title: Conformation and glycosylation of a megalin fragment correlate with nephritogenicity in Heymann nephritis.
abstract: Active Heymann nephritis (AHN), a rat model of autoimmune glomerulonephritis, is induced by immunization with autologous megalin, a 600-kDa cell surface glycoprotein isolated from crude renal extracts. Recombinant proteins containing a 563-residue N-terminal sequence of megalin were obtained from Escherichia coli and baculovirus-insect cell expression systems. Rats immunized with the soluble, secreted protein encoded by a baculovirus construct elicited high titer anti-megalin autoantibodies and developed glomerular immune deposits and elevated proteinuria consistent with AHN. Rats treated with the bacterial or nonsecreted insect cell proteins produced a milder anti-megalin response and did not develop the disease. Nephritogenicity appeared to correlate with conformational or other structural features of native megalin. All three recombinant proteins were reactive in Western blots with rabbit anti-megalin antiserum, whereas the insect cell-derived proteins reacted preferentially in Western blot and ELISA with anti-megalin autoantibodies from rats with AHN induced by native megalin. Only the secreted insect cell product was stained in a lectin blot, suggesting its specific glycosylation. These observations provide evidence that a megalin N-terminal domain includes B and T cell epitopes sufficient for a pathogenic autoimmune response and that a native-like conformation and glycosylation are essential for the induction of disease. The importance of conformational B cell epitopes for pathogenic autoantibodies recapitulates observations made in other models of organ-specific autoimmune disease. Glycosidic modifications could influence the presentation of either B or T cell epitopes in AHN, consistent with emerging evidence of the role of post-translational modifications in pathogenic autoimmune responses.
authors.affiliation: Department of Pediatrics, University of California School of Medicine, Davis, CA 95616, USA.
authors.forename: Alfonso
authors.initials: A
authors.lastname: Tramontano
authors.forename: Sudesh P
authors.initials: SP
authors.lastname: Makker
mesh_terms: D000818:Animals; D001323:Autoantibodies / Q000096:biosynthesis; D001324:Autoantigens / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D003001:Cloning, Molecular; D018985:Epitopes, B-Lymphocyte / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D018984:Epitopes, T-Lymphocyte / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D005260:Female; D005921:Glomerulonephritis / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D006031:Glycosylation; D007150:Immunohistochemistry; D007271:Injections, Intradermal; D026561:Low Density Lipoprotein Receptor-Related Protein-2 / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D011917:Rats, Inbred Lew; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D018985:Epitopes, B-Lymphocyte; D018984:Epitopes, T-Lymphocyte; D026561:Low Density Lipoprotein Receptor-Related Protein-2; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins