草蝦第一型轉醯麩胺酵素之分子選殖與功能分析

轉麩醯胺酵素 (transglutaminase, TG) 在血液凝集及蛋白質轉譯後的修飾作用與再建構過程中擔負有很重要的功能。然而在無脊椎動物中，本酵素的相關研究仍相當缺乏。我們自草蝦血球cDNA庫中選殖出一段轉麩醯胺酵素，並命名為STG I，以進一步瞭解本酵素在草蝦血淋巴凝集過程中所擔負的角色與其他可能的功能。草蝦轉麩醯胺酵素STG I之序列含有2271個鹼基對，預測蛋白質序列含有757個胺基酸殘基，估計分子量為84713 Da，等電點為5.56。STG I的胺基酸序列的N-端並無典型的疏水性引導序列或穿膜功能區間的存在，因此其應為一典型的胞漿蛋白。STG I具有1個Arg-Gly-Asp (RGD) motif。草蝦STG I的序列與螯蝦、其他無脊椎與脊椎動物的TG類似。所有參與催化反應或鈣離子鍵結的胺基酸殘基以及整段胺基酸序列在STG I與其他TG成員間都呈現出高度的保守性。RT-PCR與原位雜合分析顯示STG I 之mRNA僅在造血組織中具有高度的表現量，SDS-PAGE與西方點墨法顯示STG I之蛋白質在循環血球中大量存在。重組STG I (rSTG I) 在鈣離子的存在下才具有轉醯胺酵素活性。rSTG I能夠以凝集蛋白 (clottable protein) 做為受質，但無法產生凝集反應。到目前為止我們尚未能在草蝦中找出凝集蛋白以外其他可能做為STG I受質的蛋白質。rSTG I可以促進草蝦血球細胞的附著，隨著rSTG I披覆濃度的增加，草蝦血球的附著量顯著逐漸增加，其增加草蝦血球附著的能力具有劑量依存效應。rSTG I促進草蝦血球附著的能力是經由RGD motif來行使的。然而，當rSTG I濃度大於60 nM時，附著的草蝦血球出現高度延展或壞死的型態，trypan blue染色的結果顯示這些細胞通常是死亡的。rSTG I促進草蝦血球細胞附著的能力在生理上的重要性與其調控方式需要進一步的研究來釐清。

關鍵字

草蝦；轉麩醯胺酵素；凝集反應；細胞附著

並列摘要

Transglutaminase (TG) is important for blood coagulation and post-translation remodeling of proteins. For characterization of the biological function of TG in shrimp, we isolated a cDNA encoding a novel TG from a shrimp (Penaeus monodon) hemocyte cDNA library. The STG I cDNA consists of an open reading frame of 2271 bp. The deduced protein has 757 amino acid residues, a calculated molecular mass of 84,713 Da and an isoelectric point of 5.56. Neither a typical hydrophobic leader sequence nor a transmembrane domain could be identified from the deduced sequence. Thus, STG I may be a typical cytoplasmic protein. One Arg-Gly-Asp (RGD) motif was identified in STG I. The sequence of STG I was similar to crayfish, other invertebrate and vertebrate TG sequences. Residues critical for enzyme function and calcium binding are conserved between STG I with other members of TG family. High level of STG I mRNA expression was only detected in hematopoietic tissue and high level of protein was existed in circulating hemocytes. Recombinant STG I (rSTG I) exhibited the transamidation activity. The transamidation activity is calcium dependent. rSTG I used clottable protein (CP) as the substrae but lacked coagulation activity. We failed to find out other natural substrates of STG I from tiger shrimp. In vitro, rSTG1 increased hemocyte attachment and spreading dose-dependently. Adhesion of shrimp hemocytes on rSTG I is RGD-dependent. However, when concentration of rSTG I was higher than 60 nM, hemocytes showed highly spread or necrotic morphology. Trypan blue staining showed that the stimulated cells seem to be dead. Further researches are needed to establish the physiological importance and regulatory mechanism of STG I function

並列關鍵字

Tiger shrimp ； transglutaminase ； clotting ； cell adhesion

參考文獻

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草蝦第一型轉醯麩胺酵素之分子選殖與功能分析

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