Molecular cloning and functional characterization of a calreticulin gene from the sea cucumber Apostichopus japonicus

Abstract

Calreticulin (CRT) plays crucial roles in the regulation of Ca2+ homeostasis, immune responses and molecular chaperon. In this study, a new member of the CRT gene (denoted as AjCRT) was cloned and characterized from the sea cucumber Apostichopus japonicus. The full-length of the gene was of 3316 bp, which consisted of 204 bp 5’ untranslated region (UTR), 1870 bp of 3’-UTR and a putative 1242-bp open reading frame encoding a 48-kDa polypeptide (AjCRT) with a theoretical pI of 4.19. AjCRT had 59 % - 65 % sequence identity with CRTs from other species. It contained two CRT domains (residues 94 - 109 and 126 - 134) with a RING finger domain. AjCRT was ubiquitously expressed in all tissues examined, but was highly expressed in the coelomocytes. Temporal transcriptional levels in the coelomocytes revealed significant upregulation of AjCRT after the animal was challenged with Vibrio splendidus, reaching 4.97-fold the level of control at 4 h, but then decreased to 2.56-fold the level of control at 72 h. AjCRT knockdown decreased the expression level of the binding of Ca2+ to protein gene about 50 %. At the same time, intracellular concentration of Ca2+ also increased by 1.86-fold and 1.94-fold compared to that of the control. Taken together, the results suggested that AjCRT may be associated with the immune response against bacterial infection, probably through participating in the regulation of intracellular Ca2+ homeostasis in sea cucumber.