鑑定梨形鞭毛蟲中含表皮生長因子重複的囊體蛋白質

Abstract

梨形鞭毛蟲的生活史中有2個時期：滋養體和囊體。其中囊體必須與外界環境接觸，面對的環境更為險惡，因此囊體的外套蛋白常是許多人研究的目標。目前已知的囊體外套蛋白有2種：cyst wall proteins (CWP1~3)和high cysteine non-variant cyst protein (HCNCp)。CWPs在進行囊體化期間被大量表現，在進行囊體化的滋養體中CWPs位於ESVs，在囊體中則只位於囊壁上。HCNCp為最近發表過的蛋白質，它的胺基酸序列類似滋養體時期的一種外套蛋白：變異性特化表面蛋白(variant-specific surface proteins, VSPs)，但HCNCp的mRNA和蛋白質表現量在進行囊體化期間被大量表現，類似CWPs。除此之外，HCNCp在進行囊體化的滋養體中也位於ESVs，但在囊體中它不只在囊壁，更存在於細胞本體，與CWPs不同。此研究中我們利用隱孢子蟲的卵囊壁蛋白質(Cryptosporidium parvum oocyst wall protein, COWP)第一型區域在梨形鞭毛蟲基因組資料庫中搜尋，發現一個相似的蛋白質，以此蛋白質的胺基酸序列去比對美國國家生物科技資訊中心(National Center for Biotechnology Information, NCBI)所提供的資料庫後，則顯示它是一個相似tenascin的蛋白質，且大部分相似區域集中在EGF-like repeats，因此命名為Epidermal-Growth-Factor repeat-containing Cyst Protein 1 (EGFCP1)。序列分析的結果我們發現EGFCP1與HCNCp蛋白質具有一些共同的特性，像是具有較大的分子量、酸性pI值、富含半胱胺酸等。Tenascin是一個細胞外基質醣蛋白，特性是具有許多表皮生長因子相似重複(epidermal growth factor-like repeats, EGF-like repeats)，EGF-like repeats常可在許多細胞外蛋白質或是與膜結合蛋白的細胞外區域中發現。利用SMART軟體分析後發現EGFCP1具有9個EGF-like repeats，顯示EGFCP1可能是一個存在於細胞外的蛋白質或是與膜結合的蛋白。我們做實驗發現了EGFCP1的蛋白質與CWP1相似，表現量都會在進行囊體化時期提高且利用分子內雙硫鍵形成巨大複合物。但EGFCP1的mRNA表現量卻相反，進行囊體化時期會下降。而螢光免疫染色法顯示EGFCP1在囊體的位置是在囊壁和細胞本體，與HCNCp相似，因此我們推測EGFCP1與HCNCp兩者可能有相似的運輸途徑。我們也利用分泌分析發現到EGFCP1可被分泌到細胞外，推測EGFCP1中的EGF-like repeats具有功能。我們把EGFCP1其N端的信號訊列(signal peptide)刪除，發現EGFCP1蛋白質和mRNA表現量明顯的減少；之後我們以囊體計數的實驗更發現了刪除信號序列的EGFCP1細胞株的囊體數目減少，因此我們推測EGFCP1的蛋白質表現量降低可能影響囊體的生成。進一步地我們以螢光酵素分析法顯示EGFCP1的信號序列可能是一個潛在的Internal Ribosome Entry Site (IRES)，可以增強轉譯作用。我們又利用EGFCP1的胺基酸序列搜尋梨形鞭毛蟲基因組資料庫，發現還有其他5個蛋白質在序列或特性都相似於EGFCP1，經由螢光免疫染色法和西方墨點法發現它們的蛋白質表現特性和位於細胞內的位置都與EGFCP1相似，且經由囊體計數實驗也發現這5個蛋白質大量表現後囊體形成能力都有增加，因此我們認為它們是一個新的家族。此研究中我們發現了梨形鞭毛蟲中新的囊體蛋白質家族，並發現它們與囊壁蛋白具有一些相似特性，像是蛋白質表現量在囊體化時期提高和在細胞中的位置在囊壁和細胞本體，更進一步地觀察到EGFCP1可能促進囊體的生成，最後發現EGFCP1基因可能具有潛在的IRES可幫助轉譯。

There are two stages in Giardia lamblia life cycle: trophozoite stage and cyst stage. The cyst stage is protected by a special extracellular matrix named cyst wall, which can protect Giardia from hostile environment. Despite its importance, the composition, architecture, and formation of the cyst wall are poorly understood. Only three structural cyst wall proteins (CWPs) and a high cysteine non-variant cyst protein (HCNCp) have been identified. CWPs are upregulated during encystation and are exclusively transported to the cyst wall through novel regulated encystation secretory vesicles (ESVs). The amino acid sequence of HCNCp is similar to that of variant-specific surface proteins (VSPs), which are the coat proteins of trophozoites. But the expression levels of RNA and protein of HCNCp are upregulated during encystation, similar to those of CWPs. Besides, HCNCp is transported by ESVs, but it localizes not only to cyst wall but also to cell body of cysts. We have identified a novel protein named Epidermal-Growth-Factor repeat-containing Cyst Protein 1 (EGFCP1) by in silico screening of Giardia lamblia genome database using the type I domain of the Cryptosporidium parvum oocyst wall protein (COWP) as a query sequence. Sequence analysis revealed that the EGFCP1 is similar to the epidermal growth factor repeats (EGF-repeats) of tenascin family of glycoprotein that are extracellular matrix proteins highly expressed in tumors. Interestingly, EGFCP1 shares some common features with the previously reported Giardia HCNCp, such as high cysteine content (more than 10%), acidic pI, and higher molecular weight than CWPs. We used SMART program to analyze EGFCP1, and found that it has 9 EGF-like repeats, suggesting that EGFCP1 may be an extracellular protein or a membrane-bound protein. We found that EGFCP1 was highly expressed during encystation and formed similar polydisperse disulfide-bonded complexes detected in non-reducing gels, which matches the expression profiles of the CWPs. In contrast with the higher RNA levels of CWPs, the RNA levels are reduced during encystation. The immunofluorescence data showed that the EGFCP1 was localized to the cyst wall and cell body of cysts, which is similar to HCNCp. Therefore, it is suggested that EGFCP1 and HCNCp may share a similar transport pathway. Using secretion assays, we found that EGFCP1 can be released to medium, suggesting that the EGF-like repeats in EGFCP1 may be functional. When we deleted N-terminal signal peptide of EGFCP1, the protein and RNA expression level of EGFCP1 reduced obviously. In cyst count experiment, we found that the cyst number in deletion strain is less than the strain overexpressing the wild type EGFCP1, suggesting that the expression of EGFCP1 may affect the cyst formation. Furthermore, using luciferase assays, we found that the sequence of the signal peptide of EGFCP1 can enhance translation, suggesting that it may be a potential Internal Ribosome Entry Site (IRES). Finally, we BLASTed the Giardia genome database with amino acid sequence of EGFCP1 and found 5 other open reading frames with high similarity to EGFCP1 in sequence and characteristics. Interestingly, the expression levels of these five open reading frames are similar to those of EGFCP1, and all these overexpression strains can stimulate the ability of cyst formation. Therefore, we suggest that they belong to a new family of cyst proteins in G. lamblia. The results may lead to greater understanding of parasite cyst walls.