Cell surface carbohydrates are involved in cellular recognition and serve as receptors for viruses, antibodies, toxins and hormones.
The aim of these studies is to define a structural basis for the biochemical mechanisms that govern carbohydrate mediated cellular processes such as viral infectivity, immune response and cell differentiation (or malignancy). The stringent sugar specificity of lectins and their ability to mimic normal cellular function has been exploited as one approach to study determinants of receptor stimulation. Thus, structural knowledge on the atomic level is essential to understand the stereochemical requirements for their sugar specificity. We are extending our studies of the crystal complex of wheat germ agglutinin (WGA1) and the T-5 sialoglycopeptide (derived from its receptor glycophorin A), which revealed 2 new sites for binding sialic acid (NeuNAc), one of the most common cell surface target sugars. Structure refinement will be completed and this structure compared with that of native WGA1 to determine the effect of asymmetric sugar binding (only 3 of the 8 possible sites occupied) and inter-dimer crosslinkage on subunit/subunit association. In addition, we shall use recombinant techniques, NMR and x-ray crystallography to define differential fine- specificities for NeuNAc and GlcNAc at each of the four unique heterogeneous binding sites and design and clone a domain with a binding site of optimal affinity for NeuNAc and GlcNAc. We shall determine the 2.5 Angstroms structure of snowdrop lectin (GNA), a potent inhibitor of human and simian immunodeficiency viruses. GNA is alpha-Man specific (-Man-alpha1,3)Man-) and represents a new lectin family distinct from the Man/Glc specific legume family. The structure will be determined for the mono-saccharide complex by MIR methods at 2.5 Angstroms resolution, using four heavy atom derivatives, and for the Me- Man-alpha1,3-Man complex (different crystal form) by molecular replacement. To understand the mechanisms underlying molecular sorting in the plant, we propose to determine the crystal structure of barley lectin precursor by molecular replacrment techniques. This pro-protein contains 15 additional amino acids at its carboxyl terminus (as does pro-WGA), needed for correct deposition of mature lectin in the vacuoles. The folding motif of this C-terminal domain is believed to be the key to correct targeting. To further define the role a WGA-domain plays as a single unit in binding chito-oligosaccharides in vivo, structural studies will be initiated with tobacco chitinase, a monomeric anti-fungal hydrolase that acts on chitin to produce GlcNAc disaccharides, utilizing a WGA-like domain for binding at its N-terminus.
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