The effect of VH residues 6 and 23 on IgG3 cryoprecipitation and glomerular deposition

Abstract

MRL/lpr mice spontaneously develop a lupus-like autoimmune syndrome characterized by immunopathologic manifestations such as necrotizing vasculitis of the skin and glomerulonephritis. A feature of this autoimmune 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 cryoprecipitation, not all IgG3 antibodies cryoprecipitate. It has been postulated that electrostatic charge may be influential in cryoprecipitation. To investigate this problem, the VH and VL sequences of a panel of IgG3 cryoglobulins and non-cryoglobulins were compared, with particular attention to charged amino acid differences. At VH residues 6 and 23 the cryoglobulins were more positively charged than their non-cryoglobulin counterparts. To analyze further the effect of charge on cryoprecipitation, the sequence of an IgG3 monoclonal cryoprecipitating rheumatoid factor was modified by site-directed mutagenesis. The more positive residues at VH 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 VH residue 6 affects cryoprecipitation while residue 23 does not. When injected into normal BALB/c mice, the unmutated antibody produced glomerular immune deposits and focal glomerulonephritis, whereas loss of cryoprecipitability by mutating residue 6 completely abrogated glomerular immune deposition and glomerular injury. In contrast, the mutation at residue 23 which retains cryoprecipitability reduced glomerular immune deposition and prevented glomerular injury.