FUNCTION CORE 1-DERIVED O-GLYCANS VASCULAR DEVELOPMENT

Oklahoma Medical Research Foundation, Oklahoma City OK

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Abstract

Serine- or threonine-linked oligosaccharides (O-glycans) are commonly found on membrane and secreted proteins. The functions of this form of posttranslational modification are not well understood. Our experiments indicate that there is a fundamental requirement for core 1-derived O-glycans during angiogenesis. Because vessel development is essential to many physiologic and pathologic processes such as cancer, ischemic diseases, and chronic inflammation, elucidating the role of O-glycans in angiogenesis will potentially provide new insight into numerous disease processes and may lead to novel therapeutic approaches to many common human diseases. O-glycans with GalNAc in a1 linkage to serine or threonine have four main core structures. Among them, core 1 and 2 are widely expressed in many tissues. Formation of the core 1 structure (GalB1-3GalNAc-), the precursor for the core 2 structure and for many extended O-glycans (core 1-derived O-glycans), is catalyzed by the enzyme core 1 pi ,3-galactosyltransferase (T-synthase). We have engineered mice that are globally deficient for T-synthase (T-syn[-/-]). The T-syn[-/-] mice developed brain hemorrhage that was uniformly fatal by embryonic day 14. The T-syn[-/-] brains formed a disorganized microvascular network with distended endothelial cells and defective association of endothelial cells with pericytes, extracellular matrix and neural tissues. These data reveal a novel requirement for core 1-derived O-glycans during angiogenesis. We have developed mice with tissue-specific T-syn deficiencies using Cre/loxP technology. We propose to investigate the role of core 1-derived O-glycans in vascular development through three specific aims: Aim 1: Identify cell types requiring O-glycans for angiogenesis. Blood vessels are composed of endothelial cells and mural cells (pericytes and vascular smooth muscle cells) embedded in the extracelluar matrix and surrounding tissues. O-glycans associated with any of these cell types may contribute to the defective angiogenesis observed in the T-syn[-/-] mice. We will use mice with endothelial cell-specific, neural cell-specific, and mural cell-specific T-syn deficiencies to identify the cell type(s) that cause defective angiogenesis when lacking T-synthase. In addition, we have developed transgenic mouse lines expressing T-synthase specifically in endothelial cells. By breeding these mice with T-syn[-/-] mice, we will examine whether the endothelial cell specific expression of T-synthase can rescue the T-syn[-/-] embryonic lethality. Aim 2: Characterize the functional defect of T-syn[-/-] endothelial cells during angiogenesis. Endothelial cells are essential in angiogenesis. During angiogenesis, endothelial cells undergo multiple processes including migration, proliferation, apoptosis, and specialization. Our published and preliminary experiments suggest that O-glycans in endothelial cells play a key role during vessel formation. We will use in-vivo assays such as retina angiogenesis and tumor angiogenesis to identify specific defect(s) of T-syn[-/-] endothelial cells in vascular development. In addition, we have developed T-syn[+/+]and T-syn[-/-] endothelial cell lines to evaluate the function of T-syn[-/-] endothelial cells using in-vitro assays such as endothelial cell tube formation. Aim 3: Analyze the profiles and function of O-glycans in endothelial cells. We will combine lectins and mAb screening, as well as HPLC and mass spectrometry, to profile the O-glycan structures in T-syn[-/-] and T-syn[-/-] endothelial cells. We will investigate how differences in O-glycan expression affect endothelial cell function using in-vitro angiogenesis assay.