Effect of glycosylation and glucose trimming inhibitors on the influenza A virus glycoproteins.
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N-glycosylation and glucose trimming of the influenza virus hemagglutinin (HA) and neuraminidase (NA) were studied by using glycosylation inhibitor (tunicamycin; TM) and glucosidase inhibitors. TM treatment of MDCK cells infected with a reassortant virus NWS-N8 resulted in reduced transport of the viral glycoproteins to the cell surface. The degree of the effects differed between the HA and the NA (80% reduction for the HA and 97% reduction for the NA), indicating a difference in dependency on N-glycosylation between these glycoproteins. Differential dependency on glucose trimming was clearly demonstrated when the surface transport of the glycoproteins was compared after treatment of the virus-infected cells with glucosidase inhibitors. Fluorescence-activated cell sorting (FACS) analysis revealed that the surface transport of the NA reduced to 50% after castanospermine (CST) treatment but not did that of the HA. An anti-viral effect of a glucosidase inhibitor on the NWS-N8 strain was also demonstrated. The correlation between the expression of the NA on the cell surface and virus yield suggests that CST may interfere with virus release through its effect on the NA. (+info)
Folding and maturation of tyrosinase-related protein-1 are regulated by the post-translational formation of disulfide bonds and by N-glycan processing.
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In this study we have explored the endoplasmic reticulum associated events accompanying the maturation of the tyrosinase-related protein-1 (TRP-1) nascent chain synthesized in mouse melanoma cells. We show that TRP-1 folding process occurs much more rapidly than for tyrosinase, a highly homologous protein, being completed post-translationally by the formation of critical disulfide bonds. In cells pretreated with dithiothreitol (DTT), unfolded TRP-1 is retained in the endoplasmic reticulum by a prolonged interaction with calnexin and BiP before being targeted for degradation. The TRP-1 chain was able to fold into DTT-resistant conformations both in the presence or absence of alpha-glucosidase inhibitors, but folding occurred through different pathways. During the normal folding pathway, TRP-1 interacts with calnexin. In the presence of alpha-glucosidase inhibitors, the interaction with calnexin is prevented, with TRP-1 folding being assisted by BiP. In this case, the process has similar kinetics to that of untreated TRP-1 and yields a compact form insensitive to DTT as well. However, this form has different thermal denaturation properties than the native conformation. We conclude that disulfide bridge burring is crucial for the TRP-1 export. This suggests that although various folding pathways may complete this process, the native form may be acquired only through the normal unperturbed pathway. (+info)
Structural basis for catalysis and inhibition of N-glycan processing class I alpha 1,2-mannosidases.
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Endoplasmic reticulum (ER) class I alpha1,2-mannosidase (also known as ER alpha-mannosidase I) is a critical enzyme in the maturation of N-linked oligosaccharides and ER-associated degradation. Trimming of a single mannose residue acts as a signal to target misfolded glycoproteins for degradation by the proteasome. Crystal structures of the catalytic domain of human ER class I alpha1,2-mannosidase have been determined both in the presence and absence of the potent inhibitors kifunensine and 1-deoxymannojirimycin. Both inhibitors bind to the protein at the bottom of the active-site cavity, with the essential calcium ion coordinating the O-2' and O-3' hydroxyls and stabilizing the six-membered rings of both inhibitors in a (1)C(4) conformation. This is the first direct evidence of the role of the calcium ion. The lack of major conformational changes upon inhibitor binding and structural comparisons with the yeast alpha1, 2-mannosidase enzyme-product complex suggest that this class of inverting enzymes has a novel catalytic mechanism. The structures also provide insight into the specificity of this class of enzymes and provide a blueprint for the future design of novel inhibitors that prevent degradation of misfolded proteins in genetic diseases. (+info)
Novel alpha-L-fucosidase inhibitors from the bark of Angylocalyx pynaertii (Leguminosae).
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The extract of bark of Angylocalyx pynaertii (Leguminosae) was found to potently inhibit mammalian alpha-L-fucosidases. A thorough examination of the extract resulted in the discovery of 15 polyhydroxylated alkaloids, including the known alkaloids from seeds of this plant, 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), 1-deoxymannojirimycin (DMJ) and 2,5-imino-1,2,5-trideoxy-D-mannitol (6-deoxy-DMDP). Among them, eight sugar-mimic alkaloids showed the potent inhibitory activity towards bovine epididymis alpha-L-fucosidase and their Ki values are as follows: 6-deoxy-DMDP (83 microM), 2,5-imino-1,2,5-trideoxy-L-glucitol (0.49 microM), 2,5-dideoxy-2,5-imino-D-fucitol (17 microM), 2,5-imino-1,2,5-trideoxy-D-altritol (3.7 microM), DMJ (4.7 microM), N-methyl-DMJ (30 microM), 6-O-alpha-L-rhamnopyranosyl-DMJ (Rha-DMJ, 0.06 microM), and beta-L-homofuconojirimycin (beta-HFJ, 0.0053 microM). We definitively deduced the structural requirements of inhibitors of alpha-L-fucosidase for the piperidine alkaloids (DMJ derivatives). The minimum structural feature of alpha-L-fucosidase inhibitors is the correct configuration of the three hydroxyl groups on the piperidine ring corresponding to C2, C3 and C4 of L-fucose. Furthermore, the addition of a methyl group in the correct configuration to the ring carbon atom corresponding to C5 of L-fucose generates extremely powerful inhibition of alpha-L-fucosidase. The replacement of the methyl group of beta-HFJ by a hydroxymethyl group reduced its inhibitory potential about 80-fold. This suggests that there may be a hydrophobic region in or around the active site. The existence or configuration of a substituent group on the ring carbon atom corresponding to the anomeric position of L-fucose does not appear to be important for the inhibition. Interestingly, Rha-DMJ was a 70-fold more potent inhibitor of alpha-L-fucosidase than DMJ. This implies that the lysosomal alpha-L-fucosidase may have subsites recognizing oligosaccharyl structures in natural substrates. (+info)
Enhanced survival in Sandhoff disease mice receiving a combination of substrate deprivation therapy and bone marrow transplantation.
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Sandhoff disease is a lysosomal storage disorder characterized by G(M2) ganglioside accumulation in the central nervous system (CNS) and periphery. It results from mutations in the HEXB gene, causing a deficiency in beta-hexosaminidase. Bone marrow transplantation (BMT), which augments enzyme levels, and substrate deprivation (using the glycosphingolipid biosynthesis inhibitor N-butyldeoxynojirimycin [NB-DNJ]) independently have been shown to extend life expectancy in a mouse model of Sandhoff disease. The efficacy of combining these 2 therapies was evaluated. Sandhoff disease mice treated with BMT and NB-DNJ survived significantly longer than those treated with BMT or NB-DNJ alone. When the mice were subdivided into 2 groups on the basis of their donor bone marrow-derived CNS enzyme levels, the high enzyme group exhibited a greater degree of synergy (25%) than the group as a whole (13%). Combination therapy may therefore be the strategy of choice for treating the infantile onset disease variants. (+info)
Stemming the tide: glycosphingolipid synthesis inhibitors as therapy for storage diseases.
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Glycosphingolipids (GSLs) are plasma membrane components of every eukaryotic cell. They are composed of a hydrophobic ceramide moiety linked to a glycan chain of variable length and structure. Once thought to be relatively inert, GSLs have now been implicated in a variety of biological processes. Recent studies of animals rendered genetically deficient in various classes of GSLs have demonstrated that these molecules are important for embryonic differentiation and development as well as central nervous system function. A family of extremely severe diseases is caused by inherited defects in the lysosomal degradation pathway of GSLs. In many of these disorders GSLs accumulate in cells, particularly neurons, causing neurodegeneration and a shortened life span. No effective treatment exists for most of these diseases and little is understood about the mechanisms of pathogenesis. This review will discuss the development of a new approach to the treatment of GSL storage disorders that targets the major synthesis pathway of GSLs to stem their cellular accumulation. (+info)
Role of complex asparagine-linked oligosaccharides in the expression of a functional thyrotropin receptor.
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To evaluate the functional role of complex asparagine-linked oligosaccharides of the human thyrotropin receptor (TSHR), a Chinese hamster ovary cell line (JP09) and a K562 cell line (K562-TSHR) expressing this receptor were treated with deoxymannojirimycin (dMM), a mannosidase I inhibitor. dMM blocks the formation of complex-type structures and leads to the formation of high-mannose-type structures. Treatment of cells with dMM led to a decrease in the number of thyrotropin (TSH)-binding sites at the cell surface. Detection of the TSHR at the cell surface using a monoclonal antibody directed against the A subunit showed that this decrease was not due to a decrease in the number of TSHRs expressed at the cell surface. However the recognition of TSHR by a monoclonal antibody directed against the C peptide was greatly decreased. On immunoblotting, after deglycosylation using peptide N-glycanase F, the A subunit was visualized as a doublet (36 and 41 kDa). In control cells the species of higher molecular mass was more abundant whereas after dMM treatment the species of lower molecular mass became more abundant. This difference in molecular mass between the two peptides is compatible with the removal of the C peptide. In conclusion, the results show that inhibition of complex-type structure formation leads to (i) an incapacity for TSHR to bind TSH, without affecting its intracellular transport and (ii) an increase of TSHR susceptibility to proteases that remove the C peptide. We then hypothesized that removal of the C peptide could contribute to the formation of a non-functional TSHR. (+info)
Antiviral effect of N-butyldeoxynojirimycin against bovine viral diarrhea virus correlates with misfolding of E2 envelope proteins and impairment of their association into E1-E2 heterodimers.
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The iminosugar N-butyldeoxynojirimycin (NB-DNJ), an endoplasmic reticulum alpha-glucosidase inhibitor, has an antiviral effect against bovine viral diarrhea virus (BVDV). In this report, we investigate the molecular mechanism of this inhibition by studying the folding pathway of BVDV envelope glycoproteins in the presence and absence of NB-DNJ. Our results show that, while the disulfide-dependent folding of E2 glycoprotein occurs rapidly (2.5 min), the folding of E1 occurs slowly (30 min). Both BVDV envelope glycoproteins associate rapidly with calnexin and dissociate with different kinetics. The release of E1 from the interaction with calnexin coincides with the beginning of E1 and E2 association into disulfide-linked heterodimers. In the presence of NB-DNJ, the interaction of E1 and E2 with calnexin is prevented, leading to misfolding of the envelope glycoproteins and inefficient formation of E1-E2 heterodimers. The degree of misfolding and the lack of association of E1 and E2 into disulfide-linked complexes in the presence of NB-DNJ correlate with the dose-dependent antiviral effect observed for this iminosugar. (+info)