The long term objective of this project is to understand the pathogenesis of human membranous glomerulonephropathy (MGN). Heymann Nephritis (HN) in rats is the accepted model of MGN. Although a glycoprotein isolated form the brush border (gp330) is generally recognized as the putative antigen of HN, it's normal function has yet to be determined. We now have preliminary evidence showing the binding of serum proteins (76 and 80kD) to gp330. Interestingly, we have also shown the reactivity of the HN nephritogenic autoantibody to these same serum proteins.
The specific aims are: (1) To identify, isolate and characterize the circulatory proteins (serum reactive proteins) which show reactivity with gp330 and nephritogenic autoantibodies. This will be accomplished using standard purification techniques. Analysis of the samples will be by ELISA and SDS-PAGE/Western analysis and isoelectric focusing. cDNA clones will be isolated using the proposed polyclonal and monoclonal antibodies in order to determine the amino acid sequence for comparison to other proteins. Rat monoclonal antibodies would help define epitopes on the serum reactive proteins win comparison to the nephritogenic autoantibody epitopes. (2) to demonstrate that these serum proteins bind to glomeruli, glomerular epithelia cells and gp330. Purified serum reactive proteins will be analyzed for binding both in vivo and in vitro by iodination of the proteins or by detection with avidin- biotin or colloidal gold antibodies for immunofluorescence and electron microscopy. These experiments will help determine if the serum reactive proteins are ligands for gp330. (3) To identify if complexes of serum reactive proteins and nephritogenic autoantibodies are formed in vivo during the induction of active HN and if they play an important role in the development of HN. This would be accomplished with the use of specific polyclonal and monoclonal antibodies to serum reactive proteins using both gel exclusion chromatography and sucrose density gradient centrifugation analysis of sera from diseased rats. Diseased kidney section will also be analyzed for serum reactive protein in the glomerulus by immunofluorescence and electron microscopy. The proposed studies will provide new information regarding the relationship of the serum reactive proteins to gp330, establish the possible function of the HN autoantigen and determine the role of these serum proteins, if any, in the pathogenesis of HN.
| Kanalas, J J; Makker, S P (1993) Analysis of a 45-kDa protein that binds to the Heymann nephritis autoantigen GP330. J Biol Chem 268:8188-92 |
| Kanalas, J J (1993) Effect of the nephritogenic autoantibody of Heymann's nephritis on plasminogen-binding to gp330 and activation by urokinase. Biochim Biophys Acta 1225:101-6 |
| Kanalas, J J (1992) Analysis of plasmin binding and urokinase activation of plasminogen bound to the Heymann nephritis autoantigen, gp330. Arch Biochem Biophys 299:255-60 |
| Kanalas, J J; Makker, S P (1991) Identification of the rat Heymann nephritis autoantigen (GP330) as a receptor site for plasminogen. J Biol Chem 266:10825-9 |
