, fragment II was used to prepare proteins sequentially trunc ated from the C- and N-terminal ends by in vitro translation. Analysis of the truncated translation products by immunoprecipitation with ant i-megalin IgG revealed that the fifth ligand-binding repeat (amino aci ds 1160-1205) contains the major epitope recognized. This suggests tha t a 46-amino acid sequence in the second cluster of LDLR ligand bindin g repeats contains a major pathogenic epitope that plays a key role in pHN. Identification of this epitope will facilitate studies on the pa thogenesis of HN.