A Broad Role for Cysteines in Bovine Antibody Diversity

Abstract

Ab diversity in most vertebrates results from the assortment of amino acid side chains on CDR loops formed through V(D)J recombination. Cows (Bos taurus) have a low combinatorial diversity potential because of a small number of highly homologous V, D, and J gene segments. Despite this, a subset of the Ab repertoire (;10%) contains exceptionally long CDR H chain (HC) 3 (H3) regions with a rich diversity of cysteines and disulfide-bonded loops that diversify through a single V-D-J recombination event followed by massive somatic hypermutation. However, the much larger portion of the repertoire, encoding shorter CDR H3s, has not been examined in detail. Analysis of germline gene segments reveals noncanonical cysteines in the HC V regions and significant cysteine content in the HC D regions. Deep sequencing analysis of naturally occurring shorter CDR H3 (,40 aa) Ab genes shows that HC V and HC D regions preferentially combine to form a functional gene with an even number of total cysteines in the final V region, suggesting that disulfide bonds contribute to diversity not only in ultralong CDR H3 bovine Abs but in shorter CDR H3 bovine Abs as well. In addition to germline "hard-coded" cysteines, the bovine Ab repertoire can produce additional cysteine codons through somatic hypermutation, further diversifying the repertoire. Given the limited combinatorial diversity at the bovine Ig loci, this helps to explain how diversity is created in shorter CDR H3 Abs and potentially provides novel structural paratopes in bovine Ab combining sites. ImmunoHorizons, 2019, 3: 478-487.

Formation of ultralong DH regions through genomic rearrangement

“…Cows, in particular, have few VH and DH regions, but have very long CDR H3 regions. The homologous DH regions are cysteine rich, and diversity can be generated through both germline and somatically generated cysteines, which can form a diverse array of potential disulfide bonded loops[23, 35, 36]. In the knob region of ultralong CDR H3s, a diversity of disulfide bond patterns has been observed in several crystal structures [23, 25, 26], and mutations to and from cysteine have been confirmed through deep sequence analysis.…”

Formation of ultralong DH regions through genomic rearrangement

“…Some species, however, have fewer functional V, D, and J segments and may use additional mechanisms to add diversity to their repertoires. Cows, in particular, have only 10-12 functional VH regions in only one VH1 family [14,17,29], and 16 functional DH regions [29,35], but have very long CDR H3 regions. The homologous DH regions are cysteine rich, and diversity can be generated through both germline and somatically generated cysteines, which can form a diverse array of potential disulfide bonded loops [23,35,36].…”

“…Cows, in particular, have only 10-12 functional VH regions in only one VH1 family [14,17,29], and 16 functional DH regions [29,35], but have very long CDR H3 regions. The homologous DH regions are cysteine rich, and diversity can be generated through both germline and somatically generated cysteines, which can form a diverse array of potential disulfide bonded loops [23,35,36]. In the knob region of ultralong CDR H3s, a diversity of disulfide bond patterns has been observed in several crystal structures [23,25,26], and somatic mutations to and from cysteine have been confirmed through deep sequence analysis.…”

Molecular Dissection of the Antibody Response: Opportunities and Needs for Application in Cattle

“…This mechanism has been proposed to be a means of generating structural diversification through modification of the pattern of disulfide bond formation (41). This is facilitated by an unusual codon bias in HCDR3, which predicates mutation to cysteine (particularly in the IGHD8-2 segment codons) during bovine VDJ recombination, resulting in the generation of diversity in structure due to the making and breaking of di-sulfide bonds between paired/unpaired cysteines (2,47). This diversification mechanism has also been shown to operate in conventional length bovine antibodies (47).…”

“…This is facilitated by an unusual codon bias in HCDR3, which predicates mutation to cysteine (particularly in the IGHD8-2 segment codons) during bovine VDJ recombination, resulting in the generation of diversity in structure due to the making and breaking of di-sulfide bonds between paired/unpaired cysteines (2,47). This diversification mechanism has also been shown to operate in conventional length bovine antibodies (47). Analyses of the sequences of rearranged bovine Ig genes has additionally indicated evidence of a low frequency of gene conversion events in both light and heavy chains that involves short nucleotide segments from light and heavy chain pseudogenes (48,49), potentially providing a further means of generating sequence diversity-although current data are limited and its importance has yet to be fully determined.…”