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.

Glycosylation reactions in Plasmodium falciparum, Toxoplasma gondii, and Trypanosoma brucei brucei probed by the use of synthetic peptides.

Synthetic peptides were used to probe O- and N-glycosylation reactions in cell-free systems of the parasitic protozoa Plasmodium falciparum, Toxoplasma gondii, and Trypanosoma brucei brucei. O-Glycosylation of the peptide Pro-Tyr-Thr-Val-Val was observed with lysates from all organisms. However, the spectrum of sugars transferred from their respective nucleotide or dolichol-phosphate derivatives to the peptide varied greatly according to the parasite. N-glycosylation of the peptides N-Bz-Asn-Gly-ThrNH2 and DNP-Arg-Asn-Ala-Thr-Ala-ValNH2 by exogenous radioactive dolichol-pyrophosphate linked oligosaccharide donors was observed only when lysates of T. gondii or T. b. brucei were used, but not in P. falciparum. To assay for endogenous N-glycosylation donors, the radiolabeled tripeptide [3H]Ac-Asn-Gly-ThrNHMe was used as acceptor. The peptide was N-glycosylated only by T. gondii and T. b. brucei preparations. Only in these latter two parasites dolichol-cycle mannosyltransferase activity was demonstrated by the elongation of exogenous radiolabeled dolichol-PP-chitobiose. The data substantiate the occurrence of protein O-glycosylation in parasitic protozoa and the exceptional absence of protein N-glycosylation in the asexual intraerythrocytic stage of the malaria parasite, P. falciparum.

Oligosaccharide trimming Man9-mannosidase is a resident ER protein and exhibits a more restricted and local distribution than glucosidase II.

The subcellular distribution of a recently described neutral trimming mannosidase, the Man9-mannosidase, has been investigated by immunoelectron microscopy in pig hepatocytes. This enzyme processes asparagine-linked N-acetylglucosamine2 mannose9 oligosaccharides by specifically cleaving three of the four alpha 1,2-linked mannose residues and is distinct from the presumptive ER alpha-mannosidase and the two Golgi alpha-mannosidases. Specific polyclonal antibodies for Man9-mannosidase have been prepared, affinity-purified and characterized by immunoblotting. Immunolabeling using these antibodies was observed in the rough (rER) and smooth endoplasmic reticulum (sER) as well as transitional elements of the rough endoplasmic reticulum and smooth membrane profiles close to the Golgi apparatus while the cisternal stack of the Golgi apparatus was unlabeled. This indicates that Man9-mannosidase is an ER trimming mannosidase which acts on glycoproteins after glucosidase II trimming and before their transport to the Golgi apparatus. A comparison with glucosidase II by double immunolabeling showed a more restricted and local distribution of Man9-mannosidase since it was undetectable in the nuclear envelope and although present throughout the rER and sER, large portions of rER cisternae and many sER profiles were unlabeled. This local distribution of Man9-mannosidase might provide a morphological basis for its involvement in selective trimming reactions in the ER. The catabolic pathway for Man9-mannosidase, as for glucosidase II, may involve degradation in autophagic vacuoles.

N-methyl-N-(5-carboxypentyl)-1-deoxynojirimycin, a new affinity ligand for the purification of trimming glucosidase I.

The paper describes the synthesis of a new type of affinity resin containing N-methyl-N-(5-carboxypentyl)-1-deoxynojirimycin as the ligand attached to AH-Sepharose 4B, which allows the purification of trimming glucosidase I from a detergent extract of pig liver crude microsomes in one step and with high yield. The structure of the affinity ligand was designed on the basis of the observation that N,N-dialkylated derivatives of 1-deoxynojirimycin do strongly inhibit trimming glucosidase I, but not nonspecific aryl-alpha-glucosidases, including glucosidase II. The specific binding of glucosidase I eliminates the need of additional purification steps with their associated losses which were required with the previously synthesized N-5(-carboxypentyl)-AH-Sepharose 4B resin in order to achieve a homogenous enzyme preparation.

Oligosaccharyltransferase is highly specific for the hydroxy amino acid in Asn-Xaa-Thr/Ser.

Pig liver oligosaccharyltransferase (OST), which is involved in the en bloc transfer of the Dol-PP-linked GlcNAc(2)-Man(9)-Glc(3) precursor on to asparagine residues in the Asn-Xaa-Thr/Ser sequence, is highly stereospecific for the conformation of the 3-carbon atom in the hydroxy amino acid. Moreover, substitution of the hydroxy group by either SH as in cysteine, or NH(2) as in beta,gamma-diamino-butanoic acid as reported previously [Bause, E. et al., Biochem. J. 312 (1995) 979-985], followed by the determination of the pH optimum for enzymatic activity, indicates that neither a negative nor a positive charge in the hydroxy amino acid position is tolerated by the enzyme. Binding of the threonine beta-methyl group by OST is also specific, with serine, L-threo-beta-hydroxynorvaline and L-beta-hydroxynorleucine containing tripeptides all bound much less efficiently than the threonine peptide itself. The data are interpreted in terms of a highly stereospecific hydrophobic binding pocket for the threonine CH(3)-CH(OH) group.

Structure and expression of adenovirus type 12 E1B 58K protein in infected and transformed cells: studies using antibodies directed against a synthetic peptide.

We have studied the kinetics of synthesis of the early adenovirus type 12 (Ad12) E1B 58K tumor antigen during lytic infection and analysed its half-life, intracellular localization and phosphorylation in infected KB and transformed hamster (HA12/7) cells. Our analysis has been based on immunoprecipitations using antibodies directed against a synthetic peptide corresponding to the carboxy-terminal end of the E1B 58K protein. Its synthesis was first detectable approximately 8 h after infection and reached a maximum at about 20 h. There is a slight decrease of synthesis late after infection although its level of production is rather high throughout the infectious cycle. The half-life of the Ad12 E1B 58K polypeptide is 2-3 h in infected cells, but strikingly higher (less than 10 h) in the Ad12-transformed cell line HA12/7. Pulse-chase experiments combined with cell fractionation and immunofluorescence studies suggested that about 50% of the amount of the 58K polypeptide accumulates in the nucleus of infected KB cells at least at late times after infection, but only approximately 10% in Ad12-transformed cells. The 58K polypeptide is phosphorylated in both infected and transformed cells. Analysis of the products of acid hydrolysis indicates phosphorylation to equal amounts of serine and threonine. The implications of all these findings for possible roles of the E1B 58K tumor antigen in lytic infection and transformation are discussed.

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.

In vitro studies on the subcellular location of glucosidase I and glucosidase II in dog pancreas.

When programmed with yeast prepro-alpha-factor mRNA, the heterologous reticulocyte/dog pancreas translation system synthesizes two pheromone related polypeptides, a cytosolically located primary translation product (pp-alpha-Fcyt, 21 kDa) and a membrane-specific and multiply glycosylated alpha-factor precursor (pp-alpha-F3, 27.5 kDa). Glycosylation of the membrane specific pp-alpha-F3 species is competitively inhibited by synthetic peptides containing the consensus sequence Asn-Xaa-Thr as indicated by a shift of its molecular mass from 27.5 kDa to about 19.5 kDa (pp-alpha-F0), whereas the primary translation product pp-alpha-Fcyt is not affected. Likewise, only the glycosylated pp-alpha-F3 structure is digested by Endo H yielding a polypeptide with a molecular mass between pp-alpha-F0 and pp-alpha-Fcyt. These observations strongly suggest that the primary translation product is proteolytically processed during/on its translocation into the lumen of the microsomal vesicles. We believe that this proteolytic processing is due to the cleavage of a signal sequence from the pp-alpha-Fcyt species, although this interpretation contradicts previous data from other groups. The distinct effect exerted by various glycosidase inhibitors (e.g. 1-deoxynojirimycin, N-methyl-dNM, 1-deoxymannojirimycin) on the electrophoretic mobility of the pp-alpha-F3 polypeptide indicates that its oligosaccharide chains are processed to presumably Man9-GlcNAc2 structures under the in vitro conditions of translation. This oligosaccharide processing is most likely to involve the action of glucosidase I and glucosidase II as follows from the specificity of the glycosidase inhibitors applied and the differences of the molecular mass observed in their presence.(ABSTRACT TRUNCATED AT 250 WORDS)

Active-site-directed inhibition of asparagine N-glycosyltransferases with epoxy-peptide derivatives.

The hexapeptide Arg-Asn-Gly-epoxyethylglycine-Ala-Val-OMe specifically inactivates membrane-bound N-glycosyltransferases. The specificity is demonstrated by the inability of peptides containing 2,3-epoxypropyl-, allyl- and vinyl-glycine in the epoxyethylglycine position to function as inhibitors. The inhibition is concentration-dependent and follows first-order kinetics, but requires disruption of the membrane vesicles by detergents to achieve accessibility to the transferase. The enzyme can be protected partially against inactivation by the addition of the acceptor peptide Arg-Asn-Gly-Thr-Ala-Val-OMe, pointing to an active-site-directed reaction. Exhaustion of the endogenous pool of glycosyl donor molecules by preincubation of the membrane vesicles with the acceptor peptide before inhibitor application is accompanied by an additional decrease in the inhibition rate. This suggests that inactivation occurs only under conditions where glycosyl transfer is catalysed. A mechanism of inactivation is proposed in which the transferase catalyses its own inactivation by a kind of 'suicide' mechanism.

Structural requirements of N-glycosylation of proteins. Studies with proline peptides as conformational probes.

Conformational aspects of N-glycosylation have been investigated with a series of proline-containing peptides as molecular probes. The results demonstrate that, depending on the position of the imino acid in the peptide chain, dramatic alterations of glycosylation rates are produced, pointing to a critical contribution of the amino acids framing the 'marker sequence' triplet Asn-Xaa-Thr(Ser) on the formation of a potential sugar-attachment site. No glycosyl transfer at all was detectable to those peptides containing a proline residue either in position Xaa or in the next position beyond the threonine of the Asn-sequon on the C-terminal side, whereas the hexapeptide Pro-Asn-Gly-Thr-Ala-Val was glycosylated at a high rate. (Emboldened residues denote the 'marker sequence' that is identical in all the peptides; italicized residues distinguish the positions of proline in the various peptides.) Studies with space-filling models reveal that the lack of glycosyl-acceptor capabilities of Ala(Pro)-Asn-Gly-Thr-Pro-Val might be directly related to their inability to adopt and/or stabilize a turn or loop conformation which permits the catalytically essential interaction between the hydroxy amino acid and the asparagine residue within the 'marker sequence' [Bause & Legler (1981) Biochem. J. 195, 639-644]. This conclusion is supported by circular-dichroism spectroscopic data, which suggest structure-forming potentials in this type of non-acceptor peptides dominating over those that favour the induction of an appropriate sugar-attachment site in the acceptor peptides. The lack of acceptor properties of Tyr-Asn-Pro-Thr-Ser-Val indicates that even small modifications in the 'recognition' pattern are not tolerated by the N-glycosyltransferases.

Purification and enzymatic properties of endo-alpha 1, 2-mannosidase from pig liver involved in oligosaccharide processing.

An endo-alpha 1,2-mannosidase, which is involved in N-linked oligosaccharide processing, has been purified to homogeneity from crude pig liver microsomes using conventional techniques. Two catalytically active polypeptides, of 48 kDa, have been isolated which degrade [14C]Glc3-1-Man9,-GlcNAc2 to [14C]Glc3-1-Man and a specific Man8-GlcNAc2 isomer. They are not, however, active on synthetic alpha-mannosides. [14C]Glc1-Man9-GlcNAc2 was found to be approximately sevenfold more rapidly hydrolyzed than the [14C]Glc2- and [14C]Glc3-homologues. The 48 kDa and 50 kDa proteins are not N-glycosylated and ran on Superdex 75 as monomers. Kinetic studies showed that these proteins had similar catalytic properties: (i) the pH optima were found to be close to 6.5; (ii) neither activity was metal ion dependent; (iii) hydrolysis of [14C]Glc3-Man9-GlcNAc2 was inhibited strongly by Glc-alpha 1,3-Man (app. Ki approximately 120 microM), but not by 1-deoxymannojirimycin or swainsonine. Other evidence, including immunological data, strongly suggests that the 48 kDa and 50 kDa polypeptides are proteolytic degradation products of a single endo-alpha 1,2-mannosidase, rather than distinct subunits of an oligomeric complex. Possible functions of the endo-alpha 1,2-mannosidase in N-linked oligosaccharide processing are discussed.

Man9-mannosidase from human kidney is expressed in COS cells as a Golgi-resident type II transmembrane N-glycoprotein.

Man9-mannosidase, an alpha 1,2-specific exo-enzyme involved in N-linked oligosaccharide processing, has been cloned recently from a human kidney cDNA library [Bause, E., Bieberich, E., Rolfs, A., Völker, C. & Schmidt, B. (1993) Eur. J. Biochem. 217, 533-540]. Transient expression in COS 1 cells of the enzyme resulted in a more than 20-fold increase of a catalytic activity cleaving specifically alpha 1,2-mannosidic linkages in [14C]Man9-GlcNAc2 or [14C]Man5-GlcNAc2. Man9-mannosidase is expressed as a N-glycoprotein with a molecular mass of 73 kDa. Its enzymic activity is metal ion dependent and inhibited strongly by 1-deoxymannojirimycin (50% at 100 microM). Proteolytic studies with the membrane-associated form of Man9-mannosidase support the view that the enzyme is a type II transmembrane protein as predicted from its cDNA sequence. Several lines of evidence suggest that Man9-mannosidase, as expressed, is N-glycosylated at one of three potential Asn-Xaa-Thr/Ser/Cys acceptor sites. Approximately 50% of the N-linked oligosaccharide chains are removed by endoglycosidase H treatment, whereas complete deglycosylation of the enzyme is observed, when transfected cells were cultured in the presence of the Golgi mannosidase II inhibitor swainsonine, indicating that the sugar moiety of Man9-mannosidase is processed partially by Golgi-resident enzymes. This observation is consistent with the results of indirect immunofluorescence studies, pointing to a localization of the Man9-mannosidase predominantly in the juxtanuclear Golgi region. This localization clearly differs from that of pig liver Man9-mannosidase which appears to be located in the endoplasmic reticulum and transient vesicles.

Affinity purification and characterization of glucosidase II from pig liver.

Glucosidase II has been purified from crude pig liver microsomes by a convenient procedure involving DEAE-Sephacel, Con A-Sepharose and affinity chromatography on N-5-carboxypentyl-1-deoxynojirimycin-AH-Sepharose. Specific binding of glucosidase II to the affinity matrix required its prior separation from glucosidase I, which was accomplished by fractional Con A-Sepharose chromatography. The three-step procedure yielded, with approximately 15% enzyme recovery, a > 190-fold enriched glucosidase II, consisting of two proteins (107 kDa and 112 kDa). Both polypeptides are N-glycosylated with probably one glycan chain, in line with their binding to Con A-Sepharose. Immunological cross-reactivity and other experimental data indicate that the 107 kDa N-glycoprotein is derived from the 112 kDa species by partial proteolysis. The occasional presence of a 60 kDa peptide co-eluting with the catalytic activity suggests that glucosidase II may be associated with other protein subunit(s) in a heteromeric membrane complex. Glucosidase II hydrolyzes the alpha1,3-glucosidic linkages in Glc(2-1)-Man9-GlcNAc2, as well as synthetic alpha-glucosides, efficiently but does not remove the distal alpha1,2-linked glucose in Glc3-Man9-GlcNAc2. The enzyme has a pH optimum close to 6.5 and is not metal ion-dependent. Catalytic activity is strongly inhibited by basic sugar analogues including 1-deoxynojirimycin (dNM; app. Ki approximately 7.0 microM), N-5-carboxypentyl-dNM (app. Ki approximately 32 microM) and castanospermine (app. Ki approximately 40 microM). Substitution of the 3-OH or 6-OH group in dNM by a fluoro group reduces the inhibitory potential drastically. We conclude from these observations that the two hydroxy groups are essential for inhibitor/substrate binding due to their ability to interfere as hydrogen bond donors. A polyclonal antibody raised against the 107 kDa polypeptide reacted specifically with two proteins from different cell types on Western blots. Their molecular masses were identical with those from pig liver microsomes, pointing to a highly conserved amino acid sequence of glucosidase II. This suggests that the variance in molecular mass for glucosidase II reported for the enzyme from other tissues and species may be due to partial proteolysis.

Characterization of trimming Man9-mannosidase from pig liver. Purification of a catalytically active fragment and evidence for the transmembrane nature of the intact 65 kDa enzyme.

An alpha 1,2-mannosidase (Man9-mannosidase) involved in N-linked oligosaccharide processing has been purified about 16,000-fold from pig liver crude microsomes (microsomal fractions) by CM-Sepharose and DEAE-Sephacel chromatography, concanavalin A (Con A)-Sepharose chromatography and, as the key step of the procedure, affinity chromatography on immobilized N-5-carboxypentyl-l-deoxymannojirimycin (CP-dMM). On SDS/polyacrylamide-gel electrophoresis under reducing conditions, the isolated enzyme migrated as a single protein band with a molecular mass of 49 kDa. The enzyme does not bind Con A and is not susceptible to glycopeptidase F, indicating that it lacks N-linked oligosaccharides of the high-mannose or complex type. Purified Man9-mannosidase has a pH optimum close to 6.0 and requires bivalent cations for activity, with Ca2+ being most effective. The enzyme is inhibited strongly by basic sugar analogues of mannose such as 1-deoxymannojirimycin (dMM, Ki approximately 5 microM), N-methyl-dMM (Ki approximately 55 microM) and CP-dMM (Ki approximately 150 microM), whereas NN-dimethyl-dMM and the mannosidase II inhibitor swainsonine were hardly or not at all inhibitory. A homogeneous preparation of the 49 kDa enzyme cleaves specifically three of the four alpha 1,2-mannosidic linkages in the natural Man9-GlcNAc2 (M9) substrate. The relative rates by which the parent and intermediate structures are hydrolysed were found to be about 3:2:5 for M9, M8 and M7 respectively. The enzyme displays only marginal activity toward the remaining alpha 1,2-mannosidic linkages in the Man9-GlcNAc2 oligosaccharide (relative rate of M6 hydrolysis approximately 0.02) and is not active against nitrophenyl and methylumbelliferyl alpha-mannosides. This unique substrate specificity suggests that Man9-mannosidase processing differs from that catalysed by other trimming alpha 1,2-mannosidases hitherto reported. A polyclonal antibody raised against the denatured 49 kDa polypeptide not only recognizes a protein band of similar size in Western blots of crude microsomes, but also reacts strongly with a 65 kDa protein species. On trypsin treatment of detergent-solubilized microsomes, the 65 kDa protein is converted specifically into a stable 49 kDa fragment, indicating a precursor-product relationship between the two proteins. We conclude from this observation that the 65 kDa protein represents the intact form of Man9-mannosidase from which the 49 kDa enzyme which we have isolated has been generated, with retention of catalytic activity, by proteolysis during purification. Proteolytic studies with sealed microsomes suggest that the intact 65 kDa enzyme is a protein with a membrane-spanning domain, as well as a cytosolic polypeptide domain of size at least 3 kDa.