利用定點突變研究幽門螺旋桿菌的岩藻糖轉移酶之活性區

Abstract

幽門螺旋桿菌 (Helicobacter pylori，簡稱H. pylori)感染世界上50%的人口，目前被證實感染幽門桿菌會造成消化性潰瘍、胃淋巴瘤等相關疾病。這株致病菌可以在LPS (lipopolysaccharide) 結構末端的O抗原上表現路易士抗原 (Lewis antigen)，這種路易士抗原可以在感染週遭的胃上皮細胞看到，如此一來，幽門桿菌就可以藉模擬而躲過宿主細胞的免疫系統攻擊。在路易士抗原合成途徑中，岩藻醣轉移酶負責催化最後幾步的反應，可以將GDP-Fuc的fucose轉移到受質上。α-1,3-fucosyltransferase (簡稱α-1,3-FucT)以LacNAc為受質，經轉糖作用後形成Lex。α-1,3-FucT在C端有兩性螺旋序列，可以幫助蛋白質附著於膜上。本實驗室之前將蛋白質從C端做45個胺基酸的截短，得到水溶性蛋白質。此種長度的截短我們稱之為C45-FucT，與具有全長的α-1,3-FucT得到具有相同的高活性。從兩性螺旋序列往N端找，可以看到十組七個胺基酸的重複序列。C115-FucT從C端截短115個胺基酸，包含兩性螺旋序列與可以形成Leu zipper的組七個胺基酸的重複序列；由於C115-FT失去可以幫助形成雙體的Leucine zipper，故此種長度的截短酵素為單體且活性下降到6.5 %。 由於缺乏FucT的結構資訊，所以要了解此酵素的活性區與催化機制，是件不容易的事。利用化學修飾法找出His和Arg可能參與受質的結合後，我們將10個His與10個Arg分別利用定點突變並做活性測試，討論與GDP-Fuc和LacNAc結合有關的胺基酸。 DEPC (diethylpyrocarbonate) 和PGO (phenylglycoxal) 分別會與His和Arg形成共價鍵進而抑制酵素反應。但是當α-1,3-FucT先與GDP-Fuc結合達到保護donor sugar結合區後，再加入DEPC或PGO，抑制的效果卻會大大減低。由於GDP-Fuc的保護作用，我們可以推測在GDP-Fuc結合區，有某些His和Arg幫助GDP-Fuc跟蛋白質結合。 我們將C45-FucT的10個Arg與10個His突變成Ala並分別測試活性。活性測試是利用FucT的副產物GDP的生成量會造成NADH的減少，藉由測量NADH的量而達到測試FucT活性的目的。其中R79A、 R118A、R180A、R195A 和R354A的突變株活性完全消失，這五個胺基酸對α-1,3-FucT的活性是非常重要的。R89A、H60A、H131A、H282A、H298A和H345A則是有活性下降。我們將這六個活性下降的突變株更進一步測Km。由Km的數據得知H282、H298和H354會幫助GDP-Fuc結合；H60與R89會幫助LacNAc結合。 最近，王惠鈞教授實驗室將FucT做出結晶解出結構。目前為止只有C115-FucT這種截短的酵素可以得到結晶。從結構上，我們看到R195、N240、E249和K250會直接跟GDP-Fuc有氫鍵結合。但是在活性測試的實驗中卻發現N240會幫助LacNAc與酵素作用。利用結構疊合，我們推斷E95為catalytic base。另外藉由酵素動力學測試和C115-FucT的結構，我們推測出更接近野生型α-1,3-FucT的反應機制與構型。

Helicobacter pylori (H. pylori) is considered to be a primary cause of gastritis, duodenal ulcer and gastric cancer. This pathogenic bacterium produces Lex and Ley epitopes in the O-antigens of lipopolysaccharides (LPS) to mimic the carbohydrate antigens on the surface of gastric epithelial cells. The molecular mimicry avoids the detection by the host immune system. The enzyme α-1,3-fucosyltransferase (α1,3-FucT) from H. pylori catalyzes the glycosyl addition of fucose from the donor GDP-fucose to the acceptor LacNAc. It is a membrane associated protein. Previously, our lab carried out a systematic truncation of the C terminus of the α-1,3-FucT to improve the protein solubility. C-Terminal 45 amino acid residues include the amphiphilic helices that anchor the protein to the cell membrane. The corresponding protein, C45-FucT, was water soluble and had the same activity as that of the full-length enzyme. Next to the amphiphilic helices is the heptad repeats. Deletion of 115 residues removed both helices and the heptad repeats in the C terminal end. Because heptad repeats were expected to form the leucine zipper and facilitated the formaitnon of a dimeric structure, therefore C115-FucT was found to be a monomer with low activity. Due to lack of structural imformation of fucosyltransferase, it is not easy to understand the catalytic mechanism. In order to study the residues involed in catalysis and substrate binding, this thesis lays a special emphasis on His and Arg. The methods include the use of modifiers and preparation of different mutant proteins by site-directed mutagenesis. DEPC (diethylpyrocarbonate) and PGO (phenylglycoxal) were shown to specifically react with His and Arg, respectively. Both of these modifier can inactivate the α-1,3-FucT of H. pylori. When α-1,3-FucT was incubated with GDP-Fuc before the addition of PGO or DEPC, the higher concentration of GDP-Fuc was used in the study, the higher level of activity was retained. The protection effect of GDP-Fuc indicated that His and Arg were likely located at either the active site or the binding site of the donor substrate. Ten Arg residues and ten His residues were individually mutated to Ala and the resulting mutants were analyzed by the activity assay. The activity assay was a coupling assay carried out based on the production of the side product GDP. The result indicated the mutants R79A, R118A, R180A, R195A and R354A were completely inactive. R89A, H60A, H131A, H282A, H298A and H345A were found to have reduced activities. The Km and Vmax of these mutants were also measured. According to Km, H282, H298 and H354 were found to be involved in the binding with GDP-Fuc, Whereas H60 and R89 were related to the binding with LacNAc. The structure of was solved by x-ray crystallograpgy in the collaboration with Dr. Andrew Wang’s group of this institute. So far only C115-FucT can be crystallized for prove the structural imformation. The results suggest that R195, N240, E249, K250 directly interact with GDP-Fuc. In contrast, the kinetic analysis supports the idea that N240 is involved in the binding with LacNAc. The superimposition of C115-FucT to the structure of BGT (β-Glucosyltransferase) revealed that E95 may act as the catalytic base in general catalytic base. We also try to figure out the real structure of the C45-FucT.