GENETIC MODULATION OF BLOOD AND VASCULAR GLYCOSYLATION

寡核苷酸，如糖蛋白的N-和0-连接链 蛋白聚糖的硫酸乙酰肝素链是主要的结构 细胞和组织的组成部分，其复杂性可与 核酸和蛋白质。该计划旨在探索 两种阴离子在血液和脉管系统中的生物学作用 这些糖链的修饰：唾液酸和N-硫酸化。 许多血浆中N-和O-连接糖链的结构 糖蛋白和血细胞，并且是众所周知的。特异性凝集素 例如选择素（在白细胞、血小板和内皮上）， I型凝集素（在B细胞、巨噬细胞或骨髓前体上）， 和补体途径中的H蛋白， 在唾液酸残基的背景下识别这些链。 硫酸乙酰肝素糖胺聚糖链的N-硫酸酯是 被参与血液凝固的各种蛋白质、细胞 生长、血小板相互作用和白细胞迁移。尽管有许多 提示，唾液酸化和N-硫酸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 在模式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基因。我们 实验室是第一批从基因上解剖体内 糖基化在小鼠中的作用，并开发新技术 用于有效分析小鼠组织中的寡糖。因此，在本发明中， 这项建议的中心主题是基因操纵， 唾液酸和N-硫酸酯。当 如果需要，我们将使用我们最近开发的方法， 以细胞类型特异性方式扩增小鼠基因。更换 野生型等位基因与携带loxP靶标的重组等位基因 在无害位置的位点允许选择性基因缺失， 与Cre重组酶构建体转基因小鼠交配 通过特定的转录控制序列。这使得具体 专注于血细胞、内皮细胞和血浆蛋白， 在系统性基因失活模型是 无法存活我们也有必要的专业知识来分析 基因操作对结构的影响 造血、血管和淋巴组织， 止血，寡糖的结构，以及 造血和淋巴细胞生物学的功能后果。 这些研究预计将揭示许多重要的功能， 低聚糖对健康和疾病的影响。

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.