SYNTHESIS AND STRUCTURE OF GLYCOCONJUGATE SELECTIN LIGANDS

被招募到炎症部位的白细胞是关键成分 人类的炎症反应。这些细胞的异常募集 可以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黏附相互作用 在细胞黏附分子的选择素家族成员之间，以及 白细胞表面的反受体。可溶性类似物 因此，这些反受体代表了候选分子 异常白细胞募集的药理学操作。近期 研究已经确定了一类复杂的寡糖决定因素， 以唾液酸基Lewis x(唾液酸基)四糖分子为代表， 其功能与E-选择素和P-选择素的受体相反。克隆的 岩藻糖基转移酶基因最近也被分离出来，它们代表着 构建这些配体的工具。尽管如此，目前只有 对生物合成、结构和功能的基本了解 这些寡糖分子的复杂性，无论是作为自由实体，还是作为 它们通常与所展示的蛋白质以共价复合体的形式存在 它们在细胞表面。更全面地了解这些 复杂性是对这些潜力的逻辑探索所必需的 免疫细胞募集的药理学操作中的分子。 本节的具体目标将涉及以下问题 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 结构定义的配体分子足以用于功能测试 模拟人类炎症反应的体外和体内试验。 将对三类此类分子进行研究。在具体目标1中， 从唾液酸法Lex阳性细胞系中分离出的复合寡糖将 被细分并在结构上定义。这些分馏的分子 也将提供给第2-4节进行功能测试。具体而言 目的#2，克隆的重组岩藻糖基转移酶将用于构建 毫克量的唾液酸法四糖，将提供 到第2-4节进行功能测试。在具体目标#3中，重组 携带唾液酸糖蛋白的糖蛋白(L-选择素、LAMP-1和白涎素/CD43) 在培养的细胞系中将产生莱克斯寡糖决定簇 其糖基化表型已通过转基因修饰 岩藻糖基转移酶基因。这些分子的葡聚糖部分将是 然后从结构上定义。这些研究将提供对 蛋白质和寡糖受体分子影响的机制 岩藻糖基转移酶依赖的唾液酸法生物合成。这些分子将 也提供给第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.