Novel atypical nucleotide insertions specifically at VH―DH junction generate exceptionally long CDR3H in cattle antibodies

Some IgM cattle antibodies are amongst the largest known to exist in jawed vertebrates where CDR3H size may extend up to 61 amino acids. To understand the origin of such an exceptionally long CDR3H, bovine DH gene locus was completely characterized from Holstein cattle that revealed the presence of a total of eight DH genes, including DHQ52, with a distinct organization in sub-clusters. However, a total of 10 DH genes are identified at the polymorphic D-gene locus in cattle that are classified into four families, designated as BovDHA, BovDHB, BovDHC and BovDHD. In fetal B-cells, VDJ recombinations encoding long CDR3H (>50 codons) are directly encoded by the single germline VH gl.110.20, the longest DH2 and the JH1 genes, apart from few N- and P-nucleotide additions at the junctions. Further, non J-proximal DH7 gene is preferentially expressed in fetal B cells. The adult VDJ recombinations, however, are distinctly remarkable for ‘conserved short nucleotide sequence’ (‘CSNS’; 13–18 nucleotides), of non-VH or DH gene origin, inserted specifically at VH–DH junctions resulting in extension of CDR3H size up to 61 codons. Together with P-nucleotides, N-additions (1–9 nucleotides) are noted at both the VH–DH and DH–JH junctions. Such ‘CSNS’ insertions at VH–DH junction of adult VDJ recombinations encoding exceptionally long CDR3H provide novel mechanism of antibody diversification in cattle, not yet observed in other species. Further, analysis of VH–DH–JH recombinations originating from fetal B-cells reveals the presence of substitution, deletion or addition mutations without prior exposure to external antigen. Thus, somatic hypermutations may contribute towards diversification of the developing nascent antibody repertoire in cattle. In conclusion, the outlined experiments provide novel antibody diversification mechanism via ‘CSNS’ insertions, specifically at the VH–DH junction, in generating exceptionally long CDR3H extending up to 61 codons in cattle antibodies.