Control of Leukocyte Biology by Fucosylated Glycans

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Fucose is a monosaccharide that modifies N- and O-linked glycans on leukocytes, endothelial cells and epithelia. Alpha1,3fucosylated glycans are essential components of the counter-receptors for E-, P-, and Lselectin, and thus control leukocyte trafficking in health and disease. Fucose linked to serine and threonine (O-linked fucose) is characteristic of some EGF-like repeats of Notch and its ligands, and contributes to Notch-dependent signal transduction events in development and immunity. These fucosylated glycans, and the genes that control their expression represent pivotal targets for therapeutic regulation of the formation of leukocytes that contribute to selectin-dependent immunity and inflammation, and that may be used for cell-based treatment of cancer. Analyses of mice with targeted deficiencies in genes that control alpha1,3fucosylation (FucT-IV/VII-null mice) disclose a role for alpha1,3fucosylated glycans in the control of myelopoiesis. Mechanisms to account for such control are not yet known, and are a primary focus of this proposal. Analyses of mice with a targeted deficiency in a gene (FX) that allows conditional deficiency of general fucosylation (FX null mice) infer a role for O-fucosylation in the control of myelopoiesis. Mechanisms to account for such control are not yet known, and are also a primary focus of this proposal. Studies in Project II will focus in part on identifying the alpha1,3fucosylated selectin ligand-dependent signal transduction pathways that control myelopoiesis, using approaches that involve gene expression arrays and proteomics analyses. This work will include collaborations with Projects I (Dr. Minoru Fukuda) and III (Michiko Fukuda) to determine if glycan sulfation and N-glycosylation, respectively, contribute to the control of myelopoiesis. Project II will rely on the Intravital Microscopy Core to develop temporal and spatial morphological correlates for the molecular events that underlie normal and aberrant myelopoiesis. Project II will also study the role of O-fucosylation of Notchl in controlling myelopoiesis, using Notchdependent myelopoietic assays, and antibodies specific for 0-fucosylated peptides (from the Synthesis Core). Project II will also use atomic force microscopy methods with recombinant Notchl and Notch ligands to define the molecular basis for how the interactions between Notchl and its ligands are modulated by Olinked fucose, and by the glycans that modify O-linked fucose.