The Research On Innate Immunity Function About Glutaminyl-peptide Cyclotransferase(QPCT) In Scylla Paramamosain

In enzymology,a glutaminyl-peptide cyclotransferase is an enzyme that catalyzes the chemical reaction,which is involved in the post translational modification by converting N-terminal glutamate of protein to pyroglutamate,which is resistant to protease degradation,more hydrophobic,and prone to aggregation and neurotoxic.The function and role mechanism of glutaminyl peptide cyclase in human osteoporosis and schizophrenia has been extensively studied,but the function and role mechanism of glutathione-peptidyl-transferase has not been studied in crustacean crustaceans.In this study,we aimed to determine the function of QPCT in Scylla paramamosain,white spot syndrome virus(WSSV)or Vibrio alginolyticus infection.The entire 2445 bp QPCT gene sequence with a 1113 bp open reading frame(ORF)encoding a 370-aa-long protein was cloned from S.paramamosain.Sequence homology and phylogenetic analysis showed that S.paramamosain QPCT gene showed the closest relationship with that of Trichinella spiralis.The real-time quantitative PCR(RT-q PCR)results showed that QPCT gene was primarily expressed in digestive gland,little higher in gills,hemocytes and gonad,and low expression in muscles and heart.By the double-strand RNA(ds RNA)interference technology,we constructed the ds RNA prokaryotic expression vector in vitro and the expression of QPCT gene was successfully inhibited by the addition of ds RNA.The results of important crab immune genes expression detection found that a number of known immune-related genes in crab innate immune system were affected when QPCT gene was silenced,and partial immune pathway signal transduction related genes such as prophenoloxidase(pro PO)and crustin antimicrobial peptide were down-regulated significantly while myosin-Ⅱ-essential-light-chain-like-protein were up-regulated significantly in the hemocytes at 24 h after QPCT-ds RNA treatment.Crab immunocompetence experiments showed that the total hemocytes counts(THC)increased,and phenoloxidase(PO)activity enhanced after QPCT gene knocked down.These results suggested that QPCT may play an important role in phagocytosis,apoptosis and some major immune factors.V.alginolyticus challenged experiment showed that QPCT was over-expressed in crab hemocytes after challenged with V.alginolyticus.The results of crab mortality experiment showed that cumulative mortality of crab was significantly increased after QPCT gene silenced.It suggested that crab QPCT might has an obviously antibacterial effect.The phagocytosis and apoptosis experiment showed there were both higher phagocytosis rate of hemocytes and the apoptosis rate of hemocytes after QPCT-ds RNA treatment.The result indicated that V.alginolyticus may be able to use QPCT to avoid phagocytosis of crab hemocytes.The results showed that the QPCT could inhibit the apoptosis,when S.paramamosain treated with V.alginolyticus.The RT-q PCR results showed that QPCT was under-expressed in crab haemocytes after challenged with WSSV.WSSV copies expriment and mortality expriment showed that after QPCT gene knocked out,WSSV copies were significantly reduced,and crab mortality rate was significantly increased.When silenced the QPCT,the results of phagocytosis showed that the phagocytic rate of S.paramamosain hemocytes on WSSV increased significantly,and the apoptotic result showed that the apoptosis of crab hemocytes decreased obviously.These findings confirmed that QPCT can promote crab hemocytes apoptosis,while WSSV can restrain the expression of QPCT to promote crab hemocytes apoptosis.This is the first time that reveals the key role of QPCT in crabs virus infection.Moreover,the research provides an important theoretical basis for studying the mechanism of innate immunity of crab and WSSV infection.