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

Abstract

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 D(H) gene locus was completely characterized from Holstein cattle that revealed the presence of a total of eight D(H) genes, including D(H)Q52, with a distinct organization in sub-clusters. However, a total of 10 D(H) genes are identified at the polymorphic D-gene locus in cattle that are classified into four families, designated as BovD(H)A, BovD(H)B, BovD(H)C and BovD(H)D. In fetal B-cells, VDJ recombinations encoding long CDR3H (>50 codons) are directly encoded by the single germline V(H) gl.110.20, the longest D(H)2 and the J(H)1 genes, apart from few N- and P-nucleotide additions at the junctions. Further, non J-proximal D(H)7 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-V(H) or D(H) gene origin, inserted specifically at V(H)-D(H) 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 V(H)-D(H) and D(H)-J(H) junctions. Such 'CSNS' insertions at V(H)-D(H) 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 V(H)-D(H)-J(H) 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 V(H)-D(H) junction, in generating exceptionally long CDR3H extending up to 61 codons in cattle antibodies.