Cloning and characterization of genes and proteins related to calcium metabolism of black tiger shrimp Penaeus monodon

Abstract

Calcium ion (Ca[superscript 2+]) plays critical role in many cellular responses of eukaryotes. The important Ca[superscript 2+] controlling organelle (Ca[superscript 2+] homeostasis) in cellular level is the Endoplasmic reticulum (ER). Therefore, studying genes that control Ca[superscript 2+] homeostasis could lead to understand cellular responses related to Ca[superscript 2+]. The focus of this study was on calreticulin (CRT), Calnexin (CNX) and Endoplasmic Reticulum protein 57 (ERp57). These genes/proteins function together to control cellular Ca[superscript 2+] homeostasis and protein folding in the ER. The full length cDNA of these genes were successfully characterized. A single form of PmCRT and PmCNX contained open reading frame (ORF) of 1221 and 1788 bp corresponding to deduce proteins of 406 and 595 amino acid residues, respectively. In contrast, two isoforms of PmERp57 was found. They contained an identical ORF of 1458 bp (corresponding to 485 amino acid residues) but two different 3' UTR length of 529 and 713 bp. Furthermore, genomic organization of the three genes was characterized. The genomic PMCRT sequence contained 4 exons and 3 introns while that of PmERp57 consisted of 10 exons and 9 introns. Genomic organization of PmCNX was partially characterized. Only 4 exons and 3 introns were completed. Tissue distribution analysis of the three gene homologues revealed their expression in eye stalk, pleopod, gill, heart, ovaries, testis, hepatopancreas, stomach, intestine, hemocytes, thoracic ganglia, lympiod organ and antennal gland of P. monodon juveniles and broodstocks. Semi-quantitative RT-PCR were carried out to examine effect of thermal stress on expression levels of CRT, CNX and ERp57 in hemocytes, hepatopancreas and gill of the juvenile black tiger shrimps. Differential expression was found only in hemocytes. The semi-quantitative result from hemocytes was confirmed by quantitative real-time PCR. Expression of the three genes in hemocytes was up-regulated within the first hour after the 30 ºC heat treatment (P < 0.05). After that, their expression levels return to the normal levels. Recombinant proteins of PmCRT, PmCNX and PmERp57 were produced using an E. coli system. All recombinant proteins were expressed in the soluble form. These recombinant proteins were purified and some protein functions were in vitro assayed. Binding of PMCNX to Ca[superscript 2+] caused change in its conformation, resulting in a mobility shift on native PAGE gel while the others did not exhibit the shift. Moreover, PmCRT and PmCNX also complexed with PmERp57, causing mobility shift of the proteins bands on native PAGE gel.