OLIGOSACCHARIDE STRUCTURE AND FUNCTION IN RECOGNITION

低聚糖结构和功能的识别

• 批准号: 3481897
• 负责人: JACQUES U BAENZIGER
• 金额: $ 24.02万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-08-01 至 1991-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3481897
• 关键词: affinity chromatography; carbohydrate sequence; cell membrane; galactose; gas chromatography; glycoproteins; glycosylation; high performance liquid chromatography; human tissue; laboratory rabbit; laboratory rat; liver cells; lysosomes; mass spectrometry; membrane model; membrane proteins; oligosaccharides; phagocytosis; posttranslational modifications; protein sequence; radiotracer; receptor; tissue /cell culture

The primary objective is to examine the role of protein glycosylation in directing glycoproteins to specific intra- and extra-cellular sites. Recognition of individual oligosaccharides requires both unique oligosaccharide structures and specific receptors. Alterations in either oligosaccharide structure or receptor specificity may lead to pathologic manifestations such as developmental defects, altered regulation of growth, or altered metabolism. Two lectins with markedly different properties form a focus for our studies and are used as prototypes for basic processes involved in recognition and transport. The Gal/GalNAc-specific receptor is a membrane protein which mediates the uptake of Gal bearing glycoproteins from plasma by hepatocytes. Endogenous ligands will be identified and characterized using a blotting technique relying on recognition of ligand by the purified receptor. The structure of the oligosaccharides, their origin and functional significance will be established. The relationship of subunit assembly to ligand binding and regulation of receptor movement during endocytosis will be examined with solubilized receptor and viable cells. Components of the endocytic vesicles mediating uptake will be identified by vectorial labeling with lactoperoxidase conjugates delivered to these vesicles through the receptor. Endocytic vesicles and plasma membrane vesicles will be isolated by high magnetic gradient separation following delivery of 20-40 nm magnetic iron-dextran particles and will be used to characterize the process of uptake by the Gal/GalNAc specific receptor. The Core-specific lectin, synthesized and secreted by hepatocytes, is a soluble lectin found in plasma. Its synthesis, post-translational modification, and assembly will be examined. The basis for the slow kinetics of movement from the Golgi to the medium will be established to determine if this lectin has an intracellular as well as an extracellular function. Endogenous ligands for this lectin will be identified in either Golgi or plasma and characterized. Structural characterization of a number of glycoproteins will be carried out including the endogenous ligands for the Gal/GalNAc-specific receptor and the core specific lectin, a platelet Alpha-granule specific membrane protein expressed at the platelet surface only after stimulation of the release reaction, and endosome specific membrane proteins. The presence of unique structures may reflect their highly restricted location and may have functional significance.

本研究的主要目的是研究蛋白质糖基化在 将糖蛋白定向到特定的细胞内和细胞外部位。 对单个寡糖的识别需要两种唯一的 低聚糖结构和特定受体。其中任何一项的更改 寡糖结构或受体特异性可能导致病理 表现为发育缺陷、生长调节改变、 或改变新陈代谢。两种性质明显不同的凝集素形式 是我们研究的重点，并被用作基本工艺的原型 参与识别和运输。Gal/GalNAc特异性受体是 一种膜蛋白，介导半乳糖蛋白的摄取 由肝细胞从血浆中分离出来。内源性配体将被确定并 使用依赖于配基识别的印迹技术来表征的 通过纯化的受体。低聚糖的结构，它们的 将确定其起源和功能意义。两国关系 亚基组装到配体结合和受体运动的调节 在内吞过程中，将用溶解的受体和活的 细胞。介导摄取的内吞囊泡的成分将是 乳过氧化物酶结合物载体标记法鉴定 通过受体传递到这些囊泡。内吞囊泡和血浆 膜囊泡将通过高磁梯度分离来分离 在交付20-40 nm磁性铁-葡聚糖颗粒后，将 用于表征Gal/GalNAc特异性的摄取过程 受体。核心特异凝集素，由合成和分泌 肝细胞，是一种存在于血浆中的可溶性凝集素。它的合成， 将检查翻译后修改和组装。其基础是 因为从高尔基体到介质的缓慢运动将是 建立以确定该凝集素是否具有细胞内以及 细胞外功能。这种凝集素的内源性配体将是 在高尔基体或血浆中鉴定并表征的。结构性 将对一些糖蛋白进行鉴定，包括 Gal/GalNAc特异性受体及其核心的内源性配体 一种血小板α颗粒特异性膜蛋白--特异性凝集素 仅在刺激释放后才在血小板表面表达 反应，以及内体特异性膜蛋白。独特的存在 结构可能反映其高度受限的位置，并可能具有 功能意义。