UCSF

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.