COBRE: OK MED RES FOUND: P4: IN VIVO FUNCTIONS OF GALACTOSYLTRANSFERASE IN MICE

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. O-glycosylation is a common post-translational modification on membrane and secreted proteins. Serine (Ser)- and threonine (Thr)-linked oligosaccharides (O-glycans) have four common subtypes named cores 1 through 4. The functions of O-glycans are not well understood. Particular attention has been paid to the actions of core 20-glycans in selectin-mediated leukocyte trafficking, but even these roles are not completely defined. Core 1, which is formed by the enzyme core 1 131,3-galactosyltransferase (C1GalT), is the precursor for many extended O-glycans, especially the core 2 structure. Using Cre/loxP-mediated gene targeting, we have developed mice with a global deficiency of C1GalT (C1GalT -/-) and mice with the C1GalT gene flanked by loxP sites (C1GalTta¿xed), which can be used to create conditional or tissue-specific deficiencies of C1GalT. Global deficiency of C1GalT causes embryonic bleeding and death at 14 days post coitum (dpc). We propose to use our gene-targeted mice to study the functions of core 1-based O-glycans in vivo through four specific aims: First: We will analyze C1GalT-deficient embryos to determine if bleeding is the cause of embryonic lethality and to determine whether bleeding results from defects in vascular integrity, abnormalities in endothelial or hematopoietic cells, or regulation of blood coagulation. Second, we will develop mice with tissue-specific C1GalT deficiencies by breeding the C1GalT ta¿xedmice with different Creexpressing transgenic mice. Third, we will examine the expression of O-glycans in wild-type and C1GalTdeficient mice by lectin and mAb staining and by glycan structural analysis using exoglycosidases and mass spectrometry. Fourth, we will assess the significance of C1GalT in leukocyte trafficking and lymphocyte homing. These studies will elucidate the contributions of O-glycans in development, inflammation, hemostasis, immune responses, and other biological functions in vivo.