Molecular Cloning, Chromosomal Localization, and Expression Analysis ofCYRN1andCYRN2, Two Human Genes Coding for Cyritestin, a Sperm Protein Involved in Gamete Interaction

Abstract

Germ cell cyritestin is a membrane-anchored protein belonging to the ADAM family of proteins. Sequencing of eight human cyritestin cDNA clones revealed that they are identical at their 5' and 3' ends but differ from each other in the length of an internal deletion, suggesting that the human cyritestin mRNA is alternatively spliced. Internal deletions that are present in some cDNA isoforms do not cause a frameshift in the C-terminal coding region. Analysis of the predicted amino acid sequences demonstrated that the human cyritestin is a polymorphic protein that could include membrane-anchored and soluble forms. Southern blot analysis and characterization of human cyritestin genomic fragments revealed that the human genome contains two copies of the cyritestin gene instead of one as in the mouse. The human CYRN1 and CYRN2 genes were assigned to the region p12-21 of chromosome 8 and q12 of chromosome 16, respectively. Northern blot and RT-PCR analyses revealed that both human genes are expressed in the testis. Amino acid sequence comparisons between cyritestin and other members of the metalloprotease-disintegrin family of proteins suggested that human and mouse cyritestin and monkey tMDCI are homologous molecules.

The gene for the human tMDC I sperm surface protein is non-functional: implications for its proposed role in mammalian sperm–egg recognition

“…Recently, Adham and co-workers [17] have reported sequences for four human tMDC I transcripts (which they call CYRN1), which all contain the same 83 bp deletion that we observe ( Figure 1, Deletion I ) at the beginning of the coding region. However, all four CYRN1 transcripts apparently lack the 1 bp deletion (Figure 1, Deletion II ) and the second in-frame termination codon within the metalloproteinase-like domain (Figure 1, Stop II) and contain an additional deletion of 2 bp within the region encoding the putative signal peptide.…”

“…This 2 bp deletion, as well as the absence of the 1 bp Deletion II (see Figure 1) are not observed in any of the independent clones, isolated from many individuals, which we report here. In addition, two of the four CYRN1 cDNA clones and four out of four characterized CYRN1 PCR products (also described by Adham et al [17]), all contained deletions or insertions associated with the disintegrin-like domain, the region implicated in egg binding.…”

“…None of these features are observed in any of the many clones we have sequenced. As a result of these differences, two of the four sequences reported by Adham and co-workers [17] apparently contain an open reading frame roughly comparable in size with that found in other tMDC I species orthologues, leading those authors to propose that a functional protein is expressed in the human testis. However, we believe that these sequences are either erroneous or represent rare transcripts which are not found in the majority of individuals.…”

“…All contain the same sequence. The sequence reported by Adham et al [17] was derived from a human testis cDNA library, but it is not clear whether this was derived from a single individual. Although they have deposited sequence data for four different CYRN1 cDNA clones in the EMBL database (X89654, X89655, X89656 and X89657), somewhat surprisingly all four sequences start and end at exactly the same nucleotide.…”

Importance of the Porcine ADAM3 Disintegrin Domain in Sperm-Egg Interaction

“…Moreover, the sperm of the cyritestin null male mouse is incapable of binding to the ZP. Interestingly, several researchers have reported that there are two human cyritestin genes, CYRN1 and CYRN2, localized on chromosomes 8p12-21 and 16q12, respectively [18]. Evidence suggests that human cyritestin genes are non-functional, as judged by Western blot analysis using rabbit antisera against human and macaque CYRN1 and the presence of a variety of deletions, insertions and premature termination in humans [19].…”