Identification and molecular analysis of a stress-inducible Hsp70 from Sciaenops ocellatus.
Hsp70 proteins are a family of molecular chaperones that are involved in many aspects of protein homeostasis. In this study, an Hsp70 homologue (SoHsp70) was identified from red drum Sciaenops ocellatus and analyzed at molecular level. The open reading frame of SoHsp70 is 1920 bp and intronless, with a 5'-untranslated region (UTR) of 399 bp and a 3'-UTR of 241 bp. The deduced amino acid sequence of SoHsp70 shares 84-92% overall identities with the Hsp70s of a number of fish species. In silico analysis identified in SoHsp70 three conserved Hsp70 domains involved in nucleotide and substrate binding. The coding sequence of SoHsp70 was subcloned into Escherichia coli, from which recombinant SoHsp70 was purified and, upon ATPase assay, found to exhibit apparent ATPase activity. Expressional analysis showed that constitutive expression of SoHsp70 was detectable in heart, liver, spleen, kidney, brain, blood, and gill. Experimental challenges with poly(I:C) and bacterial pathogens of Gram-positive and Gram-negative nature induced SoHsp70 expression in kidney to different levels. Stress-responsive analysis of SoHsp70 expression in primary cultures of red drum hepatocytes showed that acute heat shock treatment elicited a rapid induction of SoHsp70 expression which appeared after 10 min and 30 min of treatment. Exposure of hepatocytes separately to iron, copper, mercury, and hydrogen peroxide significantly upregulated SoHsp70 expression in time-dependent manners. Vaccination of red drum with a Streptococcus iniae bacterin was also found to induce SoHsp70 expression. Furthermore, recombinant SoHsp70 enhanced the immunoprotective effect of a subunit vaccine. Taken together, these results suggest that SoHsp70 is a stress-inducible protein that is likely to play a role in immunity and in coping with environmental and biological stresses.
Dang
Wei
W
Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, PR China.
Hu
Yong-hua
YH
Zhang
Min
M
Sun
Li
L
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000276:immunology; D001424:Bacterial Infections / Q000276:immunology / Q000662:veterinary; D001428:Bacterial Vaccines / Q000276:immunology; D001483:Base Sequence; D005393:Fish Diseases / Q000276:immunology / Q000382:microbiology; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000494:pharmacology; D022781:Hepatocytes / Q000276:immunology; D006358:Hot Temperature; D019216:Metals, Heavy / Q000633:toxicity; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D010473:Perciformes / Q000276:immunology*; D011994:Recombinant Proteins / Q000378:metabolism; D013312:Stress, Physiological / Q000187:drug effects / Q000276:immunology*
D000276:Adjuvants, Immunologic; D001428:Bacterial Vaccines; D018840:HSP70 Heat-Shock Proteins; D019216:Metals, Heavy; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases