MOLECULAR-CLONING AND SEQUENCING OF CALNEXIN-T - AN ABUNDANT MALE GERM CELL-SPECIFIC CALCIUM-BINDING PROTEIN OF THE ENDOPLASMIC-RETICULUM

A mouse testis cDNA expression library was screened using a monoclonal antibody (1C9) that recognized an abundant testis-specific 101-kDa en doplasmic reticulum-associated protein. The screening resulted in the isolation of a 2.3-kilobase cDNA clone (A2/6). The sequence encoded 61 1 amino acids with a calculated mass of 69,454 Da, that was 60% simila r to mouse calnexin. A high affinity calcium binding domain, present i n both calnexin and calreticulin, and one transmembrane domain similar to that of calnexin were found in the A2/6 protein domain. Northern b lot analysis of total RNA from seven different tissues showed hybridiz ation only to testis RNA. Southern blot analysis indicated that A2/6 w as a single copy gene. The calculated molecular mass for A2/6 was unex pectedly lower than the 101-kDa protein recognized by 1C9 on Western b lot analysis of total testis protein. However, Escherichia coil and in vitro translation products of A2/6 cDNA yielded a similar 100-kDa pro tein. Finally, using the recombinant protein, calcium binding activity was detected by a Ca-45(2+) overlay assay. These results suggest that spermatogenic cell endoplasmic reticulum has a unique calcium binding protein, calnexin-t, which appears to be a calnexin variant.