Organization and genomic complexity of bovine lambda-light chain gene locus.

Complete characterization and physical mapping of bovine lambda (lambda) light chain locus, spanning 412kbp, on chromosome 17, has revealed twenty-five V(lambda) genes, seventeen being functional, organized in three sub-clusters 23.7kbp 5' of the J(lambda)-C(lambda) units. Three V(lambda) sub-clusters are separated by two large introns of 126.8 and 138.3kbp. The predominantly expressed V(lambda)1 genes are present in the two 5' sub-clusters, while J(lambda)-proximal V(lambda) sub-cluster comprises rarely expressed V(lambda)2 and V(lambda)3 genes. The preferential expression of V(lambda)1 genes in the bovine immunoglobulin repertoire is influenced by the composition of recombination signal sequences (RSS). Of the J(lambda)-C(lambda) cluster, it is mainly J(lambda)3-C(lambda)3 unit that is expressed in reading frame 2, though J(lambda)2 and J(lambda)3 have identical RSS. The predominant expression of J(lambda)3-C(lambda)3 genes over J(lambda)2-C(lambda)2 is likely due to endogenous counter selection for J(lambda)2 encoded CDR3 and framework 4 regions. Differences in the genomic complexity of V(lambda) genes in Hereford and Holstein cattle are due to polymorphism at the lambda-light chain gene locus. Despite more potential germline encoded combinatorial diversity, restricted V(lambda)1-J(lambda)3-C(lambda)3 recombinations encode the most lambda-light chain repertoire in cattle.

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 beta2 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 VDJCmu and 25 VDJCgamma 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 VDJCmu and VDJCgamma 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.

Analysis of the expressed heavy chain variable-region genes of Macaca fascicularis and isolation of monoclonal antibodies specific for the Ebola virus' soluble glycoprotein.

The cynomolgus macaque, Macaca fascicularis, is frequently used in immunological and other biomedical research as a model for man; understanding it's antibody repertoire is, therefore, of fundamental interest. The expressed variable-region gene repertoire of a single M. fascicularis, which was immune to the Ebola virus, was studied. Using 5' rapid amplification of cDNA ends with immunoglobulin (Ig)G-specific primers, we obtained 30 clones encoding full-length variable, diversity, and joining domains. Similar to the human V(H) repertoire, the M. fascicularis repertoire utilized numerous immunoglobulin heavy variable (IGHV) gene fragments, with the V(H)3 (41%), V(H)4 (39%), and V(H)1 (14%) subgroups used more frequently than the V(H)5 (3.9%) or V(H)7 (1.7%) subgroups. Diverse immunoglobulin heavy joining (IGHJ) fragments also appeared to be utilized, including a putative homolog of JH5beta gene segment identified in the related species Macaca mulatta, Rhesus macaque, but not in humans. Although the diverse V region genes in the IgG antibody repertoire of M. fascicularis had likely undergone somatic hypermutations (SHMs), they nevertheless showed high nucleotide identity with the corresponding human germline genes, 80-89% for IGHV and 72-92% for IGHJ. M. fascicularis and human V(H) genes were also similar in other aspects: length of complementarity-determining regions and framework regions, and distribution of consensus sites for SHMs. Finally, we demonstrated that monoclonal antibodies (mAbs) specific for an Ebola protein could be obtained from M. fascicularis tissue samples by phage display technology. In summary, the study provides new insight into the M. fascicularis V region gene repertoire and further supports the idea that macaque-derived mAbs may be of therapeutic value to humans.

Unusually long germline DH genes contribute to large sized CDR3H in bovine antibodies.

We demonstrated earlier the existence of an exceptionally long third complementarity-determining region of the heavy chain (CDR3H) (up to 61 amino acids (aa)), with multiple cysteine residues, in some functional IgM antibodies of cattle. To understand the origin of such a long CDR3H, we have now characterized the germline diversity gene (D(H)) of the cattle. A 2.3kb genomic DNA fragment hybridizing with a newly developed DNA probe to putative bovine D(H) gene sequences was isolated, cloned and its nucleotide sequence determined. Inspection of the nucleotide sequence led to identification of three bovine germline D(H) gene segments of varying size: 42bp (14 possible codons), 58bp (19 possible codons) and 148bp (49 possible codons). The characteristic repetitive GGT and TAT codons, remarkable in the CDR3H region of fetal VDJ rearrangements likely encoded by germline genes, are noted in two of the identified germline D(H) genes. These D(H) genes are preferentially expressed in the third reading frame to encode hydrophilic glycine and tyrosine residues in the CDR3H region. Phylogenetic analysis suggests that bovine D(H) genes are closest to rabbit and chicken D(H) genes. Thus, both short and long germline D(H) genes exist in cattle and these are capable of directly contributing to CDR3H size heterogeneity including the exceptionally long CDR3H region, apart from recombination associated mechanistic factors.

Bovine IgM antibodies with exceptionally long complementarity-determining region 3 of the heavy chain share unique structural properties conferring restricted VH + Vlambda pairings.

Naturally occurring antibody repertoires of cattle (Bos taurus) include a group of IgMlambda antibodies with exceptionally long complementarity-determining region 3 of the heavy chain (CDR3H) segments, containing multiple Cys residues. These massive CDR3H segments will greatly influence the tertiary and quaternary structures of the bovine IgM combining sites. As an antibody's combining site is formed by both heavy and light chains, we have analyzed the nucleotide sequences and structural properties of the lambda-light chains that pair with micro -heavy chains containing exceptionally long CDR3H. There appears to be an exquisite selective pressure for the use of three V(lambda)1 genes (V(lambda)1x and two new V(lambda)1d and V(lambda)1e genes) in IgM with unusually long CDR3H. The V(lambda)1d and V(lambda)1e genes are similar to each other, but diverge from the other V(lambda)1 genes into two closely related subfamilies. The available bovine V(lambda) genes are classified into three V(lambda) gene families: V(lambda)1, V(lambda)2 and V(lambda)3 based on nucleotide similarity >/=80%. Further, analysis of total Ser content and positions of Ser residues in the sequences was found to be sufficient to classify the cattle V(lambda)1 subfamilies. Patterns of Ser residues differ for V(lambda) domains from ruminant species (e.g. cattle, sheep and goats) and other mammals (e.g. humans and mice). These 'Ser signatures' can be used to track divergent evolution in lambda-light chains. Interestingly, Ser90L in complementarity-determining region 3 of the light chain (CDR3L) occurred in all V(lambda) domains that pair with V(H) regions containing exceptionally long CDR3H. A structural role for Ser90L was revealed in homology models of V(lambda) domains, i.e. to hold the ascending polypeptide of CDR3L in a relatively tight space between the N-terminal segment and residues from CDR1L. The CDR3L of V(lambda) domains also occupied smaller volumes if paired to V(H) domains with extremely long CDR3H (>/=48 residues), and were more variable in their conformation and filled larger volumes if CDR3Hs were