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 endoplasmic reticulum-associated protein. The screening resulted in the isolation of a 2.3-kilobase cDNA clone (A2/6). The sequence encoded 611 amino acids with a calculated mass of 69,454 Da, that was 60% similar to mouse calnexin. A high affinity calcium binding domain, present in both calnexin and calreticulin, and one transmembrane domain similar to that of calnexin were found in the A2/6 protein domain. Northern blot analysis of total RNA from seven different tissues showed hybridization only to testis RNA. Southern blot analysis indicated that A2/6 was a single copy gene. The calculated molecular mass for A2/6 was unexpectedly lower than the 101-kDa protein recognized by 1C9 on Western blot analysis of total testis protein. However, Escherichia coli and in vitro translation products of A2/6 cDNA yielded a similar 100-kDa protein. Finally, using the recombinant protein, calcium binding activity was detected by a 45Ca2+ 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.
Ohsako
S
S
Department of Veterinary Biosciences, University of Illinois, Urbana 61801.
Hayashi
Y
Y
Bunick
D
D
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D037281:Calnexin; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013094:Spermatozoa / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D008565:Membrane Proteins; D018832:Molecular Chaperones; D012333:RNA, Messenger; D011994:Recombinant Proteins; D037281:Calnexin; C087341:calmegin; D002118:Calcium