小白鼠貯精囊自體抗原生化性質的研究

Abstract

本實驗先前發現，在純系(Balb/c)小白鼠的貯精囊中，存在一種蛋白質，將其注射到雄性或雌性的小白鼠，都可以引起自體免疫反應，因此命名為貯精囊自體抗原(Seminal Vesicles Autoantigen，SVA)(Yu et al., 1993)。它是一個未被研究過的醣蛋白，經HPLC純化後，可得二個波峰，分別命名為SVA-Ⅰ與SVA-Ⅱ，二者的胺基酸組成幾乎相同，分子量皆為19 KDa左右，SVA的cDNA定序工作已完成，SVA只存在於貯精囊(seminal vesicle)中。 針對SVA的特性及可能扮演的生理功能是本論文的重點。分析由SVA所測得的圓振二向性(circular dichroism ,CD)光譜，可知SVA的二級結構主要由β-structure構成，此蛋白分子幾乎不含有helical structure。SVA的立體結構不因其與鋅離子的結合而改變，但會稍微影響到含苯基胺基酸的旋光值。經由分析而知SVA的醣基種類含有galactose、(L)-fucose、mannose、glucose及N-acetylglucos amine、NGNA、NANA。SVAⅠ和SVAⅡ酸性醣量的差別，可以解釋SVAⅠ和 SVAⅡpI值不同的原因(SVA ⅠpI為6.1;SVA ⅡpI為5.9)，由外源凝集素(lectin)-醣蛋白(glycoprotein)雙向免疫擴散法也證實SVA最外圍的醣基含有galactose和(L)-fucose。在SVA可能的生物功能方面，經由抗磷酸化胺基酸抗體及蛋白激?的測試，則否定了SVA蛋白質序列所推測可被磷酸化的潛在位置。另外，利用免疫染色法及西方墨點法初步證實SVA可結合在精蟲尾部前端(midpiece)的部位。

Mouse seminal vesicle autoantigen (SVA) has been identified recently from our laborary. It is a 19 KDa glycoprotein and the primary structure of its protein core has been established by cDNA cloning and protein sequencing. The main task of this work is to study the structure and the function of SVA. The protein was estimated to contain 25% β-structure and no helical structure on the basis of its CD. The secondary structure was stable in pH 7.4-10.5. The complex formation of SVA and Zn2+ caused no change in the protein conformation. The carbohydrate moeity of SVA composed of galactose, (L)-fucose, mannose, glucose, N-acetylglucosamine, NGNA and NANA. The results of lectin agglutination test suggested galactose and (L)-fucose as terminal residues of the conjugated carbohydrate. SVA Ⅰand SVA Ⅱ differed in the amount of acidic carbohydrates like NGNA and NANA, and this might explain their different isoelectric points (pI): SVA Ⅰ, pI 6.1; SVAⅡ, pI5.9. SVA could not be detectable with antiserum angist phosphoserine, phosphothreonine, phosphotyrosine, nor was phosphorylated by protein kinase C and endogenous kinase. This revealed that SVA is not a phosphoprotein, despite that there are potential phosphorylation sites on SVA. The results of immunolocation and Western blotting showed that SVA could bind specifically on the midpiece of sperm.