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Open Access Publications from the University of California

Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UC San Francisco Electronic Theses and Dissertations (2013)

The addition of sulfate to glycan structures can regulate their ability to serve as ligands for glycan-binding proteins. While sulfate groups present on the monosaccharides glucosamine, uronate, N-acetylglucosamine, and N-acetylgalactosamine are recognized by defined receptors that mediate important functions, the functional significance of galactose-6-O-sulfate (Gal6S) is not known. However, in vitro studies using synthetic glycans and sulfotransferase overexpression suggest that Gal6S is a recognition determinant for the lymphocyte homing receptor, L-selectin. Additionally, glycan array studies have implicated Gal6S as a recognition determinant for the inhibitory sialic acid-binding receptor, Siglec-F. Only two sulfotransferases have been shown to generate Gal6S, namely keratan sulfate galactose 6-O-sulfotransferase (KSGal6ST) and chondroitin 6-O-sulfotransferase-1 (C6ST-1). I have used mice deficient in KSGal6ST and C6ST-1 to test whether Gal6S contributes to ligand recognition by L-selectin and Siglec-F in vivo.

First, I establish that KSGal6ST is selectively expressed in lymph node high endothelial venules (HEVs), and generates Gal6S-containing glycans in lymph nodes, including 6,6'-disulfo-3'sLN (Sia2,3[6S]Gal1,4[6S]GlcNAc) in HEVs. Nevertheless, short-term homing of lymphocytes to lymph nodes is normal in KSGal6ST-deficient mice, indicating that the Gal6S-containing structures I detected do not contribute to L-selectin ligand recognition in this setting.

Next, I characterize Siglec-F ligand expression on leukocyte populations and find that this expression is largely restricted to cell types also expressing Siglec-F, namely eosinophils, neutrophils, and alveolar macrophages. I also demonstrate the presence of Siglec-F ligand activity in bronchoalveolar lavage fluid fractions containing polymeric secreted mucins. Surprisingly, none of the ligands I describe are diminished in mice lacking KSGal6ST and C6ST-1, arguing against the prevailing view that Gal6S is a critical recognition determinant for this receptor. These results refine our understanding of the biological ligands for L-selectin and Siglec-F, and introduce a mouse model for investigating the functions of Gal6S in other contexts.

Lastly, I explore the mechanisms controlling eosinophil accumulation in response to parasitic nematodes in vitro and in vivo. My studies reveal that sensing and synthesis of leukotriene B4 is required for normal eosinophil accumulation in response to N. brasiliensis. Thus, nematodes induce leukotriene production in eosinophils, and this mediator is subsequently required to amplify eosinophil accumulation.

    Cover page: CARBOHYDRATE BINDING RECEPTORS IN LEUKOCYTE MIGRATION AND ACTIVATION
    • Article
    • Peer Reviewed
    UC Santa Cruz Previously Published Works (2024)
    The intestinal lumen is a turbulent, semi-fluid landscape where microbial cells and nutrient-rich particles are distributed with high heterogeneity. Major questions regarding the basic physical structure of this dynamic microbial ecosystem remain unanswered. Most gut microbes are non-motile, and it is unclear how they achieve optimum localization relative to concentrated aggregations of dietary glycans that serve as their primary source of energy. In addition, a random spatial arrangement of cells in this environment is predicted to limit sustained interactions that drive co-evolution of microbial genomes. The ecological consequences of random versus organized microbial localization have the potential to control both the metabolic outputs of the microbiota and the propensity for enteric pathogens to participate in proximity-dependent microbial interactions. Here, we review evidence suggesting that several bacterial species adopt organized spatial arrangements in the gut via adhesion. We highlight examples where localization could contribute to antagonism or metabolic interdependency in nutrient degradation, and we discuss imaging- and sequencing-based technologies that have been used to assess the spatial positions of cells within complex microbial communities.
      Cover page: Bacterial interactions on nutrient-rich surfaces in the gut lumen.
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2013)
      Eosinophil accumulation is a characteristic feature of the immune response to parasitic worms and allergens. The cell surface carbohydrate-binding receptor Siglec-F is highly expressed on eosinophils and negatively regulates their accumulation during inflammation. Although endogenous ligands for Siglec-F have yet to be biochemically defined, binding studies using glycan arrays have implicated galactose 6-O-sulfate (Gal6S) as a partial recognition determinant for this receptor. Only two sulfotransferases are known to generate Gal6S, namely keratan sulfate galactose 6-O-sulfotransferase (KSGal6ST) and chondroitin 6-O-sulfotransferase 1 (C6ST-1). Here we use mice deficient in both KSGal6ST and C6ST-1 to determine whether these sulfotransferases are required for the generation of endogenous Siglec-F ligands. First, we characterize ligand expression on leukocyte populations and find that ligands are predominantly expressed on cell types also expressing Siglec-F, namely eosinophils, neutrophils, and alveolar macrophages. We also detect Siglec-F ligand activity in bronchoalveolar lavage fluid fractions containing polymeric secreted mucins, including MUC5B. Consistent with these observations, ligands in the lung increase dramatically during infection with the parasitic nematode, Nippostrongylus brasiliensis, which is known to induce eosinophil accumulation and mucus production. Surprisingly, Gal6S is undetectable in sialylated glycans from eosinophils and BAL fluid analyzed by mass spectrometry. Furthermore, none of the ligands we describe are diminished in mice lacking KSGal6ST and C6ST-1, indicating that neither of the known galactose 6-O-sulfotransferases is required for ligand synthesis. These results establish that ligands for Siglec-F are present on several cell types that are relevant during allergic lung inflammation and argue against the widely held view that Gal6S is critical for glycan recognition by this receptor.
        Cover page: Galactose 6-O-Sulfotransferases Are Not Required for the Generation of Siglec-F Ligands in Leukocytes or Lung Tissue*
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2013)
        The addition of sulfate to glycan structures can regulate their ability to serve as ligands for glycan-binding proteins. Although sulfate groups present on the monosaccharides glucosamine, uronate, N-acetylglucosamine and N-acetylgalactosamine are recognized by defined receptors that mediate important functions, the functional significance of galactose-6-O-sulfate (Gal6S) is not known. However, in vitro studies using synthetic glycans and sulfotransferase overexpression implicate Gal6S as a binding determinant for the lymphocyte homing receptor, L-selectin. Only two sulfotransferases have been shown to generate Gal6S, namely keratan sulfate galactose 6-O-sulfotransferase (KSGal6ST) and chondroitin 6-O-sulfotransferase-1 (C6ST-1). In the present study, we use mice deficient in KSGal6ST and C6ST-1 to test whether Gal6S contributes to ligand recognition by L-selectin in vivo. First, we establish that KSGal6ST is selectively expressed in high endothelial venules (HEVs) in lymph nodes and Peyer's patches. We also determine by mass spectrometry that KSGal6ST generates Gal6S on several classes of O-glycans in peripheral lymph nodes. Furthermore, KSGal6ST, but not C6ST-1, is required for the generation of the Gal6S-containing glycan, 6,6'-disulfo-3'sLN (Siaα2→3[6S]Galβ1→4[6S]GlcNAc) or a closely related structure in lymph node HEVs. Nevertheless, L-selectin-dependent short-term homing of lymphocytes is normal in KSGal6ST-deficient mice, indicating that the Gal6S-containing structures we detected do not contribute to L-selectin ligand recognition in this setting. These results refine our understanding of the biological ligands for L-selectin and introduce a mouse model for investigating the functions of Gal6S in other contexts.
          Cover page: KSGal6ST generates galactose-6-O-sulfate in high endothelial venules but does not contribute to L-selectin-dependent lymphocyte homing
          • Article
          • Peer Reviewed
          UC Davis Previously Published Works (2021)
          Greater understanding of the spatial relationships between members of the human gut microbiota and available nutrients is needed to gain deeper insights about community dynamics and expressed functions. Therefore, we generated a panel of artificial food particles with each type composed of microscopic paramagnetic beads coated with a fluorescent barcode and one of 60 different dietary or host glycan preparations. Analysis of 160 Bacteroides and Parabacteroides strains disclosed diverse strain-specific and glycan-specific binding phenotypes. We identified carbohydrate structures that correlated with binding by specific bacterial strains in vitro and noted strain-specific differences in the catabolism of glycans that mediate adhesion. Mixed in vitro cultures revealed that these adhesion phenotypes are maintained in more complex communities. Additionally, orally administering glycan beads to gnotobiotic mice confirmed specificity in glycan binding. This approach should facilitate analyses of how strains occupying the same physical niche interact, and it should advance the development of synbiotics, more nutritious foods, and microbiota-based diagnostics.
            Cover page: Strain-level functional variation in the human gut microbiota based on bacterial binding to artificial food particles
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