GENETIC MODULATION OF BLOOD AND VASCULAR GLYCOSYLATION

低聚糖，如糖蛋白的N-链和0-链 蛋白多糖的硫酸乙酰肝素链是主要的结构 细胞和组织的组成，其复杂性可与 核酸和蛋白质。该计划的重点是探索 两种阴离子在血液和血管系统中的生物学作用 这些糖链的修饰：唾液酸和N-硫酸。 许多血浆中N-和0-连接糖链的结构 糖蛋白和血细胞，是众所周知的。特定凝集素 如选择素(在白细胞、血小板和内皮细胞上)， I型凝集素(在B细胞、巨噬细胞或髓系前体细胞上)， 和补体途径的H蛋白，可以区别 根据唾液酸残基识别这些链。 硫酸乙酰肝素糖胺多糖链的N-硫酸酯为 由多种参与凝血的蛋白质识别，细胞 生长、血小板相互作用和白细胞迁移。尽管有很多人 线索，唾液酸化和N-硫化的大多数生理作用不是 在培养的细胞中观察得很好，必须在完整的 有机体。然而，哺乳动物糖基化的这些特征并不是 在模型无脊椎动物中有很好的代表性，几乎没有遗传缺陷 在完整的哺乳动物中已经描述了影响这些途径的因素。我们的 实验室是最早对活体进行基因解剖的实验室之一。 糖基化在小鼠体内的作用及开发新技术 用于高效分析小鼠组织中的低聚糖。因此， 这项提议的中心主题是基因操纵 完整小鼠体内的唾液酸和N-硫酸酯。什么时候 需要，我们将使用我们最近开发的方法来选择性地 以特定细胞类型的方式使小鼠基因失活。更换 野生型等位基因与携带loxP靶标的重组等位基因的关系 位于无害位置的位置允许选择性基因缺失，通过 与转基因Cre重组酶构建体驱动的小鼠交配 通过特定的转录控制序列。这允许特定的 关注血细胞、内皮和血浆蛋白，并将 在系统性基因失活模型 行不通的。我们还拥有必要的专业知识来分析 基因操作对细胞结构的影响 造血、血管和淋巴组织的作用机制 止血，低聚糖的结构，以及 对造血和淋巴细胞生物学的功能影响。 这些研究预计将揭示许多重要的功能 低聚糖与健康和疾病。

Oligosaccharides such as the N- and 0-linked chains of glycoproteins and the heparan sulfate chains of proteoglycans are major structural components of cells and tissues, with a complexity rivaling that of nucleic acids and proteins. This Program is focused upon exploring the biological roles in the blood and vasculature of two anionic modifications of these sugar chains: sialic acid and N-sulfation. The structures of the N- and 0-linked sugar chains of many plasma glycoproteins and blood cells and are well known. Specific lectins such as the selectins (on leucocytes, platelets and endothelium), the I-type lectins (on B cells, macrophages or myeloid precursors), and the H protein of the complement pathway, can differentially recognize these chains in the context of their sialic acid residues. The N-sulfate esters of heparan sulfate glycosaminoglycan chains are recognized by various proteins involved in blood coagulation, cell growth, platelet interactions and leucocyte migration. Despite many clues, most physiologic roles of sialylation and N-sulfation are not well observed in cultured cells, and must be explored in the intact organism. However, these features of mammalian glycosylation are not well represented in model invertebrates, and few genetic defects affecting these pathways have been described in intact mammals. Our labs have been among the first to genetically dissect the in vivo roles of glycosylation in the mouse and to develop new technologies for efficient analysis of oligosaccharides from mouse tissues. Thus, the central theme of this proposal is the genetic manipulation of sialic acids and N-sulfate esters in the intact mouse. When required, we will use our recently developed method to selectively inactivate mouse genes in a cell type-specific manner. Replacement of wild type alleles with recombinant ones carrying loxP target sites at innocuous positions allows selective gene deletion, by mating with mice transgenic for Cre recombinase constructs driven by specific transcriptional control sequences. This allows specific focus on blood cells, endothelium and plasma proteins, and will be important in instances where systemic gene inactivation models are non-viable. We also have the necessary expertise to analyze the consequences of the genetic manipulations on the structure of hematopoietic, vascular and lymphoid tissues, the mechanisms of hemostasis, the structure of the oligosaccharides, and the functional consequences to hematopoiesis and lymphocyte biology. These studies are expected to reveal many important functions for oligosaccharides in health and disease.