小白鼠貯精囊自體抗原生化特性的研究

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 )。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 ，二者的氨基酸組成幾乎相同，分子量皆約為 19KDa 左右， SVA 的 cDNA 定序工作已完成， SVA 只存在於貯精囊（ Seminal Vesicle ）中，且為雄性素所調控。 針對 SVA 的特性及可能扮演的生理功能是本論文的重點，我們得知， SVA 具有一個鋅離子的結合部位，在鋅離子存在下， SVA 蛋白的螢光強度在 330－350nm 區域出現降低且藍移（ blue－shift ）的現象，利用此螢光特性加以分析而算出其結合常數為 1.35 × 105M-1。 SVA 內含有兩個色氨酸，分別是 Trp2與 Trp82 ，根據螢光消光劑的影響而知，二者皆暴露於 SVA 蛋白表面，其中一個存在於正淨電環境，另一則存於負淨電環境，另外利用螢光免疫法觀察得 SVA 蛋白可結合於精蟲尾部 Midpiece 的部位，這顯示 SVA 蛋白可能與精蟲的運動功能有關。 最後，利用化學灰化方法定 SVA 蛋白含磷酸根的量，證明 SVA - I 不含磷酸根，而 SVA－II是帶有兩個磷酸根。

A 19KDa autoantigen in mouse seminal vesicle secretion has been recently discovered in our laboratory. This protein is named as seminal vesicle autoantigen (SVA). SVA is a glycoprotein and the primary structure of its core protein has been established by cDNA cloning and protein sequencing. SVA is androgen-dependent and exists exclusively in seminal vesicles. The main task of this work is to find the function of SVA. ZnCl2 could perturb specifically the SVA fluorescence due to tryptophan in 330-350nm region. Analysis of the effect of ZnCl2 on SVA fluorescence revealed a single Zn2+-binding domain in SVA. The association constant for the complex formation of Zn2+ and SVA was determinated to be 1.35×10 5M-1 at pH 7.4. SVA has two tryptophan residues, namely Trp2 and Trp82. The effect of quencher such as CsCl, Kl, and acrylamide on the SVA fluorescence suggested that both Trp2 and Trp82 were exposed on the protein surface. One tryptophan was surrounded by the negative charge while the other was surrounded by the positive charge. Together with the primary structure, it suggested that Trp82 is more likely close to the Zn2+ binding site. The results of indirect fluorescence immunolocation revealed the binding of SVA on the midpiece of sperm, suggesting the association of SVA with the regulation of sperm motility. HPLC on a DEAE column resolved SVA to two components, SVA- I and SVA- II. We found that SVA- I contained no phosphate but SVA- II contained two phosphates per protein molecule.