title: Molecular characterization of two novel molecular chaperones in bacterial-challenged Apostichopus japonicus.
abstract: Molecular chaperones of 78 kDa glucose-regulated protein (GRP78) and protein disulfide isomerase (PDI) are involved in protein folding and assembly in the endoplasmic reticulum (ER). Increasing evidences also suggest that these two molecules play an important role in immune response. In the present study, we cloned and characterized GRP78 and PDI genes from Apostichopus japonicus by RNA-seq and RACE approaches (designated as AjGRP78 and AjPDI, respectively). The AjGRP78 cDNA was of 2355bp including an open reading frame (ORF) of 2013 bp encoding a protein of 670 amino acids with three heat shock protein 70 (HSP70) family signatures. AjGRP78 contained a 23-amino acid signal peptide at the N-terminus and a HDEL motif at the C-terminus, which supported the location of the protein in the ER. The full length cDNA of AjPDI was of 1893 bp with a 5' untranslated region (UTR) of 153 bp, a 3' UTR of 228 bp and an ORF of 1512 bp encoding a protein of 503 amino acids. A 17-amino acid signal peptide, two thioredoxin domains with two active sites of CGHC, and KDEL retention signal were totally conserved in the deduced amino acid of AjPDI. Phylogenic analysis and multiple alignments have shown that both genes shared remarkably higher degree of structural conservation and sequence identities with other counterparts from invertebrates and vertebrates, further supporting that the two proteins were novel members of molecular chaperone family. Spatial expression analysis revealed that AjGRP78 mRNA transcripts were dominantly expressed in the tentacle, while AjPDI mRNA levels were abundant in the muscle, intestine and respiratory trees. For Vibrio splendidus challenged sea cucumber, the peak expression of AjGRP78 and AjPDI mRNAs in coelomocytes were detected at 24h with 1.73-fold increase and at 6h with 1.83-fold increase compared with the control group, respectively. Similarly, a significant increase in the relative mRNA levels of AjGRP78 and AjPDI was also identified in 1 μg mL(-1) LPS exposed primary cultured coelomocytes. These results collectively suggested that AjGRP78 and AjPDI were ER chaperones of A. japonicus, of which expression is induced upon bacterial infection.
authors.affiliation: School of Marine Sciences, Ningbo University, Ningbo, Zhejiang 315211, PR China.
authors.forename: Haihong
authors.initials: H
authors.lastname: Wang
authors.affiliation: School of Marine Sciences, Ningbo University, Ningbo, Zhejiang 315211, PR China.
authors.forename: Yina
authors.initials: Y
authors.lastname: Shao
authors.affiliation: School of Marine Sciences, Ningbo University, Ningbo, Zhejiang 315211, PR China.
authors.forename: Weiwei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: School of Marine Sciences, Ningbo University, Ningbo, Zhejiang 315211, PR China. Electronic address: lichenghua@nbu.edu.cn.
authors.forename: Chenghua
authors.initials: C
authors.lastname: Li
authors.affiliation: School of Marine Sciences, Ningbo University, Ningbo, Zhejiang 315211, PR China.
authors.forename: Zhimeng
authors.initials: Z
authors.lastname: Lv
authors.affiliation: School of Marine Sciences, Ningbo University, Ningbo, Zhejiang 315211, PR China. Electronic address: jinchunhua@nbu.edu.cn.
authors.forename: Chunhua
authors.initials: C
authors.lastname: Jin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000276:immunology; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D007113:Immunity, Innate; D008070:Lipopolysaccharides / Q000494:pharmacology; D008969:Molecular Sequence Data; D009928:Organ Specificity; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism*; D047528:Stichopus / Q000378:metabolism* / Q000382:microbiology; D014733:Vibrio / Q000276:immunology
chemical_list: D006360:Heat-Shock Proteins; D008070:Lipopolysaccharides; D019704:Protein Disulfide-Isomerases
keywords: 78kDa glucose-regulated protein; Apostichopus japonicus; Protein disulfide isomerase; Vibrio splendidus; qPCR