蛋白質體學方法測出血清中第三補體因子會與甲型去氧核醣核酸水解酶結合及其生理意義

Abstract

後基因體時代的來臨，蛋白質體學成為目前熱門的研究焦點。其中共價層析法可分離出帶有硫醇基之蛋白質，已經應用於蛋白質體研究。本論文進一步應用共價層析法於蛋白質交互作用，分析牛小腸中與DNase I結合之蛋白質。牛胰臟甲型去氧核醣核酸水解酶 (bpDNase) 為目前被研究最為透徹之DNase I。前人的研究中意外發現，在牛小腸中具有與DNase I結合之蛋白質，而至今僅知actin為唯一與DNase I結合之蛋白質，且會抑制DNase I之活性。本論文利用bpDNase上非必須雙硫鍵 (Cys101-Cys104) 之突變株bpDNase C101A，帶有Cys104游離硫醇基，可與Thiopropyl Sepharose 6B共價層析膠體共價鍵結，以pull down方式進行蛋白質交互作用分析，配合質譜儀技術，鑑定牛小腸中與brDNase I結合之蛋白質身分，並探討兩者結合之生理意義。 首先，我們利用大腸桿菌BL21 (DE3) pLysE大量表現突變酵素brDNase C101A後，分別通過陰離子交換管柱SOURCE 15Q及陽離子交換管柱S-hyperD得到純化均質之產物，並以CD spectrum確定突變株與野生型兩者於二級結構上並未因突變造成差異。測試brDNase C101A與共價層析膠體反應時發現，當緩衝液中含有0.5 mM之β-MSH時，可避免brDNase C101A分子間產生二聚體而得到最好的反應效率。為證實此方法可應用於蛋白質交互作用分析，我們利用H293T細胞萃取物，與已共價鍵結上brDNase C101A的膠體進行pull down assay，結果確實可偵測到actin之存在，說明此方法亦可用於捕捉與DNase I結合之蛋白質。接著我們收取牛小腸粗抽液，以膠體過濾Sephadex G-100進行初步分離後，同樣地進行pull down assay，將染色差異色帶以LC-ESI/MS/MS進行蛋白質身份鑑定，結果分別為補體因子complement 3 (C3) 與α-macroglobulin，之後以西方墨點法再次證實C3的確會與DNase I結合。由於補體因子分布於血清之中，因此後續取牛血清進行相同之pull down assay，而質譜儀與西方墨點分析結果亦相同偵測出C3的存在。在證實C3確實會與DNase I結合之後，我們利用電腦演算法進行C3與DNase I之分子間docking模擬，計算出兩者可能結合之介面位置，及可能作用之胺基酸。 由於C3會與DNase I作用之結果，我們推測當免疫系統要清除入侵外來物或死亡的細胞時，訊息傳遞使得C3活化後，活化型之C3b可帶著血清中之DNase I結合至欲清除之外來物或死亡細胞的表面，C3b會造成下游之補體活化，形成membrane-attack complex可於細胞膜上穿孔，使得DNase I順利進入細胞中執行DNA切除的動作，避免DNA釋放至血液中造成免疫反應。

Bovine pancreatic deoxyribonuclease I (bpDNase I) is the best-characterized DNase. Previous studies in this laboratory have shown that there was a DNase I-binding proteins (DBP) in bovine small intestine and this DBP is not the DNase I inhibitor, actin. Until today, actin is the only known DNase I binding protein The covalent chromatography was utilized for isolation of the DNase I-binding protein in the extract from bovine small intestine. To achieve this goal, first, the mutant brDNase I C101A , contained a free sulfhydryl group on Cys104, was constructed and expressed in BL21 (DE3) pLysE . The expressed protein was subsequently purified to homogeneity through a SOURCE 15Q anion and a S-hyperD cation-exchange columns. CD spectra analysis revealed the mutation did not alter secondary structures and the presence of 0.5 mM β-MSH in the buffer could avoid the brDNase I C101A dimer formation. The purified brDNase I C101A was then covalently linked to Thiopropyl Sepharose 6B beads. When H293T cell lysates were added to brDNase I C101A conjugated beads, the result showed that the DNase I-Sepharose beads could bind actin indicating this method is feasible for isolation of the DNase I-binding proteins. Crude extracts from bovine small intestine were than collected and subjected to a gel filtration Sephadex G-100 column. Fractions with DNase I activities were pooled and used for pull down assay. The interacted DBPs were eluted from the beads. The DBPs were identified from the SDS-PAGE using the tandem mass spectroscopy approach. Two proteins identified were complement 3 (C3) and α-macroglobulin and further confirmed by Western blot analysis. Since C3 is distributed in serum, we assumed the interaction of C3 and DNase I existed in serum. Therefore, bovine serum was used for pull down assay and the result showed, C3 in serum could interact with DNase I. Finally, we performed computational algorithm to simulate molecular docking between C3 and DNase I. The result provided the predicted interface and possible interacting amino acid residues. Here, we proposed that when infection occurred and necrotic cells were needed for clearance, C3 could be activated and bring serum DNase I to surface of dying cells. Activated C3 could cause downstream effectors assembled and form membrane attack complex which could lyse cell membrane. DNase I then enter the cell and cleave disposal DNA to avoid releasing undigested DNA, which may stimulate immune response in blood.