Three Kazal-type serine proteinase inhibitors from the red swamp crayfish Procambarus clarkii and the characterization,function analysis of hcPcSPI2 |
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| Authors: | Xin-Cang Li Ran-Ran Zhang Rong-Rong Sun Jiang-Feng Lan Xiao-Fan Zhao Jin-Xing Wang |
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| Institution: | 1. School of Life Sciences, Shandong University, Jinan, Shandong 250100, China;2. The Key Laboratory of Plant Cell Engineering and Germplasm Innovation of Ministry of Education, Shandong University, Jinan, Shandong 250100, China;1. Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Salaya, Nakhonpathom 73170, Thailand;2. Shrimp-Virus Interaction Laboratory (ASVI), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Rama VI Rd., Bangkok, Thailand;1. Unidad de Biotecnología 6-UB6, IIB-INTECH, CONICET-UNSAM, Chascomús, Argentina;2. Unidad de Biotecnología 3-UB3, IIB-INTECH, CONICET-UNSAM, Chascomús, Argentina;3. Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET), Universidad Nacional de Rosario, Rosario, Argentina;4. Instituto Nacional de Parasitología “Dr Mario Fatala Chaben”, ANLIS-Malbrán, Ministerio de Salud, Buenos Aires, Argentina;1. Tianjin Key Laboratory of Animal and Plant Resistance, School of Life Sciences, Tianjin Normal University, Tianjin, 300387, PR China;2. Institute of Entomology, College of Life Sciences, Nankai University, Tianjin, 300071, PR China;3. State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin 300071, PR China;1. Council of Scientific and Industrial Research- Unit for Research and Development of Information Products, “Tapovan” NCL Campus, S.No.113, 114, Pashan, Pune 411 008, India;2. Department of Biotechnology, Faculty of Science, Jamia Hamdard, New Delhi 110062, India;1. Center of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Phyathai Road, Bangkok 10330, Thailand;2. Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok 10110, Thailand |
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| Abstract: | Three Kazal-type serine proteinase inhibitors, hcPcSPI2, hpPcSPI3, and hpPcSPI4, with complete cDNA sequences, were identified from a cDNA library of the red swamp crayfish, Procambarus clarkii. Semi-quantitative RT-PCR shows that hcPcSPI2 exists mainly in hemocytes while both hpPcSPI3 and hpPcSPI4 were detected in the hepatopancreas and the heart. Homology comparison and phylogenic analysis indicate that hpPcSPI3 and hpPcSPI4 shared high identity and formed the same group, and both of them were different from other hepatopancreas type inhibitors in crustaceans forming a large group, while hcPcSPI2 as well as other hemocyte type inhibitors belonged to another cluster. In addition, the temporal expression profiles of these three inhibitors were studied with quantitative real-time PCR and the results suggest that hcPcSPI2 and hpPcSPI3 are likely to be involved in antiviral immune response, and all these three inhibitors respond to Vibrio anguillarum challenge in different degrees. Further study was done on hcPcSPI2. 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 Bacillus subtilis and Bacillus thuringiensis, possessing MIC50 of 30.4 and 25.0 μM, respectively. These studies reveal that hcPcSPI2 may also play an important role in the anti-bacterial immunity of the crayfish. |
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