Genetic Modulation of Blood and Vascular Glycosylation

碳水化合物链（聚糖）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。该计划的重点是在细胞表面糖萼的最外层发现的两大类阴离子聚糖-乙酰肝素和硫酸皮肤素蛋白聚糖的唾液酸（Sins）和糖胺聚糖（GAG）链。血细胞和血浆糖蛋白的N-和O-连接聚糖的结构是迄今为止描述最好的。特异性聚糖结合蛋白可区别识别这些链上的Sins，包括选择素（白细胞、血小板和内皮细胞上）和Siglecs（在特定血细胞类型上发现的具有胞质信号传导基序的I型凝集素）。一些13-半乳糖苷特异性凝集素也可以检测Sias的缺乏。参与止血和血栓形成的一些蛋白质的唾液酸化的变化可以改变它们的周转和功能。乙酰肝素和硫酸皮肤素蛋白聚糖的GAG链参与调节过程，例如血液凝固，生长因子调节， 内皮生物学、伤口修复和白细胞迁移。Sias和GAG的大多数生理和病理作用在培养细胞中并不明显，但必须在完整的生物体中进行探索-哺乳动物聚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，已知这些分子中相对较少的人糖基化缺陷。因此，该提案的中心主题是对完整小鼠中的Sins、GAG链及其一些同源结合蛋白进行最先进的遗传操作。当系统性基因失活模型不能存活或具有混乱的表型时，我们将选择性地在细胞类型特异性和发育调控的细胞中表达小鼠基因。 方式用在无害位置携带loxp靶位点的重组等位基因替换野生型等位基因允许细胞类型特异性基因驱逐，通过与由特异性转录控制序列驱动的Cre重组酶构建体转基因小鼠交配。这也将允许特别关注血细胞、内皮和血浆蛋白的聚糖。我们已经收集了必要的专业知识，以充分分析这些遗传操作对造血和血管组织结构的影响，聚糖的结构，以及对止血，血管功能，血管生成，造血，炎症，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聚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.