Characterization, Kinetics, And Possible Function Of Kazal-type Serine Proteinase Inhibitors From Fenneropenaeus Chinensis

Kazal-type serine proteinase inhibitor plays an essential role in innate immunity by restraining the activities of endogenic or exogenic serine proteinases and involved in anti-inflammatory and antimicrobial protection of mucous epithelia. Research on Kazal inhibitor can make clear its inhibitory mechanism, functions in innate immunity, and further develop its pharmaceutical value.Four Kazal-type serine proteinase inhibitors, FcSPI-1～4, from the hepatopancreas of Chinese white shrimp, Fenneropenaeus chinensis, were cloned and identified. The open reading frames (ORFs) of FcSPIs are 1389,1236,1080, and 939 base pairs and encode the pre-proteins of 462,411,359, and 312 amino acids form 9,8,7, and 6 typical Kazal domains, respectively. When analyzing the amino acid sequences of the four inhibitors, it was found that they might have been derived from the same transcript, which is subject to alternative splicing, and none of the Kazal domains is identical within each inhibitor. Multiple alignments showed that the Kazal inhibitors are homologous with a conserved motif of Cx3Cx6VCGSDGxTYx3CxLx5Cx5ITx6GC. The results from RT-PCR indicated that the expression of FcSPIs as a whole was upregulated by bacterial challenge and no obvious change was noticed after viral challenge.FcSPIs were recombined, expressed in bacteria, and purified for further study. Recombinant FcSPI-1 was sensitive to DTT and had thermal stability. The inhibitory kinetics assay suggested that rFcSPI-1 is a mixed-type fast tight binding inhibitor with inhibitory activities against subtilisin A at a molar ratio of 1:1,1:2 against proteinase K, and 2:1 against elastase. It can firmly bound to two Gram-positive and one Gram-negative bacteria but without antibacterial ability. In addition, it inhibited the activities of both bacterial-secreted proteinases and natural chymotrypsin of Chinese white shrimp, suggesting that FcSPI-1 may participate in the immune defense response by inhibition of bacterial pathogen proteinases and possibly be involved in the regulation of shrimp proteinase activity.