草蝦含迴紋針功能區塊絲胺酸蛋白酶類似物之選殖及定性

Abstract

含迴紋針功能區塊絲胺酸蛋白酶類似物（c-SPH）與節肢動物的多項先天性免疫反應有關；像是抗菌活性、細胞附著、病原模式辨認、調理作用、以及對原酚氧化酵素系統的調控。在本篇論文中，我們從草蝦血球中選殖出一段c-SPH的cDNA。這段cDNA全長1337 bp，包含一段1068 bp的載碼區，可轉譯出一段長355 個胺基酸的蛋白質；推繹出的蛋白質包含一段迴紋針功能區塊（clip domain）及一段不具催化活性的類絲胺酸蛋白酶功能區塊（SP-like domain）。此蛋白質的分子量推測為38 kDa、等電點推測為7.9。預測的signal peptide切位位於Gly21Gln22之間。我們從12種節肢動物中挑選出15條單一clip domain的SPHs進行多重序列比對分析；clip domains間的一致性低，而SP-like domains間的一致性大約在34%-46%。序列上較保守的區域位於一些特殊的胺基酸附近，這些胺基酸在有酵素活性的絲胺酸蛋白酶中負責與受質進行交互作用。親緣分析上，草蝦c-SPH與同樣是十足目中淡水螯蝦的低分子量mas-like protein最為相似，而比較不像鋏角亞門或是昆蟲綱的c-SPHs。巢狀反轉錄聚合酶連鎖反應顯示c-SPH mRNA的訊號在血球中最為強烈。該分子的抗菌活性、調理活性以及對原酚氧化酵素系統的調控則沒有被偵測到。草蝦c-SPH mRNA的表現量在葡聚醣浸泡後12天表現量上升，而在熱去活性弧菌浸泡後半小時表現量下降。重組c-SPH能夠顯著增強血球細胞的附著。這些結果顯示草蝦c-SPH參與了先天性的免疫反應。

Abstract Clip domain serine protease homologs (c-SPHs) are involved in various innate immune functions in arthropods such as antimicrobial activity, cell adhesion, pattern recognition, opsonization, and regulation of the prophenoloxidase system. In the present study, we cloned a c-SPH cDNA from tiger shrimp (Penaeus monodon) hemocytes. It is 1337 bp in length with a coding region of 1068 bp consisting a protein of 355 amino acid residues. The deduced protein includes one clip domain and one catalytically inactive serine protease-like (SP-like) domain. Its molecular weight is estimated to be 38 kDa with an isoelectric point of 7.9. The predicted cutting site of the signal peptide is located between Gly21 and Gln22. We aligned 15 single clip domain SPH protein sequences from 12 arthropod species; the similarity of these clip domains is low and that of SP-like domains is from 34%-46%. The conserved regions are located near the amino acid residues which served as substrate interaction sites in catalytically active serine protease. Phylogenetically, the tiger shrimp c-SPH is most similar to a low molecular mass masquerade-like protein of crayfish, also a member of Decapoda, but less similar to c-SPHs in Chelicerata and Insecta. Nested reverse transcription polymerase chain reaction (RT-PCR) revealed that c-SPH mRNA is expressed most in tissues with the highest hemocyte abundance. Antimicrobial, opsonization and proPO regulation of the molecule were not detected. The expression of c-SPH mRNA in hemocytes was up-regulated at the 12 day post β-glucan immersion and down-regulated at 0.5 hr post heat-inactivated Vibrio immersion. Recombinant c-SPH could significantly enhance hemocyte adhesion. The results suggest that the shrimp c-SPH protein plays a role in innate immunity.