SYNTHESIS AND STRUCTURE OF GLYCOCONJUGATE SELECTIN LIGANDS

募集到炎症部位的白细胞代表了 人类炎症反应的一部分。 这些细胞的异常募集 可引发和维持病理性炎症和组织损伤。 白细胞募集的初始事件涉及粘附相互作用 细胞粘附分子的选择素家族成员之间，和 白细胞表面的反受体。 的可溶性类似物 因此，这些反受体代表候选分子， 异常白细胞募集的药理学操作。 最近 研究已经确定了一类复合寡糖决定簇， 以唾液酸刘易斯×（唾液酸Lex）四糖分子为代表， 作为E-选择素和P-选择素的反受体。 克隆 岩藻糖基转移酶基因最近也被分离， 构建这些配体的工具。 然而，目前只有一个 对生物合成、结构和功能的初步了解 这些寡糖分子的复杂性，无论是作为游离实体，还是作为 它们通常以共价复合物的形式与蛋白质结合， 在细胞表面。 更全面地了解这些 复杂性是逻辑探索这些潜力的关键 分子在免疫细胞募集的药理学操作。 本节的具体目标将涉及与下列方面有关的问题： 这些分子的结构和生物合成特性，并将 为本计划项目的第2-4节提供以下数量： 结构确定的配体分子足以进行功能测试， 体外和体内试验，模拟人炎症反应。 将研究三类这样的分子。 具体目标#1， 从唾液酸化Lex-positive细胞系分离的复合寡糖将 被细分和结构化定义。 这些分离的分子 还将提供给第2-4节进行功能测试。 在特定 目的#2，克隆的重组岩藻糖基转移酶将用于构建 毫克量的唾液酸莱克斯四糖，这将提供 第2-4节进行功能测试。 在具体目标#3中，重组 携带唾液酸的糖蛋白（L-选择素、lamp-1和leukosialin/CD 43） Lex寡糖决定簇将在培养的细胞系中产生 其糖基化表型已经通过转染 岩藻糖基转移酶基因。 这些分子的聚糖部分将是 然后在结构上进行定义。 这些研究将提供深入了解 蛋白质和寡糖受体分子影响 岩藻糖基转移酶依赖的唾液酸Lex-biosynthesis。 这些分子将 还提供给第2-4节进行功能测试，以便进一步 我们对寡糖结构决定因素的理解 生理条件下的选择素结合相互作用。

Leukocytes recruited to sites of inflammation represent critical components of the human inflammatory response. Aberrant recruitment of these cells can initiate and maintain pathological inflammation and tissue damage. Initial events in leukocyte recruitment involve adhesive interactions between members of the Selectin family of cell adhesion molecules, and counter-receptors on the surfaces of leukocytes. Soluble analogues of these counter-receptors thus represent candidate molecules for pharmacologic manipulation of aberrant leukocyte recruitment. Recent studies have identified a class of complex oligosaccharide determinants, represented by the sialyl Lewis x (sialyl Lex) tetrasaccharide molecule, that function as counter-receptors for E-selectin and P-selectin. Cloned fucosyltransferase genes have also been recently isolated that represent tools to construct these ligands. Nonetheless, there is currently only a rudimentary understanding of the biosynthetic, structural, and functional complexity of these oligosaccharide molecules, both as free entities, or as they typically exist in a covalent complex with the proteins that display them at cell surfaces. A more comprehensive understanding of these complexities is essential for logical exploration of the potential of these molecules in the pharmacologic manipulation of immune cell recruitment. The specific aims of this section will address issues concerning the structural and biosynthetic properties of these molecules, and will provide, for Sections 2-4 of this Program Project, quantities of structurally-defined ligand molecules sufficient for functional testing in in vitro and in vivo assays that model the human inflammatory response. Three classes of such molecules will be investigated. In Specific Aim #1, complex oligosaccharides isolated from a sialyl Lex-positive cell line will be fractionated and structurally defined. These fractionated molecules will also be provided to Sections 2-4 for functional testing. In Specific Aim #2, a cloned recombinant fucosyltransferase will be used to construct milligram amounts of the sialyl Lex tetrasaccharide, which will be provided to Sections 2-4 for functional testing. In Specific Aim #3, recombinant glycoproteins (L-selectin, lamp-1, and leukosialin/CD43) that carry sialyl Lex oligosaccharide determinants will be produced in cultured cell lines whose glycosylation phenotype has been modified by transfected fucosyltransferase genes. The glycan portions of these molecules will be then structurally defined. These studies will provide insight into mechanisms whereby protein and oligosaccharide acceptor molecules influence fucosyltransferase-dependent sialyl Lex-biosynthesis. These molecules will also be provided to Sections 2-4 for functional testing, so as to further our understanding of oligosaccharide structural determinants that influence Selectin binding interactions, under physiological conditions.