Q469对ApNGT活性和底物选择性的影响.docVIP

PAGE PAGE 26 ?Q469对ApNGT活性和底物选择性的影响 Effection of Q469 Deletion on the Activity of ApNGT 摘要 ApNGT(ApHMW1C)是膜肺炎放线杆菌来源的HMW1C-样蛋白，是一种能够将葡萄糖和半乳糖转移到HMW1粘附素已知天冬酰胺糖基化位点的GT41家族的新型N-糖基转移酶(NGT)，其效率最高的供体是UDP-Glucose。已经有文献报道，ENGases可以将复杂的多糖转移到ApNGT糖基化多肽底物的葡萄糖上，获得含有复杂多糖链的糖肽。在本实验中，由于实验室前期实验已经验证ApNGT位点Q469对其活性具有很重要的影响，所以利用多重PCR技术对Q469位点进行删除，再进行转化与表达，最后通过MALDI-TOF定性和HPLC定量的对酶的活性进行检测。这次实验我们将探索新的突变体ApNGTΔQ469活性和底物选择性方面的变化。这将使我们更加深入了解ApNGT中Q469残基的重要性，以及ApNGT的作用机制，为以后改造ApNGT获得更高糖基化效率的酶奠定基础。 关键词：糖基化；N-糖基转移酶；ApNGT。 Abstract ApNGT (ApHMW1C), a HMW1C-like protein originated from actinobacillus of pneumonia, is a new-type N-glycosyltransferase (NGT) of GT41 family which is able to transfer glucose and galactose to the known asparaginate glycosylation site of HMW1 adhesin, as its favorite sugar donor is UDP-Glucose. It has been reported that ENGases can transfer complex polysaccharide to the glucose of ApNGT glycosylation polypeptide substrate, becoming the glycopeptide with complex polysaccharide chains. In this study, since the important influence of ApNGT locus Q469 on its activity has been confirmed by the earlier experiment, the Q469 residue will be deleted by multiple PCR technology firstly; then it will be transformed into E. coli DE3 cell and expressed, as the enzyme activity will be tested by MALDI-TOF and HPLC qualitatively and quantitatively. In this experiment, we will explore the activity of new mutant ApNGTΔQ469 and the alteration of substrate selectivity. It will enable us to further knowledge about the importance of Q469 in ApNGT and ApNGT’s mechanism of action, laying a basis for getting the enzyme with higher glycosylation efficiency by transforming ApNGT. Keywords: Glycosylation; N-Glycosyltransferase; ApNGT. 第一章前言 一、糖基化 蛋白质糖基化修饰存在于各个生物域，并提供机理控制不同的细胞生理过程，包括信号传导，蛋白质折叠，筛选与稳定性，病毒-细胞相互作用和宿主免疫反应[1]。 在好久以来，认为蛋白质的糖基化只在真核生物中发生。然而，在最近几年越来越多地糖蛋白在原核生物中被发现，包括致病菌，如铜绿假单胞菌，弯曲杆菌属，奈瑟氏球菌属，以及大肠杆菌等等[2]。自从认识到原核生物能够使蛋白质糖基化，已经报道了超过70种细菌糖蛋白。大多数这些蛋白质是表面暴露的，并在细菌粘附到宿主细胞或逃避宿主免疫发挥着至关重要的作用，这表明了糖蛋白可以