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.

Nested N-Terminal Megalin Fragments Induce High-Titer Autoantibody and Attenuated Heymann Nephritis

“…All four groups elicited high-titer anti-megalin autoantibodies at 4 wk, with no further increases at 8 or 12 wk despite the booster immunization ( Figure 4); rather, titers decreased slightly during this time in all groups. These changes recapitulate similar but more marked reductions in anti-megalin titers that were noted previously in AHN rats immunized with native megalin (6) or with recombinant nM60 (13) (Figure 4).…”

“…The cells were separated by centrifugation, and the culture media were concentrated 10-fold in the presence of protease inhibitor cocktail (Boehringer-Mannheim, Mannheim, Germany) on PM-10 ultrafiltration membranes (Millipore, Billerica, MA). Recombinant proteins were purified from concentrated culture media by two rounds of affinity absorption on Ni-NTA (Novagen, Madison, WI) as described previously for nM60 fragment (13). Protein concentrations were estimated by bicinchoninic acid protein assay (Bio-Rad) and by ultraviolet absorbance at 280 nm.…”

“…As previously established, the use of supplemented CFA for both primary and booster doses was shown to be advantageous to the model. For sensitization of rats that were used in lymph node cells (LNC) proliferation assays, a single 100-g dose of megalin or nM60 (13) in CFA was distributed at four dorsal sites and one rear footpad.…”

“…Proteinuria was not reported in either of these models. We found that a 60-kD proteolytic fragment of megalin that was derived from the N-terminus could elicit AHN with attendant proteinuria (16) and also showed that a recombinant protein that represented this fragment could produce similar disease (13). Full activity in the model required expression of the fragment from insect cells as a secreted product, suggesting that posttranslational modifications or conformational determinants are essential for the pathogenic potential.…”

Intramolecular Epitope Spreading in Heymann Nephritis

“…13 Domain-specific rabbit antisera generated against recombinant polypeptides representing LBD I through IV produced glomerular ID in PHN without induction of proteinuria or other clinical consequences. 14 More recently, N-terminal fragments spanning LBD I produced either by proteolysis of native megalin 15 or by expression in baculovirus-insect cells 16 were shown to be as effective as native megalin for inducing AHN. A fragment including residues 1 to 236 induced autoAb and full-blown disease characterized by glomerular ID and severe proteinuria.…”