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Articles by David F. Smith
Total Records ( 3 ) for David F. Smith
  Robert J. Luallen , Jianqiao Lin , Hu Fu , Karen K. Cai , Caroline Agrawal , Innocent Mboudjeka , Fang- Hua Lee , David Montefiori , David F. Smith , Robert W. Doms and Yu Geng
  The glycan shield of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) protein serves as a barrier to antibody-mediated neutralization and plays a critical role in transmission and infection. One of the few broadly neutralizing HIV-1 antibodies, 2G12, binds to a carbohydrate epitope consisting of an array of high-mannose glycans exposed on the surface of the gp120 subunit of the Env protein. To produce proteins with exclusively high-mannose carbohydrates, we generated a mutant strain of Saccharomyces cerevisiae by deleting three genes in the N-glycosylation pathway, Och1, Mnn1, and Mnn4. Glycan profiling revealed that N-glycans produced by this mutant were almost exclusively Man8GlcNAc2, and four endogenous glycoproteins that were efficiently recognized by the 2G12 antibody were identified. These yeast proteins, like HIV-1 gp120, contain a large number and high density of N-linked glycans, with glycosidase digestion abrogating 2G12 cross-reactivity. Immunization of rabbits with whole Δoch1 Δmnn1 Δmnn4 yeast cells produced sera that recognized a broad range of HIV-1 and simian immunodeficiency virus (SIV) Env glycoproteins, despite no HIV/SIV-related proteins being used in the immunization procedure. Analyses of one of these sera on a glycan array showed strong binding to glycans with terminal Manα1,2Man residues, and binding to gp120 was abrogated by glycosidase removal of high-mannose glycans and terminal Manα1,2Man residues, similar to 2G12. Since S. cerevisiae is genetically pliable and can be grown easily and inexpensively, it will be possible to produce new immunogens that recapitulate the 2G12 epitope and may make the glycan shield of HIV Env a practical target for vaccine development.
  Alex S. Powlesland , Tanja Fisch , Maureen E. Taylor , David F. Smith , Berangere Tissot , Anne Dell , Stefan Pohlmann and Kurt Drickamer
  LSECtin is a member of the C-type lectin family of glycan-binding receptors that is expressed on sinusoidal endothelial cells of the liver and lymph nodes. To compare the sugar and pathogen binding properties of LSECtin with those of related but more extensively characterized receptors, such as DC-SIGN, a soluble fragment of LSECtin consisting of the C-terminal carbohydrate-recognition domain has been expressed in bacteria. A biotin-tagged version of the protein was also generated and complexed with streptavidin to create tetramers. These forms of the carbohydrate-recognition domain were used to probe a glycan array and to characterize binding to oligosaccharide and glycoprotein ligands. LSECtin binds with high selectivity to glycoproteins terminating in GlcNAcβ1-2Man. The inhibition constant for this disaccharide is 3.5 µM, making it one of the best low molecular weight ligands known for any C-type lectin. As a result of the selective binding of this disaccharide unit, the receptor recognizes glycoproteins with a truncated complex and hybrid N-linked glycans on glycoproteins. Glycan analysis of the surface glycoprotein of Ebola virus reveals the presence of such truncated glycans, explaining the ability of LSECtin to facilitate infection by Ebola virus. High mannose glycans are also present on the viral glycoprotein, which explains why DC-SIGN also binds to this virus. Thus, multiple receptors interact with surface glycoproteins of enveloped viruses that bear different types of relatively poorly processed glycans.
  Sean R. Stowell , Connie M. Arthur , Kristin A. Slanina , John R. Horton , David F. Smith and Richard D. Cummings
  Human galectins have distinct and overlapping biological roles in immunological homeostasis. However, the underlying differences among galectins in glycan binding specificity regulating these functions are unclear. Galectin-8 (Gal-8), a tandem repeat galectin, has two distinct carbohydrate recognition domains (CRDs) that may cross-link cell surface counter receptors. Here we report that each Gal-8 CRD has differential glycan binding specificity and that cell signaling activity resides in the C-terminal CRD. Full-length Gal-8 and recombinant individual domains (Gal-8N and Gal-8C) bound to human HL60 cells, but only full-length Gal-8 signaled phosphatidylserine (PS) exposure in cells, which occurred independently of apoptosis. Although desialylation of cells did not alter Gal-8 binding, it enhanced cellular sensitivity to Gal-8-induced PS exposure. By contrast, HL60 cell desialylation increased binding by Gal-8C but reduced Gal-8N binding. Enzymatic reduction in surface poly-N-acetyllactosamine (polyLacNAc) glycans in HL60 cells reduced cell surface binding by Gal-8C but did not alter Gal-8N binding. Cross-linking and light scattering studies showed that Gal-8 is dimeric, and studies on individual subunits indicate that dimerization occurs through the Gal-8N domain. Mutations of individual domains within full-length Gal-8 showed that signaling activity toward HL60 cells resides in the C-terminal domain. In glycan microarray analyses, each CRD of Gal-8 showed different binding, with Gal-8N recognizing sulfated and sialylated glycans and Gal-8C recognizing blood group antigens and polyLacNAc glycans. These results demonstrate that Gal-8 dimerization promotes functional bivalency of each CRD, which allows Gal-8 to signal PS exposure in leukocytes entirely through C-terminal domain recognition of polyLacNAc glycans.
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