探討交配栓內蛋白的共價鍵結 貯精囊蛋白SVS I被凝固腺液之轉麩胺醯酶催化的活性部位

Abstract

小鼠貯精囊分泌液以還原性 SDS-PAGE 電泳分析，可以觀察到七個明顯的蛋白，按照分子量大小命名為 SVS I-VII。而實際上在貯精囊液中幾乎沒有 SVS I、SVS II、和SVS III以單體形式存在，它們會以雙硫鍵連結成分子量非常大的不同蛋白質複合體 (high molecular weight complexes, HMWCs)。經由免疫組織染色法證明 SVS I、SVS II、SVS III蛋白是組成交配栓的蛋白成分。將交配栓以還原性SDS-PAGE sample buffer 粹取，幾乎沒有 SVS I 和 SVS II 被溶解出來，只有少量 SVS III 會被溶解出來，顯示在交配栓中 SVS I-III 之間除了雙硫鍵之外還有其他共價鍵參與其中。進一步純化凝固腺中轉麩胺醯酶 （TG4），並且與組織內的轉麩胺醯酶 (TG2) ，比較催化貯精囊分泌蛋白的酵素活性。我們發現 TG4 對於 HMWCs 具有較高的活性。然而 TG2 對於還原後貯精囊液中自由的 SVS I-III 蛋白具有較佳的活性。這種高效率的 TG4 催化在，精液凝固而形成交配栓是一重要的生化反應，其生殖的意義會加以討論。 SVS I 蛋白質序列共有 820 個胺基酸，其中 43 個為麩胺酸 43 個為離胺酸。，我們根據其序列製備 7 段重組蛋白，分成 1-78 胺基酸序列為 F1 片段、79-259 胺基酸序列為 F2 片段、260-405 胺基酸序列為 F3 片段、406-500 胺基酸序列為 F4 片段、501-650 胺基酸序列為 F5 片段、651-715 胺基酸序列為 F6 片段以及 716-796 胺基酸序列為 F7 片段。比較 F1~F7 片段被 TG4 催化後所接上 BPNH2 或 A25 peptide的數量。F2 片段對於 BPNH2 作用的活性遠大於其他片段，而 A25 peptide 對於各片段的反應活性相對較低。質譜分析經 TG4 催化，而連結上 BPNH2 的 F2 片段，鑑定出 Q232 和 Q254 是TG4 作用標的。將 F2 片段的 Q232 和 Q254 突變為三種 F2 片段 (Q232G、Q254G 和Q232G/Q254G)，BPNH2 連接上去的數量相對原始 F2 片段減少，確認 Q232 和 Q254 為 TG4 催化的主要目標。

Resolution of mouse seminal vesicle secretion (SVS) by reducing SDS-PAGE can clearly identified seven major monomer proteins tentatively designated as SVS I-VII according to the decreasing order of their molecular masses. However, no monomer forms of SVS I-III are present in SVS. Instead, they appear in various high molecular weight complexes (HMWCs) formed by inter-polypeptide disulfide bridges among SVS I, SVS II and SVS III. The HMWCs become the predominant protein in SVS. We were able to immunodetect SVS I-III in cross sections of mouse copulatory plug. Heating the clotted lump in SDS-PAGE sample buffer in the presence of DTT released none of SVS I or SVS II but only a trace of SVS III into the solution, indicative of covalent cross-links in addition to disulfide bonds among the SVS I-III proteins in the plug. Further, we purified type 4 transglutaminase (TG4) from the mouse coagulating gland to homogeneity, and compared it to type 2 transglutaminase (TG2) from guinea pig liver in terms of enzymatic ability to cross-link proteins in the SVS. We found that TG4 showed much greater activity than TG2 in catalyzing the cross-links between HMWC proteins. On the contrary, it was less active than TG2 in cross-linking any of the free SVS I-III proteins released from the reduced HMWC. The reproductive significance of more efficient TG4 catalysis is discussed. Based on the SVSI-deduced protein sequence, SVS I contains 820 amino acid residues in which there are 43 glutamine and 43 lysine residues. We produced 7 recombinant polypeptide fragments including residues 1-78/F1, residues 79-259/F2, residues 260-405/F3, residues 406-500/F4, residues 501-650/F5, residues 651-715/F6, and residues 716-796/F7, and measured the covalent incorporation of 5-(biotinamido)pentylamine (BPNH2) or biotin-TVQQEL (A25 peptide) to each of F1-to-F7 by TG4. F2 was much more active than the other fragments during the BPNH2-glutamine incorporation, Relatively, a low extent of A25 cross-linking to any one of the seven polypeptide fragments was observed. The MS analysis of BPNH2-F2 conjugate identified Q232 and Q254 as the two major TG4 cross-linking sites. This was substantiated by the result that much less BPNH2 was cross-linked to any one of three F2 mutants, including Q232G and Q254G obtained from single-site mutation, and Q232G/Q254G from double-site mutation.