The mammalian genome harbors several hundred genes that function to generate the diverse and dynamic oligosaccharide repertoire displayed on all cell surfaces. As a result of variations in glycosyltransferase gene expression, oligosaccharide diversification is highly regulated in mammalian development, among specific cell types, during normal physiologic responses, and in metastatic cell behavior. Previous studies have indicated that both T lymphocyte and neuronal cell populations are functionally affected in vitro by chances in their repertoire of asparagine (N)-linked oligosaccharides. Neuronal and T lymphocyte cell types are similar in that they engage in cellular interactions involving adhesion and trafficking among various compartments of the intact organism. This research proposal is based upon the hypothesis that N-linked oligosaccharides of hybrid or complex types are crucial for the development and function of the neural and immune systems of vertebrates. The key enzymes in the conversion of high-mannose N-linked oligosaccharides into hybrid and complex forms are Mgat1-encoded GlcNAc-TI, alpha-mannosidase-II (alphaM-II) and Mgat2- encoded GlcNAc-TII. The role of hybrid and complex type oligosaccharide structures will be investigated by inactivating these above enzymes in the intact mouse by gene-targeting and by cell-type specific gene mutagenesis using previously developed Cre recombinase transgenic lines.
The Specific Aims i nclude the (1) cell type-specific investigation of hybrid and complex N-linked oligosaccharide function by inactivating the Mgat1 gene in neuronal and T lymphoid lineages in vivo. This will involve modifying the Mgat1 locus by homologous recombination to allow for mutagenic inactivation by Cre promoters. Subsequent to Mgat1 function, alphaM-II and Mgat2 act in N-glycan diversification by converting hybrid into complex forms. In humans, dysfunction or inactivation of these enzymes occurs in Congenital Dyserythropoietic Anemia (CDA) Type II and Carbohydrate Deficient Glycoprotein Syndrome (CDGS) Type II, respectively. This proposal includes an investigation of (2) alpha-mannosidase function in dyserythropoietic anemia and N- glycan diversification by inactivation of the alphaM-II gene in vivo. As CDGS Type II involves neurologic symptoms and susceptibility to infection, this proposal will (3) investigate the physiologic role of the Mgat2 gene and complex N-linked oligosaccharides in T lymphoid and neuronal cells by gene-targeting, concurrently producing an Mgat2- deficient mouse that may also provide a model of human CDGS Type II.
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