Somatic hypermutations and isotype restricted exceptionally long CDR3H contribute to antibody diversification in cattle

Antibody diversification in IgM and IgG antibodies was analyzed in an 18-month old bovine (Bos taurus) suffering from naturally occurring chronic and recurrent infections due to bovine leukocyte adhesion deficiency (BLAD). The BLAD, involving impaired leukocyte β2 integrin expression on leukocytes, develops due to a single point mutation in conserved region of the CD18 gene resulting in substitution of aspartic acid128 with glycine (D128G). Twenty four VDJCμ and 25 VDJCγ recombinations from randomly constructed cDNA libraries, originating from peripheral blood lymphocytes, were examined for the variable-region structural characteristics in IgM and IgG antibody isotypes. These analyses led to conclude that: (a) expression of exceptionally long CDR3H is isotype restricted to cattle IgM antibody; (b) VDJ recombinations encoding IgM with exceptionally long CDR3H undergo clonal selection and affinity maturation via somatic mutations similar to conventional antibodies; (c) somatic mutations contribute significantly to both IgM and IgG antibody diversification but significant differences exist in the patterns of ‘hot spot’ in the FR1, FR3 and CDR1H and, also, position-dependant amino acid diversity; and (d) transition nucleotide substitutions predominate over transversions in both VDJCμ and VDJCγ recombinations consistent with the evolutionary conservation of somatic mutation machinery. Overall, these studies suggest that both somatic mutations and exceptional CDR3H size generation contribute to IgM and IgG antibody diversification in cattle during the development of immune response to naturally occurring chronic and multiple microbial infections.