Showing papers by "Mark R. Wormald published in 2000"
TL;DR: Modifications to imino sugar inhibitors can be suggested that allow a more selective approach for molecular inhibition of both ceramide glucosyltransferase and α-glucosidase I, leading to improved compounds for the potential treatment of lysosomal glycosphingolipid storage disorders and viral infections, respectively.
Abstract: N -Butyl-deoxynojirimycin (NB-DNJ) has been approved for clinical trials as a potential therapy for Gaucher disease, a glycolipid lysosomal storage disorder As this compound has both glycoprotein processing α-glucosidase and ceramide glucosyltransferase inhibitory activity, we have sought to determine the molecular basis for these two activities NB-DNJ is known to resemble the positively charged oxocarbonium-like transition state for α-glucosidase I and the structure–function relationships we present now help to define the recognition epitope for the enzyme Inhibition of ceramide glucosyltransferase by NB-DNJ was competitive for ceramide ( K i =74 μM) and non-competitive for UDP-glucose, indicating inhibitory activity is by ceramide mimicry The presence of an N -alkyl chain was obligatory for transferase inhibition and increases in alkyl chain length provided a modest increase in inhibitory potency By contrast, α-glucosidase inhibition was independent of the N -alkyl chain and changes in chain length The effects of ring substitutions identified the C 3 hydroxyl group as being critical for both enzymes but C 1 and C 6 modifications led to a loss of transferase inhibition only Attempts to rationalise these data for transferase inhibition using an energy minimised molecular model of NB-DNJ and ceramide predicted structural homology of three stereogenic centres and the N -alkyl chain of NB-DNJ, with the trans -alkenyl and N -acyl chain of ceramide On the basis of these studies, modifications to imino sugar inhibitors can be suggested that allow a more selective approach for molecular inhibition of both ceramide glucosyltransferase and α-glucosidase I, leading to improved compounds for the potential treatment of lysosomal glycosphingolipid storage disorders and viral infections, respectively
141 citations
TL;DR: A local folding mechanism is proposed that can explain the mechanism of inactivation of tyrosinaseN-glycosylation mutants found in certain pigmentation disorders.
119 citations
TL;DR: Detailed analysis of picomole amounts of O-glycans is presented using a novel HPLC-based strategy that provides linkage and arm specific information in addition to monosaccharide sequence and has considerable implications for the domain organization of MMP-9.
Abstract: Gelatinase B is a matrix metalloproteinase (MMP-9) expressed under strict control by many cell types including neutrophils, monocytes, macrophages, and tumor cells. MMP-9 is a key mediator in the physiological maintenance of the extracellular matrix both in tissue remodeling and development, while uncontrolled enzyme activity contributes to pathologies such as cancer and inflammation. Neutrophils release MMP-9 from granules in response to IL-8 stimulation. Human MMP-9 has three potential N-linked glycosylation sites and contains a Ser/Pro/Thr rich domain, known as the type V collagen-like domain, which is expected to be heavily O-glycosylated. Indeed, approximately 85% of the total sugars on human neutrophil MMP-9 are O-linked. This paper presents the detailed analysis of picomole amounts of these O-glycans using a novel HPLC-based strategy for O-glycan analysis that provides linkage and arm specific information in addition to monosaccharide sequence. The initial structural assignments were confirmed usin...
91 citations
TL;DR: It is found that CR3 is one of the rare recombinant peptides modified with complex glycans in insect cells and the role of glycosylation in eight-cysteine domain protein-protein interactions is discussed.
Abstract: Covalent association of LTBP-1 (latent TGF-β binding protein-1) to latent TGF-β is mediated by the third eight-cysteine (also referred to as TB) module of LTBP-1, a domain designated as CR3. Spodoptera frugiperda (Sf9) cells have proved a suitable cell system in which to study this association and to produce recombinant CR3, and we show here that another lepidopteran cell line, Trichoplusia ni TN-5B1−4 (High-Five) cells, allows the recovery of large amounts of functional recombinant CR3. CR3 contains an N-glycosylation site, which is conserved in all forms of LTBP known to date. When we examined the status of this N-glycosylation using MALDI-TOF mass spectrometry and enzymatic analysis, we found that CR3 is one of the rare recombinant peptides modified with complex glycans in insect cells. Sf9 cells mainly processed the fucosylated paucomannosidic structure (GlcNAc)2(Mannose)3Fucose, although hybrid and complex N-glycosylations were also detected. In High-Five cells, the peptide was found to be modified w...
36 citations
30 citations
TL;DR: The 2-Aminobenzamide (2AB) is a common fluorescence label attached to reducing oligosaccharides by a reductive amination procedure as discussed by the authors.
Abstract: 2-Aminobenzamide (2AB) is a common fluorescence label attached to reducing oligosaccharides by a reductive amination procedure. Chemical investigation of the published literature procedure reveals labelling occurs by the expected mechanism for both protected and unprotected glucose derivatives to yield open-chain carbohydrates rather than result in the formation of any heterocyclic materials. Pentenyl glucosides may also be readily attached to the 2AB label by a sequence of dihydroxylation, periodate cleavage and subsequent reductive amination of the resulting aldehyde. 2AB labelling is compatible with deprotection of both acetate and benzyl protecting groups.
23 citations
01 Jan 2000
TL;DR: Proteins which will be discussed include the immunoglobulins IgG and IgAl, the inhibitors of the complement pathway CD59 and DAF (CD55), and the cell adhesion molecules CD2 and CD48, which mediate the alignment of the cell surfaces of cytolytic T-lymphocytes carrying the TCR complex with those of target cells carrying loaded HLA class 1 molecules.
Abstract: A full understanding of the implications of glycosylation for the structure and function of any glycoprotein can only be reached when the molecule is viewed in its entirety. NMR solution and X-ray crystallography studies of glycoproteins do not normally yield detailed information about the sugars. Here protein structural data have been complemented by oligosaccharide analysis of the sugars released from 5-10µg of protein. The data discussed in this paper were obtained using rapid glycan sequencing technology and the linkage structure data base (both developed in the Institute), which provides the dimensions of the sugars. In this way it has been possible to obtain a more complete view of some glycoproteins in the immune system and the roles which the oligosaccharides play in their functions. Roles for the sugars include stabilising the protein structure, modifying the activity of effector functions, orienting the protein on the cell surface, shielding the protein from proteases and providing specific epitopes for recognition events. The glycoproteins which will be discussed include the immunoglobulins IgG and IgAl, the inhibitors of the complement pathway CD59 and DAF (CD55), and the cell adhesion molecules CD2 and CD48. CD2 and CD48 mediate the alignment of the cell surfaces of cytolytic T-lymphocytes carrying the TCR complex with those of target cells carrying loaded HLA class 1 molecules.
1 citations