梨形鞭毛蟲Cdk1之鑑定

Abstract

梨形鞭毛蟲 (Giardia lamblia)是種具有鞭毛的原蟲類寄生蟲，呈世界性分佈。會寄生在人類腸道造成Giardiasis，主要病徵為腹瀉及營養吸收不良。梨形鞭毛蟲生活史中有兩個型態，分別是對環境有高度耐受性的感染型囊體 (cyst)時期與具有鞭毛寄生於小腸的滋養體 (trophozoite)時期。囊體化過程中，囊壁蛋白會大量表現促進囊壁形成，目前已知有三種囊壁蛋白，CWP1-3 (cyst wall protein 1-3)。此外轉錄因子Myb2與E2F1會結合到囊壁蛋白之啟動子促進囊壁蛋白基因表現。而囊體化過程伴隨著細胞週期變化，因此本研究想探討細胞週期中之關鍵蛋白Cyclin-dependent kinase (Cdk)是否參與在囊體化過程中。 我們在梨形鞭毛蟲中找到一個相近於人類Cdk1胺基酸序列的Cdk，經由胺基酸序列分析後發現其具有可能結合cyclin的PSTAIRE-like motif，PGTAIRE，故將此蛋白命名為梨形鞭毛蟲的Cdk1。實驗結果顯示Cdk1基因之mRNA與蛋白質會在囊體化時期大量表現，已知促進囊體化過程之囊壁蛋白、轉錄因子Myb2與E2F1的蛋白質表現量會在囊體化時期大量表現，與Cdk1表現上升吻合。我們發現大量表現Cdk1之細胞株的CWP1與CWP3蛋白質表現量及囊體化相關基因之RNA表現量和囊體形成能力皆較控制組細胞強。透過免疫沉澱激酶分析，發現Cdk1可能是藉由磷酸化轉錄因子Myb2和E2F1來促進囊體化過程。 為了釐清Cdk1之功能，故將可能與cyclin結合之胺基酸序列PGTAIRE突變，命名為pPCdk1-m1。將可能與ATP結合之第159個位置上的天門冬氨酸殘基突變，命名為pPCdk1-m2。及將胺基酸序列第56至187個胺基酸去除 (deletion)來破壞kinase domain，命名為pPCdk1-m3。結果顯示，突變細胞株會降低CWP1與CWP3蛋白質表現量、囊體化相關基因mRNA表現量，且囊體形成能力較正常Cdk1差，免疫沉澱激酶分析也顯示突變重要胺基酸殘基會影響Cdk1磷酸化的能力。總結實驗結果，證實Cdk1參與囊體化過程，並扮演正向調控之角色。

The intestinal protozoan Giardia lamblia is a water-borne flagellated parasite. It causes non-bacterial diarrheal disease, also known as giardiasis, throughout the world. The life cycle stages of G.lamblia is composed of cyst and trophozoite. Cysts are the infected form and resistant to environment, trophozoites are the active form which colonizes in the small intestine. Encystation, a process of cell differentiation from trophozoites to cysts, is necessary for transmission between hosts. During encystation, transcription factors Myb2 and E2F1 up-regulate genes encoding cyst wall proteins (cwps).The initiation of encystation is associated with cell cycle progression, so we attempt to identify the role of cyclin-dependent kinases (Cdks), critical cell cycle regulators, in encystation. We have found a putative cdk gene in G. lamblia database. Amino acid sequence analysis showed the Cdk homologue is similar to human Cdk1 and it contains a PSTAIRE-like motif, PGTAIRE, so we named it Giardia Cdk1. We found that mRNA and protein levels of Cdk1 were increased during encystation. Overexpression of Cdk1 resulted in increasing ability of cyst formation. Moreover, using immunoprecipitation- kinase assay, we also found that Cdk1 was able to phosphorylate Myb2 and E2F1 proteins. We constructed a set of mutants including mutation in PGTAIRE motif that may be involved in cyclin-binding which named Cdk1-m1, the aspartic acid at residue 159 that may be involved in ATP-binding and deletion of a large segment of kinase domain which named Cdk1-m2 and Cdk1-m3, respectively. Compared to wild type Cdk1, mutations of these important regions of Cdk1 leaded to decreased the levels of cwp genes expression, kinase activity, and cyst formation. The results suggest that Cdk1 is a positive regulator in encystation of G. lamblia.