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Biomedical subjects

G Legler

Publications and source records attributed to G Legler.

At least 55 records · Page 3Linked to original sources
PubMed1984

Effect of inhibitors of glycosylation on proteolytic activation of avian influenza virus hemagglutinins: discrimination between tryptic cleavage and elimination of the connecting peptide.

The glycosylation inhibitors tunicamycin (TM), 2-deoxyglucose (2-dg), bromoconduritol (BC; 3,5/4,6-6-bromo 3,4,5-trihydroxycyclohex-1-ene), and N-methyl-deoxynojirimycin (MdN) have been used to study the role of glycosylation in the two proteolytic reactions involved in the biological activation of H7 influenza virus hemagglutinins (HAs): trypsinlike cleavage and subsequent elimination of the connecting peptide. The results obtained revealed that trypsin-like cleavage of the HAs of pathogenic strains does not require glycosylation, since these HAs were efficiently cleaved in the presence of TM and 2-dg. The elimination of the connecting peptide between HA1 and HA2, however, appears to require the transfer of oligosaccharides onto the HA polypeptide, since this activity was blocked by TM and by 2-dg. Elimination was not blocked by BC or MdN, which inhibit glucose trimming and subsequent conversion of the high-mannose type to the complex type of carbohydrate.

1-Deoxynojirimycin↗
PubMed1984

Inhibition of N-linked oligosaccharide trimming does not interfere with surface expression of certain integral membrane proteins.

The effects of 1-deoxynojirimycin (dNM) and 1-deoxymannojirimycin (dMM), inhibitors of oligosaccharide trimming glucosidase I and mannosidase I, respectively, on the biosynthesis of vesicular stomatitis virus G protein, influenza virus hemagglutinin, and human class I histocompatibility antigens were investigated. Although the oligosaccharides of these membrane glycoproteins were greatly altered, neither dNM nor dMM interferred with their surface expression, as determined by a variety of assays, including accessibility to proteases and antibodies; neither did these drugs inhibit production of infectious virus particles.

1-Deoxynojirimycin↗
PubMed1984

The effect of deoxymannojirimycin on the processing of the influenza viral glycoproteins.

Deoxymannojirimycin (dMM) was tested as an inhibitor of the processing of the oligosaccharide portion of viral and cellular N-linked glycoproteins. The NWS strain of influenza virus was grown in MDCK cells in the presence of various amounts of dMM, and the glycoproteins were labeled by the addition of 2-[3H]mannose to the medium. At levels of 10 micrograms/ml dMM or higher, most of the viral glycopeptides became susceptible to digestion by endoglucosaminidase H, and the liberated oligosaccharide migrated mostly like a Hexose9GlcNAc on a calibrated column of Bio-Gel P-4. This oligosaccharide was characterized as a typical Man9GlcNAc by a variety of chemical and enzymatic procedures. Deoxymannojirimycin gave rise to similar oligosaccharide structures in the cellular glycoproteins. In both the viral and the cellular glycoproteins, this inhibitor caused a significant increase in the amount of [3H]mannose present in the glycoproteins. Deoxymannojirimycin did not inhibit the incorporation of [3H]leucine into protein in MDCK cells, nor did it affect the yield or infectivity of NWS virus particles. However, its effect on mannose incorporation into lipid-linked saccharides depended on the incubation time, the virus strain, and the cell line. Thus, high concentrations of dMM showed some inhibition of mannose incorporation into lipid-linked oligosaccharides with the NWS strain in a 3-h incubation, but no inhibition was observed after 48 h of incubation. On the other hand, the PR8 strain was much more sensitive to dMM inhibition, and mannose incorporation into lipid-linked oligosaccharides was strongly inhibited when the virus was raised in chick embryo cells, but less inhibition was observed when this virus was grown in MDCK cells. Nevertheless, in these cases also, the major oligosaccharide structure in the glycoproteins was the Man9GlcNAc2 species.

1-Deoxynojirimycin↗
PubMed1983

Effects of the glucosidase inhibitors nojirimycin and deoxynojirimycin on the biosynthesis of membrane and secretory glycoproteins.

The glucosidase inhibitors nojirimycin (NM) and 1-deoxynojirimycin (dNM) interfere with N-linked glycosylation. The effects of NM and dNM on the biosynthesis of secretory glycoproteins (IgD and IgM) and membrane glycoproteins (HLA-A, B, C and -DR antigens) have been examined. Whereas treatment of IgD- and IgM-producing cells with NM results in the transfer of drastically shortened oligosaccharide side chains, treatment with dNM inhibits trimming, most probably through interaction with glucosidase I and/or II. A comparison of NM and dNM with tunicamycin and the mannosidase inhibitor swainsonine (SW) show that each of the inhibitors interferes with N-linked glycosylation in a distinct manner. For both Ig and HLA antigens, the effects of SW are discernible at the final stages of glycan maturation only, whereas the effects of dNM are observed quite early in the biosynthetic process. The secretion of IgD, but not IgM, was blocked in dNM-treated cells. The HLA-A, B, C heavy chains synthesized by the Daudi cell line were degraded in an accelerated fashion in dNM-treated cells, but no effects were seen on the HLA-DR antigens in these cells. Although both SW and dNM interfere with trimming, further modifications of the oligosaccharide side chains occur, and show that the two processes are not obligately coupled. Glucosidase inhibitors such as NM and dNM, as well as the mannosidase inhibitor SW, allow modification of glycan structure, and may be used to study the biological role of glycoprotein oligosaccharides and their modifications.

1-Deoxynojirimycin↗
PubMed1982

Conformational aspects of N-glycosylation of proteins. Studies with linear and cyclic peptides as probes.

Conformational aspects of N-glycosylation of glycoproteins have been studied by using a series of peptides which contained, in addition to the ;marker sequence' Asn-Gly-Thr, two cysteine residues in various positions of the peptide chain. The presence of two cysteines permitted a partial fixation of the above triplet sequence in cyclic structures of various size by intramolecular disulphide bond formation. Comparison of the glycosyl acceptor properties of the linear peptides and their corresponding cyclic analogues allows the following statements. The considerably lower acceptor capabilities of the cyclic derivatives indicate that the restriction of rotational degrees of freedom imposed by disulphide bonding results in a conformation which hinders a favourable interaction of the peptide substrate with the N-glycosyltransferase. On the other hand, the glycosylation rate of linear peptides increases with increasing chain length, suggesting that the amino acids on both the N- and C-terminal side of the ;marker sequence' may contribute to a considerable extent to the induction of an ;active' conformation. Realization of a potential sugar attachment site requires a hydrogen bond interaction within the ;marker sequence' between the oxygen of threonine (serine) as the hydrogen bond acceptor and the beta-amide of asparagine as the donor [Bause & Legler (1981) Biochem. J.195, 639-644]. This interaction is obviously facilitated when the peptide chain can adopt a conformation which resembles a beta-turn or other loop structure. The available experimental and statistical data are discussed in terms of possible structural features for N-glycosylation, with the aid of space-filling models.

Amino Acids↗
PubMed1982

The effect of flavomycin on the synthesis and transfer of lipid-linked saccharides in pig brain.

Particulate membrane fractions from pig brain catalyse the synthesis of lipid-linked sugar derivatives of the dolichyl phosphate pathway. Flavomycin, a phosphoglycolipid antibiotic produced by various species of streptomycetes, interferes with the formation of these glycolipids to a different extent. The formation of dolichyl phosphate glucose was shown to be most susceptible to the antibiotic, being blocked by about 50% in the presence of 0.2mm-flavomycin, whereas the synthesis of dolichyl diphosphate N-acetylglucosamine, dolichyl diphosphate chitobiose and dolichyl diphosphate chitobiosyl mannose required higher concentrations to achieve a comparable inhibition. Although the formation of dolichyl phosphate mannose was hardly affected, the accumulation of oligosaccharides with five to seven sugar units was observed, when dolichyl diphosphate oligosaccharides were synthesized with GDP-[(14)C]mannose in the presence of 1mm-flavomycin. This indicates that the inhibition of the synthesis of larger-sized oligosaccharides, known to be mediated by lipid-bound mannose, was not caused by an actual deficiency in dolichyl phosphate mannose. At flavomycin concentrations that inhibited the formation of dolichyl phosphate glucose by 50%, the transfer of lipid-linked saccharides to either the hexapeptide Tyr-Asn-Gly-Thr-Ser-Val or endogenous protein acceptors was hardly influenced. The mode of action of flavomycin is still obscure, but seems not to be of a competitive nature, since the inhibition was unaffected by increasing concentrations of dolichyl phosphate. Some evidence indicates that, besides a direct interaction of the antibiotic with some transferases, a non-specific incorporation into the membrane and alteration of its properties might be responsible for those inhibitory effects on all enzymes which were observed at high concentrations of flavomycin.

Animals↗
PubMed1982

Inhibition by nojirimycin and 1-deoxynojirimycin of microsomal glucosidases from calf liver acting on the glycoprotein oligosaccharides Glc1-3Man9GlcNAc2.

Particulate membrane fractions from calf liver catalyze the release of glucose from GlcNAc2-Man9-Glc1-3-oligosaccharides. Maximal oligosaccharide-glucosidase activity was obtained at pH 6.2 and a detergent concentration of 0.5% Triton X-100. This activity could be distinguished from non-specific alpha-glucosidase activity on the basis of different pH-dependence and lack of activation by detergent. The relative rates for the hydrolysis of the Glc3-, Glc2-, and Glc1-oligosaccharide, estimated from the initial velocity, was 1:12:3. There is no significant difference in the enzyme activity towards free, peptide-bound, or lipid-linked oligosaccharide. Nojirimycin and 1-deoxynojirimycin were strong inhibitors of microsomal oligosaccharide-glucosidases. Hydrolysis of Glc3-oligosaccharide was inhibited by 50% at concentrations of 0.16 mM and 2 microM, respectively. Hydrolysis of the Glc2- and Glc1-oligosaccharide was inhibited to a somewhat lower extent, suggesting the presence of at least two glucosidases, one acting on Glc3- and one acting on Glc1- and Glc2-oligosaccharide.

1-Deoxynojirimycin↗
PubMed1982

Inhibition of formation of complex oligosaccharides by the glucosidase inhibitor bromoconduritol.

The alpha-glucosidase inhibitor bromoconduritol (6-bromo-3,4,5-trihydroxycyclohex-1-ene) inhibits trimming of the innermost glucose residue from the Glc3Man9GlcNAc2 precursor of high-mannose and complex oligosaccharides. This inhibition occurs both in intact cells and with a microsomal enzyme preparation. The formation of lipid-linked oligosaccharides was increased in glucosidase-inhibited cells. Inhibition of transfer of high-mannose oligosaccharides to protein was not observed. In bromoconduritol-treated virus-infected cells, trimming of mannose can occur despite incomplete removal of glucose. The glucosylated high-mannose oligosaccharides GlcMan9GlcNAc, GlcMan8GlcNAc, and GlcMan7GlcNAc were released from viral glycoproteins after digestion with Pronase and endo-beta-N-acetylglucosaminidase H. The formation of complex oligosaccharides was concomitantly inhibited. The release of infectious fowl plague virus particles (an influenza virus) was inhibited from bromoconduritol-treated infected chicken-embryo cells.

Animals↗
PubMed1982

The Gaucher mouse.

Explore the source record for details and available documents.

Acid Phosphatase↗
PubMed1981

The role of the hydroxy amino acid in the triplet sequence Asn-Xaa-Thr(Ser) for the N-glycosylation step during glycoprotein biosynthesis.

The catalytical role of the hydroxy amino acid in the "marker sequence" Asn-Xaa-Thr(Ser) for the N-glycosylation step of glycoprotein formation was investigated by using a series of hexapeptides derived from Tyr-Asn-Gly-Xaa-Ser-Val by substituting threonine, serine, cysteine, valine and O-methylthreonine respectively for Xaa. The results, which were obtained with calf liver microsomal fractions as enzyme source and dolichyl diphosphate di-N-acetyl [14C] chitobiose as glycosyl donor showed that the threonine-, serine- and cysteine-containing derivatives could be glycosylated, although at very different rates, whereas the valine and O-methylthreonine analogues did not work as glycosyl acceptors. Replacement of threonine by serine resulted in a 4-fold decrease in Vmax, and about a 10-fold increase in Km for glycosyl transfer. Replacement of serine by cysteine again decreased acceptor activity 2-3-fold. The various results, taken together, indicate an absolute requirement for a hydrogen-bond-donor function in the side chain of the hydroxy amino acid of the "marker sequence" and furthermore, point to a considerable influence of the structure of this amino acid on binding as well as on the glycosyl transfer itself. In order to explain the observed differences in the glycosyl-transfer rates, a model is proposed with a hydrogen-bond interaction between the amide of asparagine as the hydrogen-bond donor and the oxygen of the hydroxy group of the hydroxy amino acid as the hydrogen-bond acceptor. The participation of the hydroxy group in the catalytic mechanism of glycosyl transfer in the kind of proton-relay system is discussed.

Amino Acid Sequence↗
PubMed1981

Role of sugar hydroxyl groups in glycoside hydrolysis. Cleavage mechanism of deoxyglucosides and related substrates by beta-glucosidase A3 from Aspergillus wentii.

The contribution of the hydroxyl groups at C-2 and C-4 and of the hydroxy-methyl group at C-5 of beta-glucopyranosides to their hydrolysis by beta-glucosidase A3 (beta-D-glucoside glucohydrolase, EC 3.2.1.21) from Aspergillus wentii was investigated with 4-methylumbelliferyl-beta-glucosides with appropriate structural modifications. Relative hydrolysis rates expressed by kcat/kcat (glucoside) are: 2-deoxy, 4. 10(-6); 2-deoxy-2-amino, 2.4 . 10(-4); 2-deoxy-2-ammonio, less than 1 . 10(-6); 4-deoxy, 1.8 . 10(-4); xyloside, 6.3 . 10(4); galactoside, less than 1 . 10(-6). Binding to the active site as measured by the Km value of these substrates or by the Ki value of the appropriate inhibitors is only moderately decreased by the above modifications. A temperature study with the 2-deoxyglucoside showed that the decrease in kcat is not due to a change in delta H but to a more negative delta S. The steady-state hydrolysis of the 2-deoxyglucoside is approached with a "burst" (rate constant 0.13 min-1) at pH 6 and 1 mM substrate; deglycosylation of the enzyme is partially rate-limiting. Rate constants for glycosylation and deglycosylation calculated from pre-steady-state kinetics were in good agreement with the constants calculated from experiments where the 2-deoxyglucoside was used as an inhibitor for the hydrolysis of the glucoside and where a slow approach to the steady state of the inhibited reaction is observed.

Aspergillus↗
PubMed1981

Distribution of conduritol B epoxide in the animal model for Gaucher's disease (Gaucher mouse).

The time course of the distribution of the beta-glucosidase inhibitor [3H]conduritol B epoxide was determined in various organs of mice, which had received a single interperitoneal dose of the inhibitor. The epoxide is rapidly distributed over all tissues except brain where its concentration is only one-tenth of the average. This is considered an indication that the epoxide can pass the blood/brain barrier only with difficulty. A 4-fold enrichment is seen in the kidney. The inhibitor is excreted with a half-life of about 7 h; it is not metabolized. A parallel determination of beta-glucosidase activity in the tissues showed greater than 90% inhibition within 1 and 2 h and a beginning recovery between 4 and 12 h. The only exception was brain, where no effects could be seen after 1 h and where a subsequent decrease to 37% of normal was observed after 12 h.

Animals↗
PubMed1980

Isolation and structure of a tryptic glycopeptide from the active site of beta-glucosidase A3 from Aspergillus wentii.

A radioactive glycopeptide with a molecular weight of 13 200 was isolated from beta-glucosidase A3 after labeling the active site with [3H]conduritol B epoxide and cleavage with trypsin. The glycopeptide consists of 63 amino acids and 29 +/- 1 sugar residues. Its amino acid sequence was derived from the results of sequence analysis of peptic and cyanogen bromide peptides. The radioactive inhibitor is bound to aspartic acid 12 of the sequence, the sugar residues are probably bound as N-glycosides to asparagine 48 and asparagine 56, since O-glycosidic linkages have been ruled out.

Amino Acid Sequence↗
PubMed1979

Studies on the possible identity of particulate beta-glucosidase and beta-xylosidase of mouse liver.

Mouse liver beta-glucosidase (beta-D-glucosidase glucohydrolase, EC 3.2.1.21) and beta-xylosidase (1,4-beta-D-xylan xylohydrolase, EC 3.2.1.37) activities were studied under different conditions of incubation in an attempt to determine whether these two activities are due to a single enzyme or two separate enzymes. The results showed that: (a) Particle-bound beta-glucosidase and beta-xylosidase activities exhibit similar characteristics with different buffers and at various pH values, in the presence or absence of taurocholate. (b) Both activities are inhibited by gluconolactone and conduritol B eposice. beta-Glucosidase activity is inhibited competitively by the two inhibitors, but beta-xylosidase activity is inhibited non-competitively. (c) Xylonolactone was a very poor inhibitor of both activities, but the inhibition of beta-xylosidase activity was more pronounced than that of beta-glucosidase. (d) The presence of glucosides or xylosides simultaneously in the incubation medium suggested the presence of one enzyme with both activities. These results, together with the mode of inhibition produced by gluconolactone and conduritol B epoxide also suggest the presence of two different binding sites for the beta-D-glucoside and beta-D-xyloside, respectively.

Animals↗
PubMed1978

Inhibition of beta-glucosidases from almonds by cationic and neutral beta-glucosyl derivatives.

The beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) isoenzymes B from sweet and bitter almonds showed considerable differences in their kinetic and inhibition parameters, but both were inhibited much more strongly by basic beta-glucosyl derivatives than by their neutral analogs. The additional interaction energy apparently due to the basic character ranged from 18 kJ/mol (4.3 kcal/mol) for beta-glucosylamine compared to beta-glucose to 28 kJ/mol (6.9 kcal/mol) for N-benzyl-beta-glucosylamine compared to N-beta-glucosyl-p-toluidine. N-beta-Glucosylpyridinium ion and N-beta-glucosylimidazol which both cannot be protonated at the glucosylated nitrogen are very weak inhibitors. beta-2-Amino-2-deoxyglucose is bound with half the affinity of beta-glucosylamine. The structural requirement for strong inhibition is thus the protonation of the inhibitor at the glucosylated nitrogen. The additional binding energy is assumed to be due to the electrostatic interaction of the inhibitor cation with a carboxylate group in an environment of low polarity. The failure of the pyridinium ion to show this interaction is attributed to the presence of a positively charged group at the active site which acts as proton donor. The pKa values of beta-glucosylamine and its derivatives have been determined and found to be 3.5 units lower than those of the corresponding parent amines. An exception is beta-glucosylimidazol (pKa 5.4) which is protonated on the non-glycosylated nitrogen.

Glucosidases↗