Rainbow trout (Oncorhynchus mykiss) contain two calnexin genes which encode distinct proteins.
Calnexin (IP90/P88) is an integral membrane protein of the endoplasmic reticulum that binds newly synthesized N-linked glycoproteins during their folding in the ER including MHC class I molecule. This manuscript reports the identification of two unique cDNA clones of calnexin in rainbow trout. Both encode putative mature proteins of 579 and 592 aa respectively in addition to a 24 aa signal peptide. Sequence analysis revealed that only one of the two cDNA clones encodes a putative ER retention signal, K/QEDDL, followed by a serine phosphorylation site conserved with mammalian homologs. Amino acid sequence alignment illustrated conservation of the calnexin luminal domain, which consists of a globular and a P domain, in both copies. Southern blotting revealed that there are at least two copies of the calnexin gene in the trout genome and northern blotting showed a wide tissue distribution of an estimated 3 kbp calnexin transcript with an additional minor transcript of 2.3 kbp expressed only in head kidney, spleen PBLs and strongly in RTS11. Importantly, the smaller transcript was predominantly upregulated in RTS11 after a 24h treatment with the calcium ionophore A23187. In western blots, calnexin was detected primarily as a 120 kDa protein and upon A23187 treatment; a 100 kDa band was most prominently expressed. These results suggest that in salmonids there are two differentiated versions of the calnexin gene which encode proteins that may have diverged to perform unique biological functions.
Department of Biology, University of Waterloo, 200 University Ave W., Waterloo, Ontario N2L 3G1, Canada.
Lital
L
Sever
Nguyen T K
NT
Vo
Niels C
NC
Bols
Brian
B
Dixon
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D000001:Calcimycin / Q000494:pharmacology; D061207:Calcium Ionophores / Q000494:pharmacology; D037281:Calnexin / Q000145:classification / Q000235:genetics* / Q000276:immunology; D002460:Cell Line; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000276:immunology / Q000378:metabolism; D018628:Gene Dosage; D016678:Genome; D060226:Head Kidney / Q000166:cytology / Q000378:metabolism; D015395:Histocompatibility Antigens Class I / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D017686:Oncorhynchus mykiss / Q000235:genetics* / Q000276:immunology; D020033:Protein Isoforms / Q000235:genetics; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013154:Spleen / Q000166:cytology / Q000378:metabolism
D061207:Calcium Ionophores; D015395:Histocompatibility Antigens Class I; D020033:Protein Isoforms; D037281:Calnexin; D000001:Calcimycin
Antigen presentation; Calcium ionophore; Chaperone; ER stress; MHC class I; Major histocompatibility