The cloning and inducible expression of the rainbow trout ERp57 gene

Abstract

ERp57 is a member of a protein disulfide isomerase family and is a chaperone responsible for the correct folding of newly synthesized glycoproteins in the endoplasmic reticulum and in the assembly of the major histocompatibility complex class I in the endogenous pathway of antigen presentation. This study reports the identification of a full length ERp57 cDNA in rainbow trout that encodes a putative 477aa mature protein with an additional signal sequence of 16aa. The trout protein shared 75% identity with the human homolog, but interestingly did not include either a C terminal endoplasmic reticulum retention signal, Q/KEDL in humans, or a nuclear localization signal which is highly conserved in mammals. Amino acid sequence alignment revealed conservation of four classical domains in trout ERp57 and two conserved active CXXC redox motifs. Trout ERp57 protein was identified as a single band around 57 kDa. Southern blotting analysis revealed that there two copies of the ERp57 gene in the trout genome and northern blotting showed a wide tissue distribution of gene expression in various tissues with the highest expression in liver and egg. This study showed for the first time in teleost that ERp57 transcript is upregulated in response to immune stimuli such as double stranded RNA or phytohemagglutinin. Furthermore, upon treatment with ER stress inducer A23187, trout ERp57 protein expression levels were increased both in peripheral blood leukocytes and the RTS11 macrophage like cell line after 6 and 8 h respectively. These findings suggest a possible conserved function for trout ERp57 in the ER and during the activation of the immune response.