Novel Developmental, Cellular and Biochemical Functions of Fucosylated Glycans in Mammals.

Cell surface glycans are branched structures composed of linear and branched chains of monosaccharides that include fucose. Fucose has important biological functions during ontogeny and cellular differentiation, suggested by pathological phenotypes observed in different strains of mice, including mice with a null mutation in the 3’, 5’-epimerase/4’-reductase locus (FX-/- mice). FX-/- mice are conditionally deficient in all fucosylated glycans, and exhibit server thymic atrophy. This phenotype is cell autonomous and characterized by a fucose-dependent complete deficiency of mature T cell with loss of early thymic progenitors. Interestingly, Notch1 signaling deficient mouse models have been reported with the similar phenotypes, and there are reports that O-fucosylation is required in some in vitro experiments for Notch1 signaling. These results strongly suggested that lack of Notch1 signaling in FX-/- progenitors accounts for the thymic atrophy phenotype. By introducing the Notch1 intracellular domain into fucose-deficient FX-/- bone marrow-derived lymphoid progenitors, the T cell developmental defect characteristic of FX-/- lymphoid progenitors is rescued and thus implicated a fucose-dependent requirement for Notch1 signaling in this process. In vitro, OP9 cells that are bearing Notch ligand of Delta-like1 (Dll-1), Delta-like 4 (Dll-4) or Jagged2, instruct cells to assume a T lymphoid differentiation identity, whereas while OP9 cells bearing Jagged1 or Dll3 do not. However, in fucose-deficient experimental situation, FX-/- bone marrow cells fail to assume a T lineage identity when co-cultured with OP9-Dll1, OP9-Dll4 or OP9-Jagged2, and fail to initiate Notch1 signaling events. These results indicate that fucosylation is required for Notch1 signaling-dependent T cell differentiation. In effort to define the molecular mechanisms that account for fucosylation-controlled Notch1 signaling activation, I conclude from a series of experiments that (1) fucosylation controls the strength of binding between Notch1 and its ligand Dll4, and (2) fucosylation controls Notch1 receptor density at the surface of an E2a/Pbx1 immortalized bone marrow progenitor cell line. These results indicate that fucosylation controls Notch1 signaling strength and thus regulates the development of T lymphocytes. My studies have revealed novel biochemical, cellular and developmental functions of fucosylation in the development and signaling pathways characteristic of the mammalian lymphoid lineage.Ph.D.PathologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/57652/2/many_1.pd

Evidence for a functional role of the second C5a receptor C5L2

During experimental sepsis in rodents after cecal ligation and puncture (CLP), excessive C5a is generated, leading to interactions with C5aR, loss of innate immune functions of neutrophils, and lethality. In the current study, we have analyzed the expression of the second C5a receptor C5L2, the putative â defaultâ or nonsignaling receptor for C5a. Rat C5L2 was cloned, and antibody was developed to C5L2 protein. After CLP, blood neutrophils showed a reduction in C5aR followed by its restoration, while C5L2 levels gradually increased, accompanied by the appearance of mRNA for C5L2. mRNA for C5L2 increased in lung and liver during CLP. Substantially increased C5L2 protein (defined by binding of 125Iâ antiâ C5L2 IgG) occurred in lung, liver, heart, and kidney after CLP. With the use of serum ILâ 6 as a marker for sepsis, infusion of antiâ C5aR dramatically reduced serum ILâ 6 levels, while antiâ C5L2 caused a nearly fourfold increase in ILâ 6 when compared with CLP controls treated with normal IgG. When normal blood neutrophils were stimulated in vitro with LPS and C5a, the antibodies had similar effects on release of ILâ 6. These data provide the first evidence for a role for C5L2 in balancing the biological responses to C5a.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154410/1/fsb2fj043424fje.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154410/2/fsb2fj043424fje-sup-0040.pd

O-Fucose Modulates Notch-Controlled Blood Lineage Commitment

Notch receptors are cell surface molecules essential for cell fate determination. Notch signaling is subject to tight regulation at multiple levels, including the posttranslational modification of Notch receptors by O-linked fucosylation, a reaction that is catalyzed by protein O-fucosyltransferase-1 (Pofut1). Our previous studies identified a myeloproliferative phenotype in mice conditionally deficient in cellular fucosylation that is attributable to a loss of Notch-dependent suppression of myelopoiesis. Here, we report that hematopoietic stem cells deficient in cellular fucosylation display decreased frequency and defective repopulating ability as well as decreased lymphoid but increased myeloid developmental potential. This phenotype may be attributed to suppressed Notch ligand binding and reduced downstream signaling of Notch activity in hematopoietic stem cells. Consistent with this finding, we further demonstrate that mouse embryonic stem cells deficient in Notch1 (Notch1−/−) or Pofut1 (Pofut1−/−) fail to generate T lymphocytes but differentiate into myeloid cells while coculturing with Notch ligand–expressing bone marrow stromal cells in vitro. Moreover, in vivo hematopoietic reconstitution of CD34+ progenitor cells derived from either Notch1−/− or Pofut1−/− embryonic stem cells show enhanced granulopoiesis with depressed lymphoid lineage development. Together, these results indicate that Notch signaling maintains hematopoietic lineage homeostasis by promoting lymphoid development and suppressing overt myelopoiesis, in part through processes controlled by O-linked fucosylation of Notch receptors

Notch-dependent control of myelopoiesis is regulated by fucosylation

Cell-cell contact–dependent mechanisms that modulate proliferation and/or differentiation in the context of hematopoiesis include mechanisms characteristic of the interactions between members of the Notch family of signal transduction molecules and their ligands. Whereas Notch family members and their ligands clearly modulate T lymphopoietic decisions, evidence for their participation in modulating myelopoiesis is much less clear, and roles for posttranslational control of Notch-dependent signal transduction in myelopoiesis are unexplored. We report here that a myeloproliferative phenotype in FX−/− mice, which are conditionally deficient in cellular fucosylation, is consequent to loss of Notch-dependent signal transduction on myeloid progenitor cells. In the context of a wild-type fucosylation phenotype, we find that the Notch ligands suppress myeloid differentiation of progenitor cells and enhance expression of Notch target genes. By contrast, fucosylation-deficient myeloid progenitors are insensitive to the suppressive effects of Notch ligands on myelopoiesis, do not transcribe Notch1 target genes when cocultured with Notch ligands, and have lost the wild-type Notch ligand-binding phenotype. Considered together, these observations indicate that Notch-dependent signaling controls myelopoiesis in vivo and in vitro and identifies a requirement for Notch fucosylation in the expression of Notch ligand binding activity and Notch signaling efficiency in myeloid progenitors