THE EFFECT OF V-H RESIDUE-6 AND RESIDUE-23 ON IGG3 CRYOPRECIPITATION AND GLOMERULAR DEPOSITION

MRL/lpr mice spontaneously develop a lupus-like autoimmune syndrome ch aracterized by immunopathologic manifestations such as necrotizing vas culitis of the skin and glomerulonephritis. A feature of this autoimmu ne syndrome is the production of extremely large amounts of monoclonal IgG3 cryoglobulins. The structural basis of IgG3 cryoprecipitation is not well understood. Although the IgG3 isotype is necessary for cyrop recipitation, not all IgG3 antibodies cryoprecipitate. It has been pos tulated that electrostatic charge may be influential in cryoprecipitat ion. To investigate this problem, the V-H and V-L sequences of a panel of IgG3 cryoglobulins and non-cryoglobulins were compared, with parti cular attention to charged amino acid differences. At V-H residues 6 a nd 23 the cryoglobulins were more positively charged than their non-cr yoglobulin counterparts. To analyze further the effect of charge on cr yoprecipitation, the sequence of an IgG3 monoclonal cryoprecipitating rheumatoid factor was modified by site-directed mutagenesis. The more positive residues at V-H 6 and 23 present in some of the cryoglobulin antibodies were mutated to the more negative residues found in the non -cryoglobulins. The results show that V-H residue 6 affects cryoprecip itation while residue 23 does not. When injected into normal BALB/c mi ce, the unmutated antibody produced glomerular immune deposits and foc al glomerulonephritis, whereas loss of cryoprecipitability by mutating residue 6 completely abrogated glomerular immune deposition and glome rular injury. In contrast, the mutation at residue 23 which retains cr yoprecipitability reduced glomerular immune deposition and prevented g lomerular injury.