A single WAP domain (SWD)-containing protein with antipathogenic relevance in red swamp crayfish, Procambarus clarkii.

摘要

Single WAP domain-containing proteins (SWDs) are small proteins possessing a whey acidic protein (WAP) domain at the C-terminal region. In this study, a complementary deoxyribonucleic acid (cDNA) of SWD-containing protein was isolated from the hemocytes of red swamp crayfish, Procambarus clarkii called Pc-SWD. The full-length cDNA sequence is 998 bp. The deduced amino acid sequence consists of 74 residues with a signal peptide of 20 residues. The mature peptide has a single WAP domain which contains eight conserved cysteine residues forming a "four-disulphide core" (4-DSC). The predicted molecular mass of the mature protein is 5.97 kDa, with an estimated pI of 7.71. Tissue distribution analysis by reverse-transcribed polymerase chain reaction (RT-PCR) revealed that Pc-SWD transcripts were primarily found in the hemocytes, heart, hepatopancreas, gills, and intestine. The results of time course analysis demonstrated that expression of Pc-SWD was decreased at 6 h followed by a significant upregulation from 48 h to 72 h in hemocytes after white spot syndrome virus (WSSV) injection. A similar expression pattern was found in the hepatopancreas after WSSV injection. In addition, Pc-SWD expression was visibly upregulated in the gills from 6 h to 72 h after WSSV injection. The results of Western bolt revealed that Pc-SWD was constitutively expressed in the heart, hepatopancreas, gills, and intestine of unchallenged crayfish. A weak band was detected in the hemocytes and hemolymph of unchallenged P. clarkii. The Pc-SWD expression was upregulated in the hemocytes and gills after challenging with WSSV; however, no obvious change occurred in the heart and intestine. rPc-SWD could bind to both Gram-negative and -positive bacteria strongly. Moreover, rPc-SWD exhibited specific proteinase inhibitory activity against the secretory proteinase(s) from B. subtilis and P. aeruginosa. All these findings suggest that Pc-SWD possibly functions as an immunity effector in defense against the invasion of crayfish pathogens.