Molecular Cloning, Expression And Function Analysis Of Kazal-type Serine Proteinase Inhibitors From The Red Swamp Crayfish, Procambarus Clarkii

It's well known that the crayfish has become an excellent animal model to study the innate immunity of the invertebrate, as well as an important economic animal. The studies on the innate immune responses of crayfish, especially on immune defense against the main crustacean-pathogens such as white spot syndrome virus (WSSV) or Vibrio anguillarum, will further enrich the knowledge of the innate immunity of crustaceans and provide important theory for controling the infectious diseases.WSSV is one of the main pathogens of shrimp, and it's also the pathogen of the other crustaceans such as crayfish, crab, lobster, etc. In order to investigate the distribution of WSSV in the different tissues of the infected crayfish and further provide the required data for the following studies of innate immunity of crayfish, a quantitative PCR to quantify the amount of WSSV was developed. The results indicated that this method to quantify the number of WSSV was rapid, sensitive, and precise. This developed method well satisfied the need of the following WSSV infection experiment.For crayfish, the important immunity-related proteins are synthesized mainly in hemocytes and hepatopancreas, and some of them are further secreted into the circulating hemolymph to function. To investigate the mechanism of the defense against WSSV and other pathogens, the healthy crayfish were challenged by WSSV first. At 96h after challenge the gills full of hemocytes and hepatopanceas were collected to isolate the mRNA, and then the mRNA was transcribed into cDNA to construct the cDNA library. After randomly sequencing, four Kazal-type inhibitors, namely hcPcSPI1, hcPcSPI2, hpPcSPI3, and hpPcSPI4, with full-length cDNA sequence were identified from this cDNA library.To examine the tissue distribution of these four inhibitors, four pairs of primers for these four inhibitors were designed. Using the semi-quantitative RT-PCR the tissue distribution of these four inhibitors at mRNA level were detected. The results demonstrated that hcPcSPI1 and hcPcSPI2 mainly existed in hemocytes while hpPcSPI3 and hpPcSPI4 mainly distributed in hearts and hepatopancreas. Homology comparison indicated that the identity of these four inhibitors was 42.47%, hpPcSPI3 shared higher 56.67%identify with hpPcSPI4, and hcPcSPI1 only share 23.73% identity with hcPcSPI2. The phylogenectic tree constructed using the inhibitors of the crustaceans showed that hpPcSPI3 and hpPcSPI4, both derived from the hepatopancreas, are different from other hepatopancreas type inhibitors of shrimp, formed a small group. This indicates that hpPcSPI3 and hpPcSPI4 have a relatively distant evolutionary relationship with other inhibitors from hepatopancreas. In addition, the inhibitors from hemocytes seem hard to be grouped into one meaningful cluster since the Bootstrap values are very low, which reveals that these hemocyte type inhibitors, including hcPcSPI1 and hcPcSPI2, evolve rapidly.In order to examine whether these four inhibitors were involved in the immune defense against WSSV, as well as defense against V. anguillarum for hpPcSPI3 and hpPcSPI4, real-time RT-PCR was used to detect the expression profiles of these inhibitors after WSSV or V. anguillarum challenge. The results indicated that after WSSV challenge, hcPcSPI2 and hpPcSPI3 were up-regulated, hcPcSPI1 first decreased and then gradually recovered, while there was no obvious change for hpPcSPI4. It's possible that hcPcSPI1, hcPcSPI2, and hpPcSPI3 participate in the immune defense against WSSV, and hpPcSPI3 and hpPcSPI4 were also involved in the anti-V.anguillarum immune response.In crustaceans, Kazal-type serine proteinase inhibitors in hemolymph are believed to function as regulators of the host-defense reactions or inhibitors against proteinases from microorganisms. In this paper, we further analyzed two Kazal-type serine proteinase inhibitors derived from hemocytes, namely hcPcSPI1 and hcPcSPI2, from the crayfish (Procambarus clarkia).We find that hcPcSPI1 is composed of a putative signal peptide, an RGD motif, and three tandem Kazal-type domains with the domain P1 residues L, L and E, respectively. Mainly, hcPcSPI1 was detected in hemocytes as well as in the heart, gills, and intestine at both the mRNA and protein levels. Quantitative real-time PCR analysis showed that hcPcSPI1 in hemocytes was upregulated by the stimulation of Esherichia coli (8099). In addition, hcPcSPI1 and its three independent domains were overexpressed and purified to explore their potential functions. All four proteins inhibited subtilisin A and proteinase K, but not a-chymotypsin or trypsin. Recombinant hcPcSPI1 could firmly attach to Gram-negative bacteria E. coli and Klebsiella pneumoniae; Gram-positive bacteria Bacillus subtilis, Bacillus thuringiensis and Staphylococcus aureus; fungi Candida albicans and Saccharomyce cerevisiae, and only domain 1 was responsible for the binding to E. coli and S. aureus. In addition, recombinant hcPcSPIl was also found to possess bacteriostatic activity against B. subtilis and B. thuringiensis. Domains 2 and 3 contributed mainly to these bacteriostatic activities. All results suggested that hcPcSPI1 might play important roles in the innate immunity of crayfish.The serine proteinase inhibitor hcPcSPI2 is composed of a putative signal peptide, and two tandem Kazal-rype domains with the domain PI residues L and E, respectively. The temporal expression profile of this inhibitor was studied with quantitative real-time PCR and the results suggest that hcPcSPI2 is likely to be involved in anti-F. anguillarum immune response. Western blot demonstrates that hcPcSPI2 only exists in semigranular cells. Besides, after V. anguillarum challenge, the hcPcSPI2 protein could also be detected in cell-free hemolymph. Subsequently, the biochemical characteristics and bacteriostatic activity of hcPcSPI2 were assayed. The results indicate that hcPcSPI2 shows weak inhibitory activity against subtilisin A and trypsin, and may trigger bacteriostatic activity towards B. subtilis and B. thuringiensis, with MIC50 of 30.4 and 25.0μM, respectively. These studies reveal that hcPcSPI2 may also play an important role in the anti-bacterial immunity of the crayfish.