Characterization and localization of beta-1,4-N-acetyl-galactosaminyl transferase 2 in testicular cells

Sda β-1,4-N-acetyl-galactosaminyl transferase Ⅱ(β4galNAcTⅡ,B4GALNT2)此一醣轉移酶主要催化轉移GalNAc以β-1,4的鍵結，至[Neu5Acα2-3]Galβ1-4GlcNAcβ1-3Gal之Gal上，形成sda結構。此一結構最早是在血球表面抗原結構所發現，實際上它廣泛存在於哺乳動物組織或體液中，包含胃、腸、腎、血清、尿液和乳汁等，近期研究發現胃癌和結腸癌細胞中B4GALNT2表現量會明顯下降，而使sda抗原減少並與癌細胞轉移有密切關係。由此可知B4GALNT2的重要性與生物系統的相關性，但目前為止對其生理功能研究仍有很大的空間。2003年Dell的研究團隊首先在雌性生殖系統中發現sda結構的存在，本研究目的希望能先了解B4GALNT2生物特性，才能更進一步去探討Sda結構在生殖系統中的生理意義。實驗是以公鼠為動物模式利用RT-PCR和西方墨點法確立了B4GALNT2在睪丸的表現，並且發現在性成熟公鼠睾丸B4GALNT2的表現量比性未成熟公鼠睾丸表現量多，顯示B4GALNT2也許和小鼠成長過程有關。根據Shur在1993的研究結果以及序列的預測分析，推測B4GALNT2為一膜蛋白，可能存在於內質網高爾基氏體或細胞膜上。為確認此一蛋白在細胞中位置，首先萃取睪丸組織蛋白分為細胞膜及細胞質兩部分，並利用西方墨點法以B4GALNT2抗體偵測、再以重組質體轉染293T細胞株表現具有螢光的B4GALNT2-EGFP融合蛋白，以螢光顯微鏡觀察其在細胞膜的位置。並以睪丸Leydig cell和Sertoli cell細胞株(TM3及TM4)進行免疫螢光染色以B4GALNT2抗體偵測後用螢光顯微鏡觀察，結果發現在公鼠睾丸細胞株中可表現B4GALNT2，在TM4細胞株中此一蛋白似乎存在於細胞膜較多，而TM3則是存在於細胞質較多。分別以改變細胞膜通透性之固定方法在以免疫螢光染色測量B4GALNT2在細胞的位置，推測此一蛋白可能以一段靠近N端的穿膜序列嵌在細胞膜上且主要蛋白質結構朝向細胞外側。The Sda β-1,4-N-acetyl-galactosaminyl transferase Ⅱ(B4GALNT2) catalyze the addition of GalNAc to the Gal of the [Neu5Acα2-3]Galβ1-4GlcNAcβ1-3Gal terminal structure via the β-1,4 linkage to form Sda antigen. The Sda antigen was first reported as a human blood surface antigen. This antigen was found in several tissue types, including stomach, colon, kidney, and oocytes. Its presence was also demonstrated in various body fluids, such as saliva, milk, serum, and urine. Current studies revealed that Sda antigen reduced remarkably in cancer lesions of the gastrointestinal tract. It indicated the significance of this antigen in biosystem and also associated it is importance to B4GALNT2. So far the research of B4GALNT2 physiological function is still unclear. Dell’s group first found Sda antigen in reproductive system, however, the significance of sda is unclear. Using RT-PCR and western blotting analysis to examine the expression of B4GALNT2 in mouse testis, and the results ¬¬revealed that two ¬¬¬genes expressed ¬¬higher in mature mice (12 WK) than in immature mice (3 WK). It suggested that B4GALNT2 is involved in mouse reproduction development. According to Shur’s study and the primary sequence prediction, it suggests that B4GALNT2 may be as a membrane protein. To identify the location of this membrane proteins, first we have extracted the total proteins from testis, and separated via SDS-PAGE, then analyzed by immunostaining. At the same time we overexpressed EGFP-b4galnt2 in 293T cell and observed by flurorescence microscope to detect B4GALNT2. In addition, the immunohistochemical analysis was performed in sertoli cell line (TM4) and leydig cell line (TM3) via B4GALNT2 antibody, and then observed under microscope. We found that B4GALNT2 is localized on plasma membrane by a sequence near its N-terminal, and the major structure may face to outside of the plasma membrane