Genetic Modulation of Blood and Vascular Glycosylation

糖链(多糖)是细胞和组织的主要组成部分，其复杂性和质量可与核酸和蛋白质相媲美。这个项目主要关注在细胞表面糖萼最外层发现的两大类阴离子多糖--乙酰肝素和硫酸皮肤素蛋白多糖的唾液酸(SINS)和糖胺聚糖(GAG)链。血细胞和血浆糖蛋白的N-和O-连接糖链的结构是迄今为止描述得最好的结构之一。特定的糖结合蛋白识别这些链上的SIN，包括选择素(在白细胞、血小板和内皮上)和Siglecs(具有胞浆信号基序的I型凝集素，发现于特定类型的血细胞上)。一些13-半乳糖苷特异性凝集素也可以检测到SIAs的缺失。一些参与止血和血栓形成的蛋白质唾液酸化的变化可以改变它们的周转和功能。肝素和硫酸皮肤素蛋白多糖的Gag链参与调节凝血、生长因子调节、 内皮生物学、伤口修复和白细胞迁移。大多数SIAs和GAG的生理和病理作用在培养细胞中并不明显，但必须在完整的有机体中进行探索--哺乳动物多糖的这种复杂性在模型无脊椎动物中并没有完全表现出来。另一方面，这些分子中相对较少的人类糖基化缺陷是已知的。因此，这项建议的中心主题是在完整的小鼠中对SIN、Gag链及其一些同源结合蛋白进行最先进的遗传操作。当系统基因失活模型不可行或具有令人困惑的表型时，我们将有选择地失活特定细胞类型和发育调节的小鼠基因。 举止。用在无害位置携带loxP靶点的重组等位基因替换野生型等位基因，通过与由特定转录控制序列驱动的Cre重组酶构建的转基因小鼠交配，允许细胞类型特异性基因驱逐。这也将允许特别关注血细胞、血管内皮细胞和血浆蛋白的多糖。我们已经汇集了必要的专业知识，全面分析了这些基因操作对造血和血管组织结构、多糖结构的影响，以及对止血、血管功能、血管生成、造血、炎症、对感染的先天免疫反应和伤口愈合的功能影响。这些研究有望揭示这些多糖在健康和疾病中的许多重要功能。

Carbohydrate chains (glycans) are major components of cells and tissues, with a complexity and mass rivaling nucleic acids and proteins. This program focuses on the two major classes of anionic glycans found at the outermost aspects of the cell surface glycocalyx - the sialic acids (Sins) and the glycosaminoglycan (GAG) chains of heparan and dermatan sulfate proteoglycans. The structures of the N- and O-linked glycans of blood cells and plasma glycoproteins are among the best described to date. Specific glycan-binding proteins differentially recognize Sins on these chains, including the selectins (on leukocytes, platelets and endothelium) and the Siglecs (I-type lectins with cytosolic signaling motifs, found on specific blood cell types). Some 13-galactoside-specific lectins can also detect the absence of Sias. Changes in the sialylation of some proteins involved in hemostasis and thrombosis can alter their turnover and function. The GAG chains of the heparan and dermatan sulfate proteoglycans are involved in regulating processes such as blood coagulation, growth factor modulation, endothelial biology, wound repair and leukocyte migration. Most of the physiologic and pathological roles of Sias and GAGs are not evident in cultured cells, but must be explored in the intact organism - and this complexity of mammalian glycans is not fully represented in model invertebrates. On the other hand, relatively few human glycosylation defects in these molecules are known. Therefore, the central theme of this proposal is state-of-the-art genetic manipulation of Sins, GAG chains, and some of their cognate binding proteins in the intact mouse. When systemic gene inactivation models are non-viable or have confusing phenotypes, we will selectively inactivate mouse genes in a cell type-specific and developmentally-regulated manner. Replacement of wild type alleles with recombinant alleles carrying loxp target sites at innocuous positions allows cell-type specific gene eviction, by mating with mice transgenic for Cre recombinase constructs driven by specific transcriptional control sequences. This will also allow a specific focus on glycans of blood cells, endothelium and plasma proteins. We have assembled the necessary expertise to fully analyze the consequences of these genetic manipulations on the structure of hematopoietic and vascular tissues, the structure of the glycans, and the functional consequences to hemostasis, vascular function, angiogenesis, hematopoiesis, inflammation, the innate immune response to infections, and wound healing. These studies are expected to reveal many important functions for these glycans in health and disease.