Showing papers by "Mark R. Wormald published in 2006"

Structural comparison of differently glycosylated forms of acid-β-glucosidase, the defective enzyme in Gaucher disease

Abstract: Gaucher disease is caused by mutations in the gene encoding acid-β-glucosidase. A recombinant form of this enzyme, Cerezyme®, is used to treat Gaucher disease patients by `enzyme-replacement therapy'. Crystals of Cerezyme® after its partial deglycosylation were obtained earlier and the structure was solved to 2.0 A resolution [Dvir et al. (2003), EMBO Rep. 4, 704–709]. The crystal structure of unmodified Cerezyme® is now reported, in which a substantial number of sugar residues bound to three asparagines via N-glycosylation could be visualized. The structure of intact fully glycosylated Cerezyme® is virtually identical to that of the partially deglycosylated enzyme. However, the three loops at the entrance to the active site, which were previously observed in alternative conformations, display additional variability in their structures. Comparison of the structure of acid-β-­glucosidase with that of xylanase, a bacterial enzyme from a closely related protein family, demonstrates a close correspondence between the active-site residues of the two enzymes.

Isolation synthesis and glycosidase inhibition profile of 3-epi-casuarine

Abstract: 3- epi -Casuarine, the first naturally occurring stereoisomer of casuarine, was isolated from Myrtus communis L. The glycosidase inhibition profile and NMR spectra of 3- epi -casuarine are compared with those of casuarine; the change in configuration at one of the six stereogenic centres causes a dramatic change in the conformation of the bicyclic system. The key step in the 6% overall yield synthesis of 3- epi -casuarine from d -gluconolactone is the efficient cyclization of a completely unprotected pentahydroxyaminomesylate to the pyrrolizidine nucleus. A low yield synthesis of casuarine is also reported.

Cancer-associated glycoforms of gelatinase B exhibit a decreased level of binding to galectin-3

Abstract: Gelatinase B (MMP-9) and galectin-3 are widely known to participate in tumor cell invasion and metastasis. Glycans derived from MMP-9 expressed in MCF-7 breast cancer and THP-1 myeloid leukemia cells were compared with those from MMP-9 expressed in natural neutrophils. The many O-linked glycans of neutrophil gelatinase B presented a cluster of mainly galactosylated core II structures, 46% of which were ligands for galectin-3; 11% contained two to three N-acetyllactosamine repeating units that are high-affinity ligands for the lectin. The glycan epitopes thus provide MMP-9 with both high-affinity and (presumably) high-avidity interactions with galectin-3. In contrast, the O-glycans released from MMP-9 expressed in MCF-7 and THP-1 cells were predominantly sialylated core I structures. Only 10% of MCF-7 and THP-1 gelatinase B O-glycans were ligands for galectin-3 and contained only a maximum single N-acetyllactosamine repeat. Consistent with the glycan analysis, surface plasmon resonance binding assays indicated that the cancer-associated glycoforms of MMP-9 bound galectin-3 with an affinity and avidity significantly reduced compared with those of the natural neutrophil MMP-9. Galectin-3 exists as a multimer that also binds laminin, providing a means of localizing neutrophil MMP-9 in the extracellular matrix (ECM). The analytical data presented here suggest that MMP-9 glycoforms secreted by tumor cells are unlikely to be tethered at the site of secretion, thus promoting more extensive cleavage of the ECM and providing a rationale for the contribution that gelatinase B makes to cancer cell metastasis.

Sugar amino acids at the anomeric position of carbohydrates : synthesis of spirocyclic amino acids of 6-deoxy-L-lyxofuranose

Abstract: Two anomeric spirodiketopiperazines and one spirohydantoin of 6-deoxy- l -lyxofuranose have been prepared from l -fucono-δ-lactone via ring contraction to the corresponding tetrahydrofuran carboxylate and anomeric functionalization including regioselective bromination and azide displacement.

Sialylation of urinary prothrombin fragment 1 is implicated as a contributory factor in the risk of calcium oxalate kidney stone formation

Abstract: Urinary glycoproteins are important inhibitors of calcium oxalate crystallization and adhesion of crystals to renal cells, both of which are key mechanisms in kidney stone formation. This has been attributed to glycosylation of the proteins. In South Africa, the black population rarely form stones (incidence < 1%) compared with the white population (incidence 12-15%). A previous study involving urinary prothrombin fragment 1 from both populations demonstrated superior inhibitory activity associated with the protein from the black group. In the present study, we compared N-linked and O-linked oligosaccharides released from urinary prothrombin fragment 1 isolated from the urine of healthy and stone-forming subjects in both populations to elucidate the relationship between glycosylation and calcium oxalate stone pathogenesis. The O-glycans of both control groups and the N-glycans of the black control samples were significantly more sialylated than those of the white stone-formers. This demonstrates a possible association between low-percentage sialylation and kidney stone disease and provides a potential diagnostic method for a predisposition to kidney stones that could lead to the implementation of a preventative regimen. These results indicate that sialylated glycoforms of urinary prothrombin fragment 1 afford protection against calcium oxalate stone formation, possibly by coating the surface of calcium oxalate crystals. This provides a rationale for the established roles of urinary prothrombin fragment 1, namely reducing the potential for crystal aggregation and inhibiting crystal-cell adhesion by masking the interaction of the calcium ions on the crystal surface with the renal cell surface along the nephron.

The importance of including local correlation times in the calculation of inter-proton distances from NMR measurements: ignoring local correlation times leads to significant errors in the conformational analysis of the Glcα1–2Glcα linkage by NMR spectroscopy

Abstract: The experimental determination of oligosaccharide conformations has traditionally used cross-linkage 1H–1H NOE/ROEs As relatively few NOEs are observed, to provide sufficient conformational constraints this method relies on: accurate quantification of NOE intensities (positive constraints); analysis of absent NOEs (negative constraints); and hence calculation of inter-proton distances using the two-spin approximation We have compared the results obtained by using 1H 2D NOESY, ROESY and T-ROESY experiments at 500 and 700 MHz to determine the conformation of the terminal Glcα1–2Glcα linkage in a dodecasaccharide and a related tetrasaccharide For the tetrasaccharide, the NOESY and ROESY spectra produced the same qualitative pattern of linkage cross-peaks but the quantitative pattern, the relative peak intensities, was different For the dodecasaccharide, the NOESY and ROESY spectra at 500 MHz produced a different qualitative pattern of linkage cross-peaks, with fewer peaks in the NOESY spectrum At 700 MHz, the NOESY and ROESY spectra of the dodecasaccharide produced the same qualitative pattern of peaks, but again the relative peak intensities were different These differences are due to very significant differences in the local correlation times for different proton pairs across this glycosidic linkage The local correlation time for each proton pair was measured using the ratio of the NOESY and T-ROESY cross-relaxation rates, leaving the NOESY and ROESY as independent data sets for calculating the inter-proton distances The inter-proton distances calculated including the effects of differences in local correlation times give much more consistent results

3,7,7a-Tri-epi-casuarine penta­acetate

Abstract: The relative stereochemistry at six contiguous centres in an analogue of the natural product casuarine, viz. 3,7,7a-tri-epi-casuarine penta­acetate, C18H25NO10, has been established by an analysis of a crystalline penta­acetate.