GALACTOSYLTRANSFERASES--STRUCTURE AND REGULATION

半乳糖基转移酶——结构和调控

• 批准号: 6498751
• 负责人: KEITH BREW
• 金额: $ 18.42万
• 依托单位: FLORIDA ATLANTIC UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-01 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6498751
• 关键词: X ray crystallography; blood groups; cofactor; crystallization; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; galactosyltransferases; gene mutation; isozymes; lactalbumin; lysozyme; metalloenzyme; recombinant proteins; species difference

Glycosyltransferases catalyze the synthesis of specific oligosaccharide structures on cells and extracellular molecules that have crucial roles in biological recognition processes in normal and pathological states; some also act as membrane-bound carbohydrate receptors and others are important in the regulation of development. However, there is little information available on the structures, interactions, and mechanisms of these important enzymes. Two galactosyltransferases will be investigated that catalyze reactions in which the configuration of the transferred monosaccharide is retained and inverted, respectively. However, shared sequence motifs suggest that, at least parts of these enzymes may be distantly related. Alpha- Galactosyltransferase catalyze the synthesis of a galactosyl- alpha-1,3-galactosyl-beta-OR structure; this enzyme is inactivated in humans and about 1 percent of endogenous antibodies are directed against the product of its action, the alpha-gal epitope, and represent a major barrier to the xenotransplantation of organs. The mechanism of this enzyme and its structural basis will be investigated by kinetic, mutational and collaborative X-ray diffraction analysis of available crystals that diffract to 2.0 Angstrom units resolution. The results will provide information about the basis of specificity in homologous enzymes that catalyze the synthesis of blood group A and B glycans and Forrssman glycolipid. Beta-4- Galactosyltransferase-1, is one of six homologous enzymes in humans that catalyze the transfer of beta-galactose to N- acetylglucosamine or glucose in glycoproteins and glycolipids. The binding of alpha-lactalbumin, a mammary specific protein that is homologous with lysozyme, modulates the specificity of the enzyme to allow it to catalyze lactose biosynthesis (galactose transfer to glucose) during lactation. Structural and mutational approaches will be used to investigate the basis of regulation through this protein-protein interaction. A new homologue of alpha-lactalbumin and lysozyme has been identified as a testis- expressed cDNA. This lacks the lysozyme catalytic site and has some similarities to alpha-lactalbumin, raising the possibility that it could be a novel glycosyltransferase regulatory protein. The activity and structure will be investigated using a recombinant form of this protein.

糖基转移酶催化细胞和细胞外分子上特定寡糖结构的合成，在正常和病理状态下的生物识别过程中起着至关重要的作用；一些还作为膜结合碳水化合物受体，其他在发育调节中起重要作用。然而，关于这些重要酶的结构、相互作用和机制的信息很少。将研究两种半乳糖转移酶，分别催化反应，其中转移的单糖的构型被保留和反转。然而，共享的序列基序表明，这些酶的至少一部分可能是远亲。半乳糖转移酶催化合成半乳糖- α -1,3-半乳糖- β - or结构；这种酶在人体内是失活的，大约1%的内源性抗体是针对其作用产物α -半乳糖表位的，这是器官异种移植的主要障碍。该酶的机制及其结构基础将通过动力学、突变和协同x射线衍射分析来研究，衍射分辨率为2.0埃单位。该结果将为催化A型和B型血聚糖和Forrssman糖脂合成的同源酶的特异性基础提供信息。β -4-半乳糖转移酶-1是人类六种同源酶之一，催化糖蛋白和糖脂中β -半乳糖向N-乙酰氨基葡萄糖或葡萄糖的转移。乳清蛋白是一种与溶菌酶同源的乳腺特异性蛋白，它的结合调节了酶的特异性，使其能够在哺乳期间催化乳糖生物合成（半乳糖转化为葡萄糖）。结构和突变方法将用于研究通过这种蛋白质-蛋白质相互作用的调节基础。一个新的α -乳清蛋白和溶菌酶同源序列已被鉴定为睾丸表达的cDNA。它缺乏溶菌酶催化位点，与α -乳清蛋白有一些相似之处，这提高了它可能是一种新的糖基转移酶调节蛋白的可能性。活性和结构将使用该蛋白的重组形式进行研究。