Molecular cloning and characterization of Izumo1 gene from sheep and cashmere goat reveal alternative splicing

We cloned the cDNA and genomic DNA encoding for Izumo1 of cashmere goat (Capra hircus) and sheep (Ovis aries). Analysis of 4.6 kb Izumo1 genomic sequences in sheep and goat revealed a canonical open reading frame (ORF) of 963 bp spliced by eight exons. Sheep and goat Izumo1 genes share > 99% identity at both DNA and protein levels and are also highly homologous to the orthologues in cattle, mouse, rat and human. Extensive cloning and analysis of Izumo1 cDNA revealed three (del 69, del 182 and del 217) and two (del 69 and ins 30) alternative splicing isoforms in goat and sheep, respectively. All of the isoforms are derived from splicing at typical GT-AG sites leading to partial or complete truncation of the immunoglobulin (Ig)-like domain. Bioinformatics analysis showed that caprine and ovine Izumo1 proteins share similar structure with their murine orthologue. There are a signal peptide at the N-terminus (1-22 aa), a transmembrane domain at the C-terminus (302-319 aa), and an extracellular Ig-like region in the middle (161-252 aa) with a putative N-linked glycosylation site (N-205-N-S). Alignment of Izumo1 protein sequences among 15 mammalian species displayed several highly conserved regions, including LDC and YRC motifs with cysteine residues for potential disulfide bridge formation, CPNKCG motif upstream of the Ig-like domain, GLTDYSFYRVW motif upstream of the putative N-linked glycosylation site, and a number of scattered cysteine residues. These distinctive features are very informative to pinpoint the important gene motifs and functions. The C-terminal regions, however, are more variable across species. Izumo1 cDNA sequences of goat, sheep, and cow were found to be largely homologous, and the molecular phylogenetic analysis is consistent with their morphological taxonomy. This implies the Izumo1 gene evolves from the same ancestor, and the mechanism of sperm-egg fusion in mammals may be under the same principle in which Izumo1 plays an important role.