Scallop Serine Protease Inhibitor Gene Cloning And Expression

Scallop culture is an important marine aquaculture industry in China. But since 1994, scallop aquaculture in China has been experiencing a continual large-scale mortality, which not only caused a great economic loss but also threatened the existence and development of the culture industry. Although it is still unclear about the causes for the scallop mortality, deterioration water quality and deterioration of the stock are suspected to be the most important causes. With the improvement and control of the marine environment, enhancing the disease resistance of scallop is commonly believed to be the resolution to the control of the disease. The cloning and expression of the genes involved in immune defense are now considered to be a basic solution in the disease control because of their potential use in the development of therapeutic agents, study of immune defense mechanism and genetic improvement to increase the resistance to disease. Serine proteases and their inhibitors play a central role in the invertebrate immune response. They are mainly involved in signaling and amplification cascades that lead to the activation of specific defense mechanisms, such as melanization, coagulation and induction of antimicrobial peptides. In the present study, large scale EST sequencing method together with RACE technique was used to isolate and clone the serine proteases and serine proteases inhibitors involved in the immune defence from scallop cDNA libraries. Nine full length cDNA sequences of immune-related serine proteases and serine protease inhibitors were obtained and the tissue distribution and the temporal expression of these genes after injury and microbe challenge were measured by Northern blotting and RT-PCR. The full length of serine protease genes CFPS1 and CFPS2 from scallop Chlamys farreri was 1211 and 1152-bp encoded 354 and 336 amino acids, respectively. CFPS1 and CFPS2 are believed to be the secreted protein. They have the typical signal peptide at the N-terminus and the resulting mature CFPS1 and CFPS2 lack transmembrane domains and retention signals. The deduced amino-acid sequences of CFPS1 and CFPS2 consist of two parts, a regulatory amino-terminal clip domain and a catalytic serine proteinase domain at the carboxyl terminus. A linking sequence connects the two domains. Clip domains are 37 amino acid residue sequences which are knitted together by three disulfide bonds and thus are expected to form a quite compact structure. The CFPS1 and CFPS2 are synthesized as zymogens and are activated by a specific proteolytic cleavage, in a manner similar to the activation of trypsinogen or chymotrypsinogen. The activation site is between the clip domain and the catalytic domain. An additional pair of cysteine residues links the two domains such that when the CFPS1 and CFPS2 are activated, the catalytic heavy chain remains covalently attached by a disulfide bond to the light chain, which contains the clip domain. Northern blotting analyses using CFPS1 and CFPS2 probe confirmed the presence of approximately 1.3-kb transcript for C. farreri that were most strongly expressed within collected hemocytes, respectively. The temporal expression of CFPS1 and CFPS2 were measured by RT-PCR after injury or microbe challenge, respectively. After the adductor was wounded or injected with Vibrio Anguillarum, the mRNA expression of CFPS1 in hemolymph was up-regulated and reached the maximum level at 16 h, respectively. The mRNA expression of CFPS2 in hemolymph also was up-regulated and reached the maximum level at 16 h after injected with V. Anguillarum, but there was no significant difference in CFPS2 gene expression among the control, blank, and challenged samples after M. luteus and P. pastoris challenge, respectively. These results indicated that CFPS1 and CFPS2 could play an important role in injury healing and immune response in mollusks as it could be induced by injury and microbe challenge. The full length of serine protease inhibitor genes CFPSI-1, CFPSI-2, CFPSI-3, CFPSI-4 from scallop C. farreri and AISPI-1, AISPI-2, AISPI-3 from the bay scallop Argopecten irradians was 1841, 1358, 1187 ,1064, 1020, 897 and 642-bp, encoded 508, 347, 364 323, 278, 238 and 153 amino acids, respectively. They are believed to be the secreted protein and have the typical signal peptide at the N-terminus. The deduced amino-acid sequence of CFPSI-1, CFPSI-2, CFPSI-3, CFPSI-4, AISPI-1, AISPI-2 and AISPI-3 contained 12, 8, 8, 7, 6, 5 and 3 tandem and homologous domains similar to that of kazal-type serine protease inhibitors, respectively, including the conserved sequenceC-X(7)-C-X(6)-Y-X(3)-C-X(2,3)-C and 6 cysteine residues responsible for the formation of disulfide bridges, indicating that the CFPSI-1, CFPSI-2, CFPSI-3 and CFPSI-4 protein from scallop C. farreri and AISPI-1, AISPI-2, AISPI-3 from the bay scallop A. irradians should be members of Kazal-type serine protease inhibitor family. Northern blotting analyses using CFPSI-1, CFPSI-2, CFPSI-3, CFPSI-4, AISPI-1,AISPI-2 and AISPI-3 probe confirmed the presence of approximately 1.9, 1.4 1.3 and 1.2-kb transcript for C. farreri and 1.1, 1.0 and 0.7-kb transcript for A. irradians that were most strongly expressed within collected hemocytes, respectively. The temporal expression of CFPSI-1, CFPSI-2, CFPSI-3, CFPSI-4, AISPI-1,AISPI-2 and AISPI-3 was measured by RT-PCR after injury or microbe challenge, respectively. After the adductor was wounded or injected with V. Anguillarum, the mRNA expression of CFPSI-1, CFPSI-2 in hemolymph was down-regulated, respectively. But the mRNA expression of CFPSI-3 and CFPSI-4 in hemolymph was up-regulated and reached the maximum level at 8 h after injected with V. Anguillarum and M. luteus. There was no significant difference in CFPSI-3 and CFPSI-4 gene expression among the control, blank, and challenged samples after P. pastoris challenge. After the adductor was wounded or injected with V. Anguillarum, the mRNA expression of AISPI-1 in hemolymph was up-regulated and reached the maximum level at 8 and 16 h, respectively, and then progressively dropped back to the original level. The mRNA expression of AISPI-2 and AISPI-3 in hemolymph was also up-regulated and reached the maximum level at 4, 6 and 2 h after injected with V. Anguillarum, M. luteus and P. pastoris. These results indicated that CFPSI-1, CFPSI-2, CFPSI-3, CFPSI-4, AISPI-1,AISPI-2 and AISPI-3 could play an important role in injury healing and immune response in mollusks as it could be induced or inhibited by injury and microbe challenge.