Genomic Organization Of The IgH Genes And Their Repertoire Generation In Holstein Cows

Immunoglobulins (Igs) play a pivotal role in adaptive immunity, and the membrane IgM (mIgM) is a key receptor for B lymphocyte development. In humans and mice, mutations in IgM encoding genes may cause B cell deficiency, leading to severe immunodeficiency. Previous studies reveal that the bovine IgH loci exhibit very unique structural characters-tw o IgH loci exist in the bovine genome and are located in chromosome 21 and 11, respectively. Both of them harbor a μ gene encoding IgM. The allelic exclusion is usually observed during the Ig expression, that is, a successful expression of IgH genes in one chromosome would turn off the recombination of the IgH genes in the homologous chromosome. It remains unclear how the two bovine μ genes intact with each other to secure normal B cell development and to keep B cells with mono-antibody specificity. Compared with humans and mice, cows have a very limited number of VH gene segments, so it also needs to be characterized that what mechanisms cows use to generate significant antibody diversity with the extremely limited number of the VH gene segments.In this study, the entire Holstein cow RP42-BAC library was screened using Southern blotting, which identified 30 BAC clones comprising the bovine IgH genes. FISH analyses indicated that only a single IgH locus existed in the bovine genome, which was located in chromosome 21, whereas no functional IgH locus was found in chromosome 11. Based on the sequencing of seven BAC clones and long-PCR, we obtain the genomic structure of bovine IgH locus covering partial VH region, which is arranged in the order of 5’-VHn-DHn-JHn-μl-(ψδ-ψV-DHn)3-JHn-μ2-δ-γ3-γ1-y2-ε-α-3’.This study reveals that no allelic exclusion is involved in expression of the two bovine μ genes as both genes are located in tandem in the same IgH locus. Apart from independent expression of the individual μ genes by VDJ recombination, the μ2 can also be expressed by class switch recombination (CSR). Expression analysis suggested that the IgM2 subclass was dominantly expressed in most tissues throughout different developmental stages. Since no significant differences in positive/negative selection and physical and chemical properties were observed between IgM1 and IgM2 heavy chain variable regions, it can be concluded that there is no significant difference in function between the two IgM subclasses.Only 12 functional VH gene segments were identified among the partial VH locus which extends approximately 284 Kb, and all of them exclusively belong to a single VH family with high homologies (>89%). A total number of 23 DH segments were found in the four DH loci, while 16 of which show potential function and a DH gene segment with exceptionally long coding sequence (149 bp) was identified. VDJ recombination analyses showed strong preferential usage of DH and JH segments in both IgMl and IgM2, which greatly limits the VH-DH-JH combinatorial diversity. Compared with the CDR3Hs of humans and mice, the length of bovine CDR3Hs shows the widest range (3-60+aa), while the panorama of N, P additions in cows is similar to that of mice, which allows us to deduce that the generation of junctional diversity in cows mainly depends on the CDR3H length and sequence polymorphism.Sequence analysis of cDNAs revealed that the exceptionally long CDR3Hs were only observed in IgM2 and were exclusively encoded by the DH8 segment. However, in the genomic VDJ recombinants, some other DH segments were also observed in the generation of the ultra-long CDR3Hs with unusually long N, P nucleotide additions. As the DH segments in bovine IgM2 CDR3H tend to possess relatively longer coding sequences, the mean length of bovine IgM2 CDR3Hs is significantly longer than those of humans, mice and bovine IgMl. To maintain the structural stability and to increase the spatial flexibility, the longer-prone CDR3Hs of bovine IgM2 mainly depend on disulfide bridges formed by cysteines, which are commonly observed in bovine ultra-long CDR3Hs.In summary, we confirm that only a single IgH locus exists in the bovine genome and characterize the entire structure of the locus. Although two bovine μ genes are functional, the μ2 gene is expressed dominantly in different tissues throughout the developmental stages. To compensate the deficiency in VDJ recombinatorial diversity caused by limited number of VH gene segments, cows rely mainly on increasing the junctional diversity and the mean length of CDR3Hs. Although the ultralong CDR3Hs are exclusively encoded by DH8 segment, in theory the unusual long N, P additions may also account for the ultralong CDR3H generation. This study provides a deep insight into the understanding of the antibody diversification and expression mechanism of two functional μ genes in cows.