HUMAN FUCOSYLTRANSFERASE STRUCTURE/FUNCTION ANALYSIS

描述：(改编自调查员的摘要)表达 细胞表面的碳水化合物结构被发现起到了 在许多细胞过程中起着重要作用，例如。细胞-细胞识别， 生长控制和受体结合。碳水化合物含有一种 内酯和新内酯系列核链上的A1A3连接岩藻糖被发现是 许多人类癌症中的肿瘤相关标志物。酶类能够 已经确定了岩藻糖向这些受体的催化转移，并 学习。目前，至少有六种不同的a1a3岩藻糖基转移酶 是从人类来源确认的。其中五种酶已被克隆 并测定了它们的DNA和氨基酸序列。这些酶是 以包括受体在内的酶性质的差异为特征的 专一性、动力学特性和分布。这一家族的酶 是研究结构与功能关系的理想系统 糖基转移酶。本申请中提出的研究将需要 利用酶的形式和性质的多样性来鉴定 参与底物结合的酶的特定残基和部分 和催化作用。最初的重点将是通过化学手段识别 特定残留物，以前已被确定为 基于特定部位化学试剂的结果的活性。示例 包括GDP-果糖保护的N-乙基马来酰胺敏感半胱氨酸残基 出现在例如。FT-III和-V与GDP-岩藻糖保护的吡哆醛-P 可修饰的赖氨酸残基以每种酶的形式存在。其他研究将使用 光亲和试剂探测受体碳水化合物的结合部位 和供体GDP-岩藻糖及其受体结合特性的分析 域交换变体。定点诱变与酶动力学 研究将用于分析这些位点在底物中的功能 结合和催化。这些研究将提供信息，导致 更好地理解酶的功能，也许是一个基础 受体特异性的差异。另一个目标将集中在克隆和 NCI-H69一种新型岩藻糖基转移酶的遗传特性 小细胞肺癌细胞。这种酶具有不同于 其他已知的形式。 本申请中提出的研究将导致更大的 了解酶的复杂性、结构-功能关系和 这种酶家族中的活性部位的性质具有重要的 在疾病过程中的作用。

DESCRIPTION: (adapted from the investigator's abstract) Expression of carbohydrate structures at the cell surface has been found to play an important role in many cellular processes, eg. cell-cell recognition, growth control, and receptor binding. Carbohydrates containing an a1A3-linked fucose on lacto- and neolacto-series core chains are found to be tumor-associated markers in many human cancers. Enzymes capable of catalyzing transfer of fucose into these acceptors have been identified and studied. Presently, at least six distinct a1A3 fucosyltransferases have been identified from human sources. Five of these enzymes have been cloned and their DNA and amino acid sequences determined. These enzymes are characterized by differences in enzymatic properties including acceptor specificity, kinetic properties, and distributions. This family of enzymes is an ideal system for studies of structure function relationships of glycosyltransferases. The research proposed in this application will take advantage of the multiplicity of enzyme forms and properties to identify specific residues and portions of the enzyme involved in substrate binding and catalysis. The initial focus will be on identifying by chemical means specific residues which have previously been determined to be essential for activity based upon results from site-specific chemical reagents. Examples include the GDP-frucose protected, N-ethylmaleamide-sensitive Cys residue present in eg. FT-III and -V and the GDP-fucose protected pyridoxal-P modifiable Lys residue present in each enzyme form. Other studies will use photoaffinity reagents to probe the binding sites for acceptor carbohydrates and donor GDP-fucose and analysis of acceptor binding properties through domain swapped variants. Site-directed mutagenesis and enzyme kinetic studies will be used to analyze the function of these sites in substrate binding and catalysis. These studies will provide information leading to a better understanding of enzyme function and, perhaps, a basis for differences in acceptor specificity. Another aim will focus on cloning and genetic characterization of a novel fucosyltransferase enzyme from NCI-H69 small cell lung carcinoma cells. This enzyme has properties distinct from other known forms. The research proposed in this application will lead to a greater understanding of enzyme complexity, structure-function relationships, and the nature of active sites in this family of enzymes having important function in disease processes.