無脊椎動物有一種以上的TG?草蝦第二型轉麩醯胺酵素參與凝血反應

Abstract

凝血反應的快捷，可使生物有效防止受傷或感染後體液流失及微生物侵入體腔，此機制對只具有先天性免疫反應的無脊椎動物尤為重要。凝血系統的組成與活化在不同種的無脊椎動物中差異頗大，然而轉麩醯胺酵素 (transglutaminase, TG) (EC 2.3.2.13)卻是其中唯一保守的成員。TG催化形成分子間或分子內的ε-(γ-glutamyl) lysine 鍵結，使蛋白質聚合，其已知的生理功能則包括凝血和蛋白質轉譯後的修飾。我們由草蝦血球cDNA中選殖出一段TG序列，命名為草蝦第二型轉麩醯胺酵素（Shrimp Transglutaminase II, STG II），譯碼區含2,274個核苷酸，轉譯出的蛋白質含757個胺基酸，預測分子量約85,000 Da，等電點5.48。RT-PCR結果顯示STG II主要在血球而非肝胰臟表現，組織表現型式與STG I不相同。Neighbor Joining分析指出草蝦STG II和STG I之間的遺傳距離遠大於草蝦STG II和斑節蝦TG的距離，暗示草蝦的兩個TG可能位於染色體上不同的基因座。我們利用昆蟲-桿狀病毒表現系統得到重組STG II。穿透式電顯觀察發現重組的STG II能夠聚合凝血蛋白(clottable protein, CP, 萃取自草蝦血漿)形成一長鏈，型態相似於草蝦血球萃取液作用於血漿的凝血產物。本研究顯示草蝦的第二型轉麩醯胺酵素屬於血球型TG，參與凝血反應。

Coagulation (clot formation) can form a physical barrier to prevent the loss of body fluid and dissemination of microbes into the hemocoel after injury or infection. Its quickness and efficiency is essential for survival of invertebrates that relay solely on the innate immunity. Though the components and activation of coagulation system is diversified in the invertebrates, transglutaminase (TG) (EC 2.3.2.13) is the only conserved member throughout the known species. TG catalyzes intermolecular or intramolecular ε-(γ-glutamyl) lysine bond formation, resulting in protein polymerization. Physiologically, they are known for the roles in blood coagulation and post-translational protein modification. In the present study, we cloned a TG from shrimp (Penaeus monodon) hemocyte cDNA. It was assigned shrimp transglutaminase II (STG II). The TG cDNA consists of a coding region of 2,274 bp. The deduced protein has 757 amino acid residues, a calculated molecular mass of 85,000 Da and an isoelectric point of 5.48. RT-PCR results showed a high level of STG II expression in hemocytes, but not in hepatopancreas, differently from STG I. Neighbor Joining analysis indicates the genetic distance between STG II and STG I is much larger than that between STG II and TG of kuruma shrimp (Marsupenaeus japonicus). These evidences suggest at least two types of shrimp TG encoded from different chromosomal loci. The recombinant STG II was expressed using insect-baculovirus expression system. It showed polymerization activity on clottable proteins extracted from shrimp plasma. Under TEM observation, the polymerized chain resembles structurally the reacting products from shrimp hemocyte lysate supernatants on their plasma. Conclusively, STG II is characterized to be a hemocytic TG and involved in coagulation.