Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0038362 (stomatitis)
 8,852 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Enzymes that catalyze the two successive stages of Golgi-associated processing of asparagine-linked oligosaccharides distributed differently when membranes from Chinese hamster ovary cells were centrifuged in a sucrose density gradient. A mannosidase that removes only outer, alpha-1,2-linked mannose residues from the precursor oligosaccharides of the vesicular stomatitis viral G protein (to yield a "trimmed" oligosaccharide core) was separated from enzymes (galactosyl- and sialyltransferases) that act in the later, terminal stage of glycosylation. Freshly acylated G protein with newly trimmed oligosaccharides banded in the distribution of early-acting membranes, defined by the mannosidase, whereas G protein pulse-labeled with [3H]galactose distributed in the profile of the late-acting membranes. G protein present in the early-acting membranes in crude fractions could be terminally glycosylated by incubation with exogenous Golgi membranes in vitro; G protein lost its ability to be processed in vitro as it appeared to enter the late-acting membranes in vivo. These experiments reveal the existence of two distinct compartments through which intracellularly transported proteins such as G pass in sequence as Golgi-associated processes are carried out. It is likely that these compartments consist of cisternae on the cis and trans sides of the Golgi stack.
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PMID:Early and late functions associated with the Golgi apparatus reside in distinct compartments. 680 52

The biosynthesis of the erythrocyte anion transport glycoprotein, Band III (Mr 100,000), is of interest, as its N-terminal half is hydrophilic and faces the cytoplasmic surface; the C-terminal half spans the phospholipid bilayer several times. Band III is synthesized by erythroid precursor cells obtained from the spleens of anaemic mice. Newly synthesized Band III was inserted into rough endoplasmic reticulum membranes with an asymmetric orientation which resembled that of mature Band III in erythrocyte membranes: the N-terminal portion of the molecule facing the cytoplasm. Newly made Band III contained a high-mannose asparagine-linked oligosaccharide, which was susceptible to cleavage by endoglycosidase H. During the next 20-30 min, this oligosaccharide was processed to a form resistant to endoglycosidase H degradation, presumably in the Golgi complex. The processed Band III was subsequently expressed on the cell surface, at about 30-45 min after synthesis. To study the mechanism of insertion of Band III into microsomes, we used erythroid precursor cells from the spleens of anaemic mice as a source of messenger RNA for studies in vitro in the wheat germ and reticulocyte lysate cell-free system containing dog pancreatic microsomes. Immediately after synthesis, Band III was found to be inserted into microsomal membranes in its mature configuration, with the N-terminal portion exposed to the cytoplasm and its hydrophobic C-terminal portion spanning the lipid bilayer. The newly-synthesized Band III was also provided with a high-mannose asparagine-linked oligosaccharide. Band III was found to be inserted into dog pancreatic microsomes in a co-translational manner; in synchronized translation studies microsomes could be added as late as the time when the hydrophilic N-terminal half of the protein had been synthesized and still allow normal trans-membrane insertion and glycosylation. There is no cleavage of any N-terminal peptide during membrane insertion. In many respects, therefore, the biosynthesis of Band III resembles that of co-translationally-inserted proteins whose N-terminal portions are exposed on the exterior of the cell, like vesicular stomatitis virus glycoprotein, HLA-A antigens, and glycophorin. However, our results suggest that Band III contains a sequence near the middle of the protein which directs its insertion into endoplasmic reticulum membranes.
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PMID:Synthesis and maturation of the erythrocyte anion transport protein--an internal sequence for membrane insertion. 682 84

Independently derived mutants of Chinese hamster ovary cells have been isolated and shown to exhibit a subtle glycosylation defect resulting in the premature termination of certain asparagine-linked carbohydrate moieties. This carbohydrate alteration is akin to the types of structural variation termed microheterogeneity and is thought not to affect the biological activities of glycoproteins that manifest the phenomenon. However, the carbohydrate change expressed by the mutants is stable and heritable, and 125I-lectin-binding studies suggest that it profoundly alters their surface recognition properties. The mutation appears to affect a specific subpopulation of galactose residues in asparagine-linked carbohydrate of the type found associated with the G glycoprotein of vesicular stomatitis virus. The mutant cells also exhibit morphological changes in substratum culture.
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PMID:Microheterogeneity among carbohydrate structures at the cell surface may be important in recognition phenomena. 719 40

The purpose of the present study was to analyze the post-translational and activation-dependent modifications of the G protein-coupled thrombin receptor. A human receptor cDNA was engineered to encode an epitope tag derived from the vesicular stomatitis virus glycoprotein at the COOH terminus of the receptor and expressed in human embryonic kidney 293 cells. We show here that the mature receptor is a glycosylated protein with an apparent molecular mass ranging from 68 to 80 kDa by SDS-polyacrylamide gel electrophoresis. Removal of asparagine-linked oligosaccharides with N-glycosidase F leads to the appearance of a 36-40-kDa receptor species. The current model for receptor activation by thrombin involves specific hydrolysis of the arginine-41/serine-42 (Arg-41/Ser-42) peptide bond. Cleavage of the receptor by thrombin was demonstrated directly by Western analyses performed on membranes and glycoprotein-enriched lysates from transfected cells. Whereas thrombin treatment of cells results in increased mobility of the receptor in SDS-polyacrylamide gel electrophoresis, we found that their treatment with the thrombin receptor agonist peptide leads to a decrease in thrombin receptor mobility due, in part, to phosphorylation. The serine proteases trypsin and plasmin also cleave and activate the receptor similar to thrombin, whereas chymotrypsin cleaves the receptor at a site distal to Arg-41, thus rendering it unresponsive to thrombin while still responsive to thrombin receptor agonist peptide.
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PMID:Post-translational and activation-dependent modifications of the G protein-coupled thrombin receptor. 771 46

Hepatitis delta virus (HDV) is a defective virus requiring the hepatitis B virus (HBV) to provide hepatitis B surface antigens as the envelope protein. The hepatitis B surface antigens are posttranslationally modified by N-linked glycosylation, and its significance in HDV assembly was investigated with a cotransfection system using human hepatoma cell line Huh-7. After the N-linked glycosylation of HBsAg was blocked by tunicamycin treatment, the packaging of HDV in the culture system could be suppressed to a level as low as 5-10% of the untreated control. The extent of inhibition correlated with the increased concentrations of tunicamycin. In contrast, the loss of HBsAg glycosylation did not affect the efficiency of assembly of HBV particles. When the N-linked glycosylation site of small HBsAg at amino acid 146 was mutated from asparagine to glutamine, the mutant HBsAg packaged only a modest amount of HDV particles. The quantity and kinetics of formation of HDV particles in culture system were reduced by the depletion of HBsAg glycosylation. Therefore HDV, similar to influenza and vesicular stomatitis viruses, depends on glycosylation of the envelope proteins as a signal for envelope protein maturation and for virion formation.
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PMID:N-linked glycosylation of hepatitis B surface antigens is involved but not essential in the assembly of hepatitis delta virus. 865 25

Calreticulin is a soluble, endoplasmic reticulum-resident protein and a molecular chaperone for glycoproteins. We have reconstituted the binding of recombinant calreticulin to two glycoprotein substrates, vesicular stomatitis virus G protein and influenza hemagglutinin, in vitro. The binding was found to be direct and to require monoglucosylated, asparagine-linked oligosaccharides on the substrate glycoprotein but no other cellular factors. The binding could be modulated in vitro by incubation of substrate with purified preparations of the glycan modifying enzymes glucosidase II and the UDP-glucose:glycoprotein glucosyltransferase, thus recapitulating the regulation of calreticulin-binding by glycan modification that occurs in vivo. Using the purified ER enzymes and the recombinant calreticulin, an assay was established for reconstituting a complex, multicomponent chaperone binding cycle in vitro. We demonstrated, moreover, that the acidic C-terminal 62 residues of calreticulin are dispensable for substrate binding whereas further deletions inhibit substrate binding.
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PMID:In vitro reconstitution of calreticulin-substrate interactions. 1041 84

We have previously reported a functional role associated with hepatitis C virus (HCV) E1 glycoprotein using vesicular stomatitis virus (VSV)/HCV pseudotype. In this study, we have investigated the role of glycosylation upon intracellular transport of chimeric E1-G, and in infectivity of the pseudotyped virus. Interestingly, surface expressed E1-G exhibited sensitivity to Endoglycosidase H (Endo H) treatment, which was similar to full-length E1, suggesting that additional complex oligosaccharides were not added while E1-G was in transit from the endoplasmic reticulum (ER) to the mammalian cell surface. As a next step, each of the four potential N-linked glycosylation sites located at amino acid position 196, 209, 234, or 305 of the E1 ectodomain were mutated separately (asparagine --> glutamine), or in some combination. FACS analysis suggested that mutation(s) of the glycosylation sites affect the translocation of E1-G to the cell surface to different extents, with no single site being particularly essential. VSV pseudotype virus generated from glycosylation mutants exhibited a decrease in titer with an increasing number of mutations at the glycosylation sites on chimeric E1-G. In a separate experiment, N-glycosidase F treatment of pseudotype generated from the already synthesized E1-G or its mutants decreased virus titer by approximately 35%, and the neutralization activity of patient sera was not significantly altered with N-glycosidase F-treated pseudotype virus. Taken together, our results suggested that E1-G does not add complex sugar moieties during transport to the cell surface and retain the glycosylation profile of its parental E1 sequence. Additionally, the removal of glycans from the E1-G reduced, but does not completely impair, virus infectivity.
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PMID:Influence of N-linked glycans on intracellular transport of hepatitis C virus E1 chimeric glycoprotein and its role in pseudotype virus infectivity. 1520 15

The secretory membrane system is comprised of membrane-bound organelles defined by specific sets of proteins that function in sequential modification of cargo proteins and lipids. This processing of cargo proteins and lipids is coupled to their secretory transport. Here, we investigated the effect of inhibiting N-glycan processing by swainsonine, an inhibitor of Golgi alpha1,2-mannosidase-II, on secretory transport of the thermo-reversible tsO45 mutant of vesicular stomatitis virus glycoprotein tagged with green fluorescent protein (VSVG-FP). Quantitative analysis using kinetic modeling combined with live cell imaging was used to derive the rate coefficients that delineate secretory transport of VSVG-FP. We found that neither inhibition of N-glycan processing nor elimination by mutagenesis of the first of the two asparagine-linked glycans had any significant effect on the rate of VSVG-FP transport through the Golgi. These data suggest that at least for VSVG, the multi-enzymatic process of N-glycan modification does not comprise a rate-limiting step for its Golgi efflux.
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PMID:Processing of VSVG protein is not a rate-limiting step for its efflux from the Golgi complex. 1708 98

A temperature-sensitive mutant of vesicular stomatitis G protein is used to follow the movement of that protein from the endoplasmic reticulum to transport vesicles to cis-Golgi and finally medial/trans-Golgi by assessing the maturation of two asparagine-linked oligosaccharides. These assays can be used to identify the factors that are required for and regulate protein trafficking through these compartments.
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PMID:In vitro analysis of endoplasmic-reticulum-to-golgi transport in mammalian cells. 1822 8


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