Peter Scudder

Glycosidases in structural analysis.

Publisher Summary Structural analysis of the carbohydrate components of various molecules begins with release of the intact carbohydrate, either by chemical or enzymatic means, followed by purification and structural determination. Glycosidases have enjoyed prominence as reagents for the sequencing and structural characterization of the released oligosaccharides, in particular with respect to asparagine-linked glycans, which generally show greater uniformity in their monosaccharide composition and structural features than related serine/threonine-linked compounds. The use of enzymes for structural determinations is likely to diminish in the future with the increased sophistication of analytical methods for carbohydrate analysis such as multidimensional proton nuclear magnetic resonance (NMR) and fast atom bombardment and electrospray-mass spectrometry. The principle of oligosaccharide sequencing relies on the ability of specific exoglycosidases to remove terminal monosaccharides from the nonreducing end of oligosaccharides. To achieve complete removal of the terminal monosaccharide residues, a relatively high concentration of enzyme is used together with an extended incubation time. By using the information obtained from a series of digestion experiments involving different exoglycosidases, a structure can be deduced.

Aminosugar Attenuation of HIV Infection

The envelope glycoproteins of HIV mediate cellular uptake of virus, and fusion of infected with non-infected CD4+ T cells. Inhibitors of oligosaccharide processing have been found to attenuate HIV infections in vitro and putatively work by altering the structure and function of these glycoproteins. One such compound, SC-48334 (N-butyl-1-deoxynojirimycin) inhibits α-glucosidase I and has been reported to reduce the infectivity of virus recovered from infected cell cultures in a dose-dependent manner. The envelope precursor, gp160, is heavily glycosylated and its endoproteolytic processing to gp120 and gp41, necessary for infection, is retarded in the presence of SC-48334. However, processing is not abolished and mature envelope glycoproteins are still present. Using baculovirus-insect-derived gp120 probed with antibodies, we find suggestive evidence that a local conformational change is induced in the V3 loop of gp120 which may affect cell-surface proteolytic cleavage thought to be involved in cell-virus fusion. Finally, we present preliminary information on a prodrug form of SC-48334 which does not inhibit intestinal disaccharidases.

Studies on Selectin-Carbohydrate Interactions

Recruitment of neutrophils to sites of inflammation is now believed to occur through an initial rolling interaction at the luminal surface of activated endothelium and is mediated by a class of mammalian lectins referred to as the selectins. Selectins recognize carbohydrate determinants on co-receptors. It is generally believed that many selectin molecules must bind to many carbohydrate receptor molecules i.e. multivalent binding, to enable sufficient binding strength to elicit the rolling response between the neutrophil and the endothelial cell. One of the approaches to the generation of more potent molecular antagonists of the selectin-mediated cell-cell interaction is to mimic the multivalent interaction in a single compound. Recent experiments utilising conjugated forms of sialyl Lewisx-BSA have explored this feasibility (Welply et al., 1994). In that study, monovalent sLex (sialic acid alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc), the minimum binding determinant for E-selectin, as well as monovalent sialyllactosamine (sialic acid alpha 2-3Gal beta 1-4GlcNAc), a non-binding structure, and the corresponding multivalent BSA-conjugated forms were tested for their ability to inhibit binding of HL-60 cells to immobilised E-selectin. As expected, only sLex and sLex-BSA were found to do so. sLex16-BSA (16 mol tetrasaccharide/mol BSA) showed a dose-dependent inhibition of HL-60 binding with a measured IC50 of 1 microM; demonstrating close to a three-order of magnitude enhancement of inhibitory activity compared to free sLex. This result indicated that multivalent forms of sLex are capable of binding to E-selectin with higher affinity than do monovalent glycans. In another study, fluorescent forms of monovalent sLex were synthesized and used to measure a true thermodynamic dissociation constant for the monovalent sLex:E-selectin interaction of 120 +/- 31 microM (Jacob et.al., 1995).

Control of human cervical mucin glycosylation by endogenous fucosyl and sialyltransferases.

In many epithelial glycoproteins a reciprocal relation exiStS between levels of L-fucose and sialic acid which could be the result of their mutually exclusive incorporation during oligosaccharide synthesis1. Changes in the visco-elastic properties of human cervical mucus occurring during the menstrual cycle may be related to alterations in the structure2 or composition3 of cervical mucin involving terminal L-fucosyl and sialyl residues. In the present study the possible role of endogenous glycosyltransferases in controlling levels of L-fucose and N-acetylneuraminic acid in cervical mucin was investigated by measuring the variation in activity of s-galacto-side fucosyl and sialyltransferases in the cervical epithelium throughout the menstrual cycle.