Molecular cloning of a novel Ca(2+)-binding protein (calmegin) specifically expressed during male meiotic germ cell development.

During mammalian spermatogenesis, many specific molecules are expressed. We have recently identified a 93-kDa male meiotic germ cell-specific antigen (Meg 1) exclusively expressed in germ cells from the pachytene spermatocyte to the spermatid stage using the monoclonal antibody TRA 369 (Watanabe, D., Sawada, K., Koshimizu, U., Kagawa, T., and Nishimune, Y. (1992) Mol. Reprod. Dev. 33, 307-312). In this study, we cloned a cDNA representing this antigen from a mouse testis cDNA expression library, using the monoclonal antibody TRA 369. Northern blotting showed that this transcript was 2.3 kilobases in length and was expressed only in the testis and not in other somatic tissues or in the ovary. The expression of the mRNA was first detected at the pachytene spermatocyte stage of male germ cell development, and this expression was correlated with the expression of the protein. Sequence analysis of the cDNA revealed that the predicted protein consists of 611 amino acids, including a hydrophobic NH2 terminus characteristic of a signal peptide, two sets of internal repetitive sequences (four repeats of IPDPSAVKPEDWDD and GEWXPPMIPNPXYQ), and a hydrophilic COOH terminus. The deduced amino acid sequence has 58% homology with dog calnexin (the ER membrane phosphoprotein of pancreatic cells) and significant partial homology with calreticulin (high affinity Ca(2+)-binding protein of the ER membrane) at the repetitive sequence. Furthermore, we demonstrated the 45Ca2+ binding ability of this antigen by a 45Ca2+ overlay assay, and the name calmegin is proposed for this antigen. Calmegin is a novel Ca(2+)-binding protein that is specifically expressed in spermatogenesis. The highly regulated, specific, and abundant expression of calmegin suggests that it has important roles in spermatogenesis.