The primary objective of the proposal is to elucidate the roles of O-glycan core 2 branchings in development, formation of selectin ligands, and the control of glycosylation in normal and tumor cells. In the past few years, the investigators have made critical progress in this field. First, they have cloned cDNAs encoding core 2 and I beta-1,6-N- acetylglucosaminyltransferases which form beta-1,6-N- acetylglucosaminyl branches. The primary structure of these two enzymes revealed that they are homologous to each other in their catalytic domains. Second, they demonstrated that the formation of poly-N-acetyllactosaminyl repeats and sialyl Lex in O-glycans are entirely dependent on branches formed by the core 2 beta-1,6-N-acetylglucosaminyltransferase, core 2 GnT. Third, they discovered that the formation and disappearance of core 2 branches are closely associated with thymocyte differentiation. Based on these findings, four major areas for further study are proposed as follows: 1) Elucidation of roles of core 2 GnT in presentation of selectin ligands. They will study the expression of O-glycan sialyl Lex directed by core 2 GnT and determine the roles of such structures and carrier glycoproteins in presentation of ligands for E- and P-selectins. 2) Elucidation of the role of core 2 branching in embryonic development. The studies will determine the roles of core 2 branched O-glycans in mouse development by ectopic expression, and systematic and tissue- specific knock- out of core 2 GnT gene. 3) Subcellular localization of core 2 GnT and its role in the control of glycosylation. The studies will determine the subcellular localization of core 2 GnT and evaluate its effect on the control of glycosylation by the manipulation of its subcellular localization. 4) Isolation of core 4 GnT gene and its expression in normal and cancerous colon cells. They will isolate core 4 GnT gene, which is related to the core 2 GnT gene and determine the roles of core 2 GnT and core 4 GnT in glycosylation of normal and cancerous colon cells. These studies will allow us to understand the physiological roles of core 2- based O-glycans during development and oncogenesis.
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