Anti-gal immunoglobulin genes

Anti-Gal is a natural antibody present in all humans. It interacts specifically with the carbohydrate structure Gal[alpha]1-3Gal[beta]1-4GlcNAc-R (termed a-galactosyl or [alpha]-gal epitope). As much as 1% of circulating immunoglobulins in human display anti-Gal specificity. The overall objective of the project is to explore the molecular aspects of the anti-Gal production as a model of immune competence in humans, particularly in elderly population. The immediate goal of this study was to understand the immunoglobulin genes involved in this antibody production. This interest emerged from our first part of the study which established an age-associated decrease in anti-Gal affinity in human populations. Since the immunoglobulin concentration in sera of our samples did not change significantly between young and elderly populations, we hypothesized that the low affinity in elderly may result from the lack or excess of somatic mutations or may be caused by the use of inappropriate germline genes. As a first step toward investigating this hypothesis, the second part of our study focused on generation of human anti-Gal mAb by EBV transformation and examining the immunoglobulin genes within several monoclonal cell lines. The results revealed that the utilization of VH genes for the anti-Gal production was biased in that eight of nine anti-Gal B cell clones from seven young individuals used VH3 family genes and one used a VH1 gene. Further, the occurrence of somatic mutations in these genes was demonstrated. To obtain more information on anti-Gal V genes and enhance our ability to study anti-Gal genes, we initiated the use of phage display technology. Preliminary, two combinatorial phage display libraries constructed from young healthy individuals were screened for anti-Gal phage/Fab. Two phage clones displaying anti-Gal specificity were isolated. While the sequences of heavy chains of two clones belong to the same VH3 family, the two light chains used distinct light chain gene families, i.e. V[kappa] and V[lambda] respectively. Both clones contained somatic mutations as well. We hypothesize that the restricted use of VH3 genes for anti-Gal production may be the result of functional constraints imposed by the need of a suitable structural motif for specific interaction with the [alpha]-galactosyl epitope. This study has provided a valuable insight into the gene structures responsible for the anti-Gal production.