梨形鞭毛蟲B型細胞週期素蛋白質之鑑定

Abstract

摘要 梨形鞭毛蟲(Giardia lamblia)是世界上造成腹瀉症狀的一種常見原蟲類寄生蟲。在其生活史中，囊體化對於形成感染型的囊體(cyst)十分重要。囊體在結構上具有一層囊壁，能幫助梨形鞭毛蟲抵抗外界的逆境，目前已知囊壁主要由囊壁蛋白質(cyst wall protein,CWP)以及醣類所共同組成。在囊體形成的過程，梨形鞭毛蟲的染色體套數有明顯的增加且有細胞核分裂的現象發生，顯示在囊體化過程可能有類似細胞週期調控的機制。我們由梨形鞭毛蟲的基因庫中的16個細胞週期素相似基因中發現一個編號為GL50803_3977的基因，分析結果顯示此基因類似Cyclin B，因此我們將GL50803_3977蛋白質命名為gCyclin B1。 首先，透過螢光酵素活性測試我們發現gCyclin B1基因的轉錄作用受到梨形鞭毛蟲WRKY蛋白質的調控。RT-PCR及real-time PCR分析梨形鞭毛蟲內生性gcyclin B1基因表現量的結果顯示gcyclin B1基因在囊體化時期的RNA表現量明顯高於滋養體時期。利用人工製備帶有HA tag的gCyclin B1重組蛋白質轉染(transfect)到梨形鞭毛蟲，以免疫螢光染色偵測gCyclin B1蛋白質的表現位置，發現在滋養體(trophozoite)時期與囊體化時期主要表現在細胞質，但也有少量出現在細胞核；西方墨點法偵測結果顯示相比於控制組，gCyclin B1與CWP1蛋白質表現量有明顯提高；RT-PCR及real-time PCR分析顯示gcyclin B1基因大量表現能促進cwp1、cwp2、cwp3及gmyb2基因表現量上升。透過囊體計數方式發現大量表現gCyclin B1蛋白質能夠促進囊體形成數量上升；利用免疫沉澱激酶分析確認大量表現gCyclin B1蛋白質能夠促進cyclin-depedent kinases (CDKs) 磷酸化gMyb2蛋白質。欲瞭解gCyclin B1的功能以及其與囊體化之關係，我們製作了三個突變株: 由於Cyclin B1上的E70是與CDK1的端葉結合所需要的，我們進行E70A的點突變，命名為gCyclin B1m1；由於Cyclin B1上的P box domain是與CDK1一同作用來磷酸化受質所需要的，我們將P box domain上進行R91G、L94A、W97A的三個點突變，命名為gCyclin B1m2；由於Cyclin B1上的K146是與CDK1的PSTAIRE螺旋結合需要的，我們進行K146A的點突變，命名為gCyclin B1m3。gCyclin B1m1、gCyclin B1m2 及gCyclin B1m3主要表現位置也是在細胞質，且相較於正常的gCyclin B1，三個突變株誘導囊壁蛋白質CWP1、CWP3表現以及促使cwp1、cwp2、cwp3與gmyb2基因表現的能力下降，而且使梨形鞭毛蟲形成囊體的數目減少，還有無法提高CDKs磷酸化gMyb2蛋白質的能力。以上實驗結果推論：在囊體化過程中，gCyclin B1蛋白質與CDKs結合後，磷酸化gMyb2蛋白質而使gMyb2蛋白質活性提高並進一步調控cwp1、cwp2與cwp3基因的表現量上升。

Abstract Giardia lamblia is a common protozoan parasite which causes diarrhea. In its life cycle, encystation is an important behavior to propagate to the outside. Cysts with a heavy layer, called cyst wall, can help to resist external adversities. The component of cyst wall are known as cyst wall proteins (CWPs) and carbohydrates. During the process of the formation of cysts, the chromosome copy number of G. lamblia is increased significantly and the phenomenon of nuclear fission is occurred. These show that encystation may have a similar regulation mechanism of cell cycle. There are 16 cyclin-like genes found in G. lamblia database. Analysis of GL50803_3977 gene suggests that this gene is similar to mammalian Cyclin B, so we name it as gCyclin B1. By using the luciferase activity test, we found that the transcription of gcyclin B1 gene is regulated by WRKY protein. RT-PCR and real-time PCR data showed that the gene expression of endogenous gcyclin B1 rise in encystation stage. The artificial vectors of gCyclin B1 with HA tag were transfected into G. lamblia. By immunofluorescence assays, gCyclin B1 proteins were mainly localized in cytoplasm in both trophozoite and encystation stage, but some were expressed in nuclei, too. Relative to the levels in control, the levels of gCyclin B1 and CWP1 proteins increased significantly in the gCyclin B1 stable transfectants. RT-PCR and real-time PCR data showed that the expression of cwp1, cwp2, cwp3 and gmyb2 gene was induced by the overexpression of gCyclin B1. gCyclin B1 proteins could promote the cyst formation of G. lamblia by cyst counting method. The immunoprecipitation kinase analysis confirmed that the overexpression of gCyclin B1 protein could induce the activity of CDKs to phosphorylate the gMyb2 protein. In order to understand the function of gCyclin B1 and the relationship between gCyclin B1 and encystation, we made three mutant clones: Residue E70 of Cyclin B1 is important for interaction of the C-terminal lobe of CDK1. We made a gCyclin B1m1 construct with a E70A mutation. Residues R91, L94 and W97 of Cyclin B1 are important for phosphorylation of substrates, so we made a gCyclin B1m2 construct with R91G、L94A、W97A mutations. Residue K146 of Cyclin B1 is important for interaction of the PSTAIRE helix on CDK1, so we made a gCyclin B1m3 construct with a K146A mutation. gCyclin B1m1, gCyclin B1m2 and gCyclin B1m3 were localized in cytoplasm. Compared to wild type gCyclin B1, the levels of the cwp1, cwp2, cwp3 and gmyb2 mRNA and CWP1 and CWP3 protein in these mutant cell lines were decreased significantly. The mutant clones also had lower ability to promote the cyst formation of G. lamblia and to induce the activity of CDKs. According to the above results, we propose a model: In the process of encystation, high level of gCyclin B1/CDKs complexes phosphorylate gMyb2 protein and induce its activity. gMyb2 protein then helps the expression of cwp genes. Our results suggest that gCyclin B1 protein plays an important role in G. lamblia differentiation into cysts.