VH and VL gene usage by anti-beta-amyloid autoantibodies in Alzheimer's disease: detection of highly mutated V regions in both heavy and light chains

Abstract

In a previous study, four human IgM kappa monoclonal antibodies (mAbs) secreted by Epstein-Barr-virus-transformed B cell lines independently derived from the peripheral blood of a patient with Alzheimer's disease (AD) were found to react with a conformational epitope of beta-amyloid protein and to stain amyloid plaques in AD brain. Three of these mAbs were studied further. They did not react with an additional panel of antigens and autoantigens. By a competitive inhibition ELISA, the Kd was determined to be 5.7 x 10(-8) M. The VH and V kappa of these mAbs were sequenced at the cDNA level and found to be identical. The corresponding VH and V kappa germline counterpart genes hsigghvm148 and hsiggkvm148 were identified to be analogous to the previous reported VH3 germline gene humighvf1/dp53 and the V kappa IV germline gene hsigk18. DA1/4 and JH4b were used by the heavy chain and J kappa 4 was used by the light chain. Multiple nucleotide substitutions were seen in both VH and V kappa when their sequences were compared to the germline sequences. High replacement/silent ratios in the CDR regions of both VH and V kappa indicate positive-selective pressure. Of a total of 22 amino acid replacements in VH and V kappa, 12 were nonconservative replacements. Furthermore, 7 of these 12 resulted in charge changes. The monoreactivity, the moderately high affinity constant, the clonal expansion evident by identical VH and VL used by these B cells secreting antibodies of interest, and the excessive replacement mutations in both VH and VL segments lead to the conclusion that these antibodies have been generated due to an antigen-driven process.