梨形鞭毛蟲Pax同源蛋白質中的gPax2 對於cyst wall protein 1和2基因的轉錄調控之影響

Abstract

Pax蛋白質家族是一個很大的轉錄因子群，普遍存在於脊椎動物中，主要參與胚胎發育和器官生成。目前的研究中，在原蟲類中並未發現有類似pax基因存在；而我們在梨形鞭毛蟲資料庫中搜尋到兩個類似Pax的蛋白質，gPax1和gPax2。不同於脊椎動物Pax蛋白質家族之paired DNA-binding domain位於N端，gPax1和gPax2的paired domain位於C端。 我們發現內生性gpax2 RNA表現量在梨形鞭毛蟲囊體化24小時時期表現會較滋養體時期低。我們製作可大量表現gpax2的質體，並在其C端加有HA-tag，轉染至wild type梨形鞭毛蟲中。我們利用HA抗體作西方墨點法得知HA tagged gPax2在滋養體時期和囊體化24小時時期之蛋白質表現量差不多。其次，我們經由免疫螢光染色觀察到HA tagged gPax2在梨形鞭毛蟲滋養體時期和囊體化24小時時期皆表現於細胞核中。我們利用電泳遷移率分析發現gPax2會專一性結合於梨形鞭毛蟲兩種生活型態轉換的指標性基因-cyst wall protein 1 (cwp1)和cwp2基因之啟動子，這兩個基因會在囊體化時大量表現，皆為組成囊壁的成份之一。我們製作一個突變體，將其C端的paired domain去除，gPax2del，發現gPax2del即失去和cwp1和cwp2基因啟動子結合的能力，而且其在細胞核表現量有變少，散佈在細胞質的vesicles中。大量表現gPax2的細胞株，相較於vector control的細胞株，cwp1和cwp2基因表現的mRNA和Cwp1蛋白質的量增加。相較於大量表現gPax2細胞株，大量表現gPax2del的細胞株則會使cwp1和cwp2基因的mRNA表現量減少，Cwp1蛋白質也觀察到有減少。我們認為gPax2可能參與cwp1和cwp2基因的轉錄調控，且其paired domain也會影響cwp1和cwp2基因表現。 另外我們在paired domain中找到兩段疑似nuclear localization signal (NLS)片段，分別將其序列上的正價性胺基酸作突變體，gPax2m1和gPax2m2，大量表gPax2m1和gPax2m2的細胞株中，皆發現gPax2m1和gPax2m2在細胞核的表現量有變少，而且都明顯表現在梨形鞭毛蟲吸盤周圍，我們認為此二段區域可能是gPax2的NLS。和大量表現gPax2細胞株相比，大量表現gPax2m1的細胞株則會使cwp1和cwp2基因的RNA表現量減少，Cwp1蛋白質也觀察到有減少；而大量表現gPax2m2的細胞株則會使cwp1和cwp2基因的RNA表現量增加，Cwp1蛋白質也觀察到有增加。我們認為gPax2的paired domain中存有NLS，並且也會影響cwp1和cwp2基因的表現。 我們推論，gPax2會促進cwp1和cwp2基因的表現，而且其C端的paired domain可能參與該調控。

Pax protein family is a large group of transcription factors in vertebrates. They are mainly involved in embryogenesis and organogenesis. To date, the pax gene has still not been found in protozoa. We have found two Pax-like open reading frames, gPax1 and gPax2, in Giardia lamblia data base. Unlike Pax proteins from vertebrates which have a paired DNA-binding domain near the N terminus, the paired domains of gPax1 and gPax2 were located near the C terminus. We found that the mRNA expression levels of endogenous gpax2 gene during 24 hours encystation were lower than that during vegetative growth in G. lamblia. We transfected a construct that expressed HA-tagged gpax2 into wild type G. lamblia. Western blot showed that HA-tagged gPax2 was expressed at similar levels during vegetative growth and during 24 hr encystation stage in G. lamblia. Immunofluorescence assay showed that the HA-tagged gPax2 was located in nuclei during vegetative growth and 24 hr encystation in G. lamblia. By electrophoretic mobility shift assays, we found that gPax2 specifically bound to the promoters of cyst wall protein 1 (cwp1) and cwp2 which are important genes during stages-changing in G. lamblia. Cwp1 and Cwp2 are two components of cyst wall. The expression of the cwp1and cwp2 mRNA was induced during encystation in G. lamblia. We made a mutant, gPax2del, which lacks the C-terminal paired domain. We found that gPax2del could not bind to the cwp1 and cwp2 promoters. gPax2del was expressed in some vesicles in cytosol, and gPax2del proteins were hardly detected in nuclei. We found that the levels of the cwp1 and cwp2 mRNA and Cwp1 protein in the gPax2 overexpressing cell line increased significantly relative to the levels in the vector control cell line. In addition, we found that the levels of the cwp1 and cwp2 mRNA and Cwp1 protein in the gPax2del overexpressing cell line decreased significantly relative to the levels in the gPax2 overexpressing cell line. We suggest that gPax2 might be involved in the transcriptional regulation of the cwp1 and cwp2 genes, and the paired domain would contribute partially to this regulation. We found two stretches of basic amino acids that may be nuclear localization signals (NLS)-like sequences in the paired domain of gPax2. We made two mutants, gPax2m1 and gPax2m2, by mutating the positive charge amino acids to neutral amino acids. We found that gPax2m1 and gPax2m2 were located in cytosol, and around the sucking disk, but gPax2m1 and gPax2m2 were hardly detected in nuclei. This suggests that these two stretches of basic amino acids in the paired domain could be NLSs. We found that the levels of the cwp1 and cwp2 mRNA and Cwp1 protein in the gPax2m1 overexpressing cell line decreased significantly relative to the levels in the gPax2 overexpressing cell line. The levels of the cwp1 and cwp2 mRNA and Cwp1 protein in the gPax2m2 overexpressing cell line increased significantly relative to the levels in the gPax2 overexpressing cell line. We suggest that two NLSs may be present in the paired domain of gPax2 and they could influence the cwp1 and cwp2 gene expression. We suggest that gPax2 can up-regulate the cwp1 and cwp2 gene expression and the paired domain of gPax2 might be involved in this regulation.