title: Identification and characterization of Inositol-requiring enzyme-1 and X-box binding protein 1, two proteins involved in the unfolded protein response of Litopenaeus vannamei.
abstract: The inositol-requiring enzyme-1 (IRE1)-X-box binding protein 1 (IRE1-XBP1) pathway is the key branch of the unfolded protein response (UPR). To investigate the role of the IRE1-XBP1 pathway in reducing environmental stress and increasing anti-viral immunity in Litopenaeus vannamei, homologues of IRE1 (designated as LvIRE1) and XBP1 (designated as LvXBP1) were identified and characterized. The full-length cDNA of LvIRE1 is 4908bp long, with an open reading frame (ORF) that encodies a putative 1174 amino acid protein. The full-length cDNA of LvXBP1 is 1746bp long. It contains two ORFs that encode putative 278 amino acid and 157 amino acid proteins, respectively. LvXBP1 mRNA has the predicted IRE1 splicing motifs CNG'CNGN located within the loop regions of two short hairpins. Sequencing of the splicing fragment induced by endoplasmic reticulum (ER)-stress showed a 3bp or 4bp frame shift from the predicted sites. The spliced form LvXBP1 (LvXBP1s) contained an ORF encodes a putative 463 amino acid protein. The reporter gene assays indicated that LvXBP1s activates the promoter of L. vannamei immunoglobulin heavy chain binding protein (LvBip), an important UPR effector. RT-PCR showed that LvXBP1 was spliced during the experiments. For heat shock treatment, the total LvXBP1 expression was increased and peaked at about 36h, whereas the percentages of the two isoforms were relatively stable. For the WSSV challenge, LvXBP1 was upregulated during the experiment and the percentage of the spliced form continuously declined after 18h of infection. Knock-down of LvXBP1 by RNA interference resulted in a lower cumulative mortality of L. vannamei under WSSV infection. Furthermore, the expression profiles of LvIRE1 and LvXBP1 in the gills, hemocytes, intestines, and hepatopancreas of the WSSV-challenged shrimp were detected using real-time RT-PCR. Taken together, these results confirm that the IRE1-XBP1 pathway is important for L. vannamei environmental stress resistance, suggest that L. vannamei IRE1-XBP1 may activated by WSSV and be annexed to serve the virus.
authors.affiliation: MOE Key Laboratory of Aquatic Product Safety/State Key Laboratory for Biocontrol, School of Marine Sciences, Sun Yat-sen University, 135 Xingang Road West, Guangzhou, PR China.
authors.forename: Yi-Hong
authors.initials: YH
authors.lastname: Chen
authors.forename: Li
authors.initials: L
authors.lastname: Zhao
authors.forename: Li-Ran
authors.initials: LR
authors.lastname: Pang
authors.forename: Xiao-Yun
authors.initials: XY
authors.lastname: Li
authors.forename: Shao-Ping
authors.initials: SP
authors.lastname: Weng
authors.forename: Jian-Guo
authors.initials: JG
authors.lastname: He
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D060829:Arthropod Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004722:Endoribonucleases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D055785:Gene Knockdown Techniques; D008969:Molecular Sequence Data; D033561:Penaeidae / Q000201:enzymology / Q000378:metabolism*; D010802:Phylogeny; D000071701:Regulatory Factor X Transcription Factors; D016415:Sequence Alignment; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D056811:Unfolded Protein Response
chemical_list: D060829:Arthropod Proteins; D004268:DNA-Binding Proteins; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D004722:Endoribonucleases