Genetic Modulation of Blood and Vascular Glycosylation

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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.