Exceptionally Long CDR3H Are Not Isotype Restricted in Bovine Immunoglobulins

Abstract

Exceptionally long third complementarity determining regions of the heavy chain (CDR3H) were previously described as a specificity of bovine IgG and IgM immunoglobulins. In addition, the genomic organization of the immunoglobulin heavy chain locus remains to be elucidated with a special focus on the number of variable segments (IGHV). By analyzing the variable regions according to the isotype-specific PCR using cDNA-PCR, we were able to prove the existence of exceptional long CDR3H in all bovine isotypes. The corresponding sequences of three distinct amplicons were grouped according to the length of the CDR3H. Sequences of CDR3H possessed 5 to 10, 12 to 31 or at least 48 amino acid residues. Long and mid-length CDR3H were composed of mainly hydrophilic amino acid residues, while short CDR3H also contained hydrophobic amino acid residues. All sequences with long CDR3H were related to the germline variable segment 10. Using the current genome assembly, Bos taurus NCBI build 6.1, the genomic organization of the bovine immunoglobulin heavy-chain locus was analyzed. A main locus was investigated on BTA21. Exons coding for variable, diversity, and joining segments, as well as for the constant regions of different isotypes, were also localized on BTA7, BTA8, and BTA20. Together with the information from unplaced contigs, 36 IGHV were detected of which 13 are putatively functional. Phylogenetic analysis revealed two bovine IGHV families (boVH1, boVH2). Thus, the existence of the two bovine families suggested was demonstrated, where boVH1 comprises all functional segments. This study substantially improves the understanding of the generation of immunoglobulin diversity in cattle.

Conservation and diversity in the ultralong third heavy-chain complementarity-determining region of bovine antibodies

“…Cysteines are also commonly found in CDR H3 of chickens (6), pigs (7), and the duck-billed platypus (8). The ultralong CDR H3s are found in bovine immunoglobulins of all classes (9), arise early in fetal development (10), and are encoded by the V H BUL, D H 2 and J H 1 gene segments (2, 11–12). Their length is due to a remarkably long, germline D H 2 segment that encodes four cysteines, and also repeated glycine, serine, and tyrosine residues, whose codons are biased for mutation to cysteine with just one base change (Fig.…”

Development of a Bioinformatics Framework for the Detection of Gene Conversion and the Analysis of Combinatorial Diversity in Immunoglobulin Heavy Chains in Four Cattle Breeds

“…The second bovine IGHV family consists solely of non-functional IGHV s that have not been identified in expression analyses yet. Eleven IGHV segment pairs shared 100% sequence identity, whereas two of these pairs contain a functional segment and either an ORF or a putative functional segment, respectively [17]. The high proportion of pseudogene segments leads to the assumption of their use in gene conversion events.…”

“…IGHD are classified into four families and the IGHD exons revealed huge size differences [21, 22]. The organization of the actual bovine germline repertoire and its possible allelic variants is incomplete and needs to be investigated in more detail [17]. Since, even the organization of the extensively studied human immunoglobulin germline repertoire is questioned and requires ongoing analyses [23].…”

“…In all rearranged bovine immunoglobulin isotypes, exceptionally long complementarity determining region 3 of the heavy chain (CDR3H) possessing up to 67 aa were described [17]. Together with IGHD2 and IGHJ1 , the germline IGHV10/34 segment was found to be the only variable segment rearranged in these exceptionally long CDR3Hs [17, 24], in a non-isotype dependent manner [17, 25].…”

“…Together with IGHD2 and IGHJ1 , the germline IGHV10/34 segment was found to be the only variable segment rearranged in these exceptionally long CDR3Hs [17, 24], in a non-isotype dependent manner [17, 25]. Furthermore, those specialized CDR3H possessing several cysteine residues enabling the formation of intra-CDR3H disulfide bonds.…”