VH genes encoding the immune response to beta-(1,6)-galactan: somatic mutation in IgM molecules

Abstract

The immune response to beta-(1,6)-galactan in the BALB/c mouse has been well characterized and includes the amino acid sequence determination of 13 monoclonal antibodies. The genetic potential encoding the VH regions of these antibodies has been determined by isolation and sequencing of homologous germline genes. The germline repertoire encoding these proteins was found to consist of two closely related genes. One of these directly encodes the VH segments of seven Gal-binding proteins, and the second directly encodes one additional protein sequence. Sequence variations found in the VH regions of five other Gal-binding proteins can be explained by somatic mutations leading to single base substitutions in the more frequently used gene. Since four of the hybridoma proteins exhibiting somatic mutations are of the IgM class, these results indicate that somatic mutation, in this system, is not associated with class switching and can apparently be initiated early in B-cell development. The two Gal genes are the only members of a very restricted multigene family and probably result from a gene duplication estimated to occur 1.4-2.8 million years ago. Three other genes hybridizing at moderate stringency to a VHGal probe were also sequenced and were found to be members of two additional VHIII families. Studies of the silent to replacement substitution ratios of these and other VH genes indicate that the number of silent substitutions found in immunoglobulin VH genes is lower than expected when compared with proteins such as preproinsulin and globin. Analysis of base composition reflected in these sequences indicates a marked increase of A-T% in the first and second codon positions of complementarity determining regions (CDR) which may be important in facilitating point mutations.