Leukocytes recruited to sites of inflammation represent critical components of the human inflammatory response. Aberrant recruitment of these cells can initiate and maintain pathological inflammation and tissue damage. Initial events in leukocyte recruitment involve adhesive interactions between members of the Selectin family of cell adhesion molecules, and counter-receptors on the surfaces of leukocytes. Soluble analogues of these counter-receptors thus represent candidate molecules for pharmacologic manipulation of aberrant leukocyte recruitment. Recent studies have identified a class of complex oligosaccharide determinants, represented by the sialyl Lewis x (sialyl Lex) tetrasaccharide molecule, that function as counter-receptors for E-selectin and P-selectin. Cloned fucosyltransferase genes have also been recently isolated that represent tools to construct these ligands. Nonetheless, there is currently only a rudimentary understanding of the biosynthetic, structural, and functional complexity of these oligosaccharide molecules, both as free entities, or as they typically exist in a covalent complex with the proteins that display them at cell surfaces. A more comprehensive understanding of these complexities is essential for logical exploration of the potential of these molecules in the pharmacologic manipulation of immune cell recruitment.
The specific aims of this section will address issues concerning the structural and biosynthetic properties of these molecules, and will provide, for Sections 2-4 of this Program Project, quantities of structurally-defined ligand molecules sufficient for functional testing in in vitro and in vivo assays that model the human inflammatory response. Three classes of such molecules will be investigated.
In Specific Aim #1, complex oligosaccharides isolated from a sialyl Lex-positive cell line will be fractionated and structurally defined. These fractionated molecules will also be provided to Sections 2-4 for functional testing.
In Specific Aim #2, a cloned recombinant fucosyltransferase will be used to construct milligram amounts of the sialyl Lex tetrasaccharide, which will be provided to Sections 2-4 for functional testing.
In Specific Aim #3, recombinant glycoproteins (L-selectin, lamp-1, and leukosialin/CD43) that carry sialyl Lex oligosaccharide determinants will be produced in cultured cell lines whose glycosylation phenotype has been modified by transfected fucosyltransferase genes. The glycan portions of these molecules will be then structurally defined. These studies will provide insight into mechanisms whereby protein and oligosaccharide acceptor molecules influence fucosyltransferase-dependent sialyl Lex-biosynthesis. These molecules will also be provided to Sections 2-4 for functional testing, so as to further our understanding of oligosaccharide structural determinants that influence Selectin binding interactions, under physiological conditions.
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