S. Ohsako et al., MOLECULAR-CLONING AND SEQUENCING OF CALNEXIN-T - AN ABUNDANT MALE GERM CELL-SPECIFIC CALCIUM-BINDING PROTEIN OF THE ENDOPLASMIC-RETICULUM, The Journal of biological chemistry, 269(19), 1994, pp. 14140-14148
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.