EUKARYOTIC HYALURONAN SYNTHASES

结构元件，粘附介质和细胞外信号传导 分子。 HA多糖的数量和质量 调节发育过程中的细胞行为、细胞间粘附和 识别、运动和新血管形成。 的长期目标 本研究旨在阐明 负责产生HA的HA合酶在 人类 已经研究了脊椎动物中HA的生物合成， 几十年来几乎没有成功，直到1996年，一个真核HA家族 鉴定了大肠杆菌。 这些酶是新颖而有趣的 糖基转移酶，因为它们转移两个不同的糖基 在聚合过程中，几乎所有其他酶都只转移一种糖。 脊椎动物HA生物合成的分子和生化细节 缺乏。 因此，本项目五年期的主要目标 是表征来自非洲爪蟾和人类的几种HA酶。 的 研究人员已经生产出功能性重组爪蟾HA合酶 在无背景系统中的酶。 该项目的具体目标 1）分析真核生物HA酶的动力学。 经典的生物化学研究将揭示酶 聚合其UDP-糖前体。 2)在结构上表征 真核生物HA酶。 辐射灭活和质谱 将用于定义功能单元和单体尺寸， 分别 3)鉴定HA的前体结合位点 - 是的 将利用光亲和性和化学标记研究 与UDP-糖底物相互作用的酶的ot标签区域。 4)确定HA合酶的功能残基。 的残基 在HA酶中保守的基因将被定点改变， 诱变;特别地，某些半胱氨酸残基将由于 它们在催化和二硫键形成中的潜在作用。 目标3中确定的必需残留物也将通过 诱变研究。 酶学和结构信息可以 允许生成描述催化机制的工作模型 这些酶。

Structural element, a mediator of adhesion, and an extracellular signaling molecule. The quantity and the qualities of the HA polysaccharide modulate cell behavior during development, intercellular adhesion and recognition, locomotion, and neovascularization. The long-term goal of this investigation is to elucidate the structure and the mechanism of action of the HA synthase enzymes responsible for production of HA in humans. The biosynthesis of HA in vertebrates has been studied for decades with little success until 1996, when a family of eukaryotic HA synthases was identified. These enzymes are novel and interesting glycosyltransferases because they transfer two different sugar groups during polymerization; almost all other enzymes transfer only one sugar. The molecular and biochemical details of HA biosynthesis in vertebrates are lacking. Therefore, the major goal of this five-year project period is to characterize several HA synthases from Xenopus and humans. The investigators have produced functional recombinant xenopus HA synthase enzyme in a background-free system. The specific aims of this project are; 1) To analyze the kinetics of the eukaryotic HA synthases. Classical biochemical studies will reveal the manner in which the enzyme polymerizes its UDP-sugar precursors. 2) To characterize structurally the eukaryotic HA synthases. Radiation inactivation and mass spectrometry will be used to define the functional unit and monomer sizes, respectively. 3) To identify the precursor binding sites of the HA synthases. photoaffinity and chemical labeling studies will be utilized ot tag regions of the enzyme that interact with the UDP-sugar substrates. 4) To determine the functional residues of the HA synthase. The residues that are conserved among HA synthases will be altered by site-directed mutagenesis; in particular, certain cysteine residues will be mutated due to their potential roles in catalysis and in disulfide bond formation. The essential residues identified in aim 3 will also be confirmed by mutagenesis studies. The enzymological and structural information may allow the generation of a working model describing the catalytic mechanism of these enzymes.