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1.
Int J Mol Sci ; 20(20)2019 Oct 12.
Article in English | MEDLINE | ID: mdl-31614738

ABSTRACT

The essential role of dolichyl phosphate (DolP) as a carbohydrate carrier during protein N-glycosylation is well established. The cellular pool of DolP is derived from de novo synthesis in the dolichol branch of the mevalonate pathway and from recycling of DolPP after each cycle of N-glycosylation, when the oligosaccharide is transferred from the lipid carrier to the protein and DolPP is released and then dephosphorylated. In Saccharomyces cerevisiae, the dephosphorylation of DolPP is known to be catalyzed by the Cwh8p protein. To establish the role of the Cwh8p orthologue in another distantly related yeast species, Candida albicans, we studied its mutant devoid of the CaCWH8 gene. A double Cacwh8∆/Cacwh8∆ strain was constructed by the URA-blaster method. As in S. cerevisiae, the mutant was impaired in DolPP recycling. This defect, however, was accompanied by an elevation of cis-prenyltransferase activity and higher de novo production of dolichols. Despite these compensatory changes, protein glycosylation, cell wall integrity, filamentous growth, and biofilm formation were impaired in the mutant. These results suggest that the defects are not due to the lack of DolP for the protein N-glycosylation but rather that the activity of oligosacharyltransferase could be inhibited by the excess DolPP accumulating in the mutant.


Subject(s)
Candida albicans/metabolism , Dolichols/biosynthesis , Fungal Proteins/genetics , Polyisoprenyl Phosphate Oligosaccharides/metabolism , Protein Processing, Post-Translational , Pyrophosphatases/genetics , Candida albicans/growth & development , Cell Wall/metabolism , Dolichols/genetics , Fungal Proteins/metabolism , Glycosylation , Morphogenesis , Pyrophosphatases/metabolism
2.
J Biol Chem ; 291(21): 11042-54, 2016 May 20.
Article in English | MEDLINE | ID: mdl-27015803

ABSTRACT

The glycosylation of asparagine residues is the predominant protein modification in all three domains of life. An oligosaccharide chain is preassembled on a lipid-phospho carrier and transferred onto asparagine residues by the action of a membrane-bound enzyme, oligosaccharyltransferase. The oligosaccharide donor for the oligosaccharyl transfer reaction is dolichol-diphosphate-oligosaccharide in Eukaryota and polyprenol-diphosphate-oligosaccharide in Eubacteria. The donor in some archaeal species was reportedly dolichol-monophosphate-oligosaccharide. Thus, the difference in the number of phosphate groups aroused interest in whether the use of the dolichol-monophosphate type donors is widespread in the domain Archaea. Currently, all of the archaeal species with identified oligosaccharide donors have belonged to the phylum Euryarchaeota. Here, we analyzed the donor structures of two species belonging to the phylum Crenarchaeota, Pyrobaculum calidifontis and Sulfolobus solfataricus, in addition to two species from the Euryarchaeota, Pyrococcus furiosus and Archaeoglobus fulgidus The electrospray ionization tandem mass spectrometry analyses confirmed that the two euryarchaeal oligosaccharide donors were the dolichol-monophosphate type and newly revealed that the two crenarchaeal oligosaccharide donors were the dolichol-diphosphate type. This novel finding is consistent with the hypothesis that the ancestor of Eukaryota is rooted within the TACK (Thaum-, Aig-, Cren-, and Korarchaeota) superphylum, which includes Crenarchaea. Our comprehensive study also revealed that one archaeal species could contain two distinct oligosaccharide donors for the oligosaccharyl transfer reaction. The A. fulgidus cells contained two oligosaccharide donors bearing oligosaccharide moieties with different backbone structures, and the S. solfataricus cells contained two oligosaccharide donors bearing stereochemically different dolichol chains.


Subject(s)
Archaea/metabolism , Asparagine/metabolism , Polyisoprenyl Phosphate Oligosaccharides/chemistry , Polyisoprenyl Phosphate Oligosaccharides/metabolism , Archaea/classification , Archaeal Proteins/metabolism , Archaeoglobus fulgidus/metabolism , Asparagine/chemistry , Glycosylation , Hexosyltransferases/metabolism , Membrane Proteins/metabolism , Molecular Structure , Pyrobaculum/metabolism , Pyrococcus furiosus/metabolism , Spectrometry, Mass, Electrospray Ionization , Sulfolobus solfataricus/metabolism , Tandem Mass Spectrometry
3.
J Biol Chem ; 288(45): 32673-32684, 2013 Nov 08.
Article in English | MEDLINE | ID: mdl-24062310

ABSTRACT

Asparagine (N)-linked glycosylation regulates numerous cellular activities, such as glycoprotein quality control, intracellular trafficking, and cell-cell communications. In eukaryotes, the glycosylation reaction is catalyzed by oligosaccharyltransferase (OST), a multimembrane protein complex that is localized in the endoplasmic reticulum (ER). During N-glycosylation in the ER, the protein-unbound form of oligosaccharides (free oligosaccharides; fOSs), which is structurally related to N-glycan, is released into the ER lumen. However, the enzyme responsible for this process remains unidentified. Here, we demonstrate that eukaryotic OST generates fOSs. Biochemical and genetic analyses using mutant strains of Saccharomyces cerevisiae revealed that the generation of fOSs is tightly correlated with the N-glycosylation activity of OST. Furthermore, we present evidence that the purified OST complex can generate fOSs by hydrolyzing dolichol-linked oligosaccharide, the glycan donor substrate for N-glycosylation. The heterologous expression of a single subunit of OST from the protozoan Leishmania major in S. cerevisiae demonstrated that this enzyme functions both in N-glycosylation and generation of fOSs. This study provides insight into the mechanism of PNGase-independent formation of fOSs.


Subject(s)
Hexosyltransferases/metabolism , Leishmania major/enzymology , Membrane Proteins/metabolism , Oligosaccharides/metabolism , Protozoan Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Glycosylation , Hexosyltransferases/genetics , Leishmania major/genetics , Membrane Proteins/genetics , Oligosaccharides/genetics , Polyisoprenyl Phosphate Oligosaccharides/metabolism , Protozoan Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics
4.
J Biol Chem ; 288(28): 20616-23, 2013 Jul 12.
Article in English | MEDLINE | ID: mdl-23720757

ABSTRACT

Mature dolichol-linked oligosaccharides (mDLOs) needed for eukaryotic protein N-glycosylation are synthesized by a multistep pathway in which the biosynthetic lipid intermediate Man5GlcNAc2-PP-dolichol (M5-DLO) flips from the cytoplasmic to the luminal face of the endoplasmic reticulum. The endoplasmic reticulum membrane protein Rft1 is intimately involved in mDLO biosynthesis. Yeast genetic analyses implicated Rft1 as the M5-DLO flippase, but because biochemical tests challenged this assignment, the function of Rft1 remains obscure. To understand the role of Rft1, we sought to analyze mDLO biosynthesis in vivo in the complete absence of the protein. Rft1 is essential for yeast viability, and no Rft1-null organisms are currently available. Here, we exploited Trypanosoma brucei (Tb), an early diverging eukaryote whose Rft1 homologue functions in yeast. We report that TbRft1-null procyclic trypanosomes grow nearly normally. They have normal steady-state levels of mDLO and significant N-glycosylation, indicating robust M5-DLO flippase activity. Remarkably, the mutant cells have 30-100-fold greater steady-state levels of M5-DLO than wild-type cells. All N-glycans in the TbRft1-null cells originate from mDLO indicating that the M5-DLO excess is not available for glycosylation. These results suggest that rather than facilitating M5-DLO flipping, Rft1 facilitates conversion of M5-DLO to mDLO by another mechanism, possibly by acting as an M5-DLO chaperone.


Subject(s)
Eukaryotic Cells/metabolism , Glycoproteins/metabolism , Membrane Proteins/metabolism , Protozoan Proteins/metabolism , Trypanosoma brucei brucei/metabolism , Electrophoresis, Polyacrylamide Gel , Endoplasmic Reticulum/metabolism , Flow Cytometry , Glucose/pharmacology , Glycoproteins/genetics , Glycosylation , Lysosomal Membrane Proteins/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Membrane Proteins/genetics , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Microscopy, Fluorescence , Models, Biological , Mutation , Polyisoprenyl Phosphate Oligosaccharides/metabolism , Polysaccharides/metabolism , Protein Biosynthesis , Protozoan Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Transformation, Genetic , Trypanosoma brucei brucei/genetics , Trypanosoma brucei brucei/growth & development
5.
J Biol Chem ; 284(30): 19835-42, 2009 Jul 24.
Article in English | MEDLINE | ID: mdl-19494107

ABSTRACT

To further evaluate the role of Rft1 in the transbilayer movement of Man(5)GlcNAc(2)-P-P-dolichol (M5-DLO), a series of experiments was conducted with intact cells and sealed microsomal vesicles. First, an unexpectedly large accumulation (37-fold) of M5-DLO was observed in Rft1-depleted cells (YG1137) relative to Glc(3)Man(9)GlcNAc(2)-P-P-Dol in wild type (SS328) cells when glycolipid levels were compared by fluorophore-assisted carbohydrate electrophoresis analysis. When sealed microsomes from wild type cells and cells depleted of Rft1 were incubated with GDP-[(3)H]mannose or UDP-[(3)H]GlcNAc in the presence of unlabeled GDP-Man, no difference was observed in the rate of synthesis of [(3)H]Man(9)GlcNAc(2)-P-P-dolichol or Man(9)[(3)H]GlcNAc(2)-P-P-dolichol, respectively. In addition, no difference was seen in the level of M5-DLO flippase activity in sealed wild type and Rft1-depleted microsomal vesicles when the activity was assessed by the transport of GlcNAc(2)-P-P-Dol(15), a water-soluble analogue. The entry of the analogue into the lumenal compartment was confirmed by demonstrating that [(3)H]chitobiosyl units were transferred to endogenous peptide acceptors via the yeast oligosaccharyltransferase when sealed vesicles were incubated with [(3)H]GlcNAc(2)-P-P-Dol(15) in the presence of an exogenously supplied acceptor peptide. In addition, several enzymes involved in Dol-P and lipid intermediate biosynthesis were found to be up-regulated in Rft1-depleted cells. All of these results indicate that although Rft1 may play a critical role in vivo, depletion of this protein does not impair the transbilayer movement of M5-DLO in sealed microsomal fractions prepared from disrupted cells.


Subject(s)
Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Microsomes/metabolism , Polyisoprenyl Phosphate Oligosaccharides/analysis , Polyisoprenyl Phosphate Oligosaccharides/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Alkyl and Aryl Transferases/metabolism , Biological Transport , Dolichol Monophosphate Mannose/metabolism , Gene Expression Regulation, Bacterial , Glucose/metabolism , Hexosyltransferases/metabolism , Mannose/metabolism , Membrane Proteins/metabolism , Microsomes/chemistry , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Polyisoprenyl Phosphate Monosaccharides/metabolism , Saccharomyces cerevisiae/genetics
6.
Proc Natl Acad Sci U S A ; 106(3): 767-72, 2009 Jan 20.
Article in English | MEDLINE | ID: mdl-19129492

ABSTRACT

The oligosaccharide donor for protein N-glycosylation, Glc(3)Man(9)GlcNAc(2)-PP-dolichol, is synthesized via a multistep pathway that starts on the cytoplasmic face of the endoplasmic reticulum (ER) and ends in the lumen where the glycosylation reaction occurs. This necessitates transbilayer translocation or flipping of the lipid intermediate Man(5)GlcNAc(2)-PP-dolichol (M5-DLO) across the ER membrane. The mechanism by which M5-DLO-or any other lipid-is flipped across the ER is unknown, except that specific transport proteins or flippases are required. We recently demonstrated M5-DLO flipping activity in proteoliposomes reconstituted from detergent-solubilized ER membrane proteins and showed that it was ATP-independent and required a trypsin-sensitive protein that sedimented at approximately 4S. By using an activity-enriched fraction devoid of glycerophospholipid flippase activity, we now report that M5-DLO is rapidly flipped in the reconstituted system with a time constant tau <2 min, whereas its triantennary structural isomer is flipped slowly with tau >200 min. DLOs larger than M5-DLO are also poorly translocated, with tau ranging from approximately 10 min to >200 min. We conclude that (i) the number and arrangement of mannoses in the DLO glycan has a profound effect on the ability of the DLO to be translocated by the flippase, (ii) glycan size per se does not dictate whether a DLO will be flipped, and (iii) the flippase is highly specific for M5-DLO. Our results suggest a simple structural model for the interaction between the DLO head group and the flippase.


Subject(s)
Endoplasmic Reticulum/metabolism , Lipid Bilayers/metabolism , Phospholipid Transfer Proteins/physiology , Polyisoprenyl Phosphate Oligosaccharides/metabolism , Animals , Biological Transport , Glycosylation , Proteolipids/metabolism , Rats
7.
Glycobiology ; 12(11): 749-62, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12460943

ABSTRACT

N-glycosylation in nearly all eukaryotes proceeds in the endoplasmic reticulum (ER) by transfer of the precursor Glc(3)Man(9)GlcNAc(2) from dolichyl pyrophosphate (PP-Dol) to consensus Asn residues in nascent proteins. The Saccharomyces cerevisiae alg (asparagine-linked glycosylation) mutants fail to synthesize oligosaccharide lipid properly, and the alg12 mutant accumulates a Man(7)GlcNAc(2)-PP-Dol intermediate. We show that the Man(7)GlcNAc(2) released from alg12Delta-secreted invertase is Manalpha1,2Manalpha1,2Manalpha1,3(Manalpha1,2Manalpha1,3Manalpha1,6)-Manbeta1,4-GlcNAcbeta1-4GlcNAcalpha/beta, confirming that the Man(7)GlcNAc(2) is the product of the middle-arm terminal alpha1,2-mannoslytransferase encoded by the ALG9 gene. Although the ER glucose addition and trimming events are similar in alg12Delta and wild-type cells, the central-arm alpha1,2-linked Man residue normally removed in the ER by Mns1p persists in the alg12Delta background. This confirms in vivo earlier in vitro experiments showing that the upper-arm Manalpha1,2Manalpha1,6-disaccharide moiety, missing in alg12Delta Man(7)GlcNAc(2), is recognized and required by Mns1p for optimum mannosidase activity. The presence of this Man influences downstream glycan processing by reducing the efficiency of Ochlp, the cis-Golgi alpha1,6-mannosyltransferase responsible for initiating outer-chain mannan synthesis, leading to hypoglycosylation of external invertase and vacuolar protease A.


Subject(s)
Endoplasmic Reticulum/metabolism , Glycoproteins/metabolism , Golgi Apparatus/metabolism , Mannosyltransferases/metabolism , Polyisoprenyl Phosphate Oligosaccharides/metabolism , Polysaccharides/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Carbohydrate Sequence , Chromatography, Ion Exchange , Glycoproteins/chemistry , Glycosylation , Magnetic Resonance Spectroscopy , Mannosyltransferases/chemistry , Mannosyltransferases/genetics , Molecular Sequence Data , Molecular Structure , Mutation/genetics , Protein Processing, Post-Translational , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics
8.
Biochemistry ; 41(24): 7670-6, 2002 Jun 18.
Article in English | MEDLINE | ID: mdl-12056898

ABSTRACT

Nisin is an example of type-A lantibiotics that contain cyclic lanthionine rings and unusual dehydrated amino acids. Among the numerous pore-forming antimicrobial peptides, type-A lantibiotics form an unique family of post-translationally modified peptides. Via the recognition of cell wall precursor lipid II, nisin has the capacity to form pores against Gram-positive bacteria with an extremely high activity in the nanomolar (nM) range. Here we report a high-resolution NMR spectroscopy study of nisin/lipid II interactions in SDS micelles as a model membrane system in order to elucidate the mechanism of molecular recognition at residue level. The binding to lipid II was studied through (15)N-(1)H HSQC titration, backbone amide proton temperature coefficient analysis, and heteronuclear (15)N[(1)H]-NOE relaxation dynamics experiments. Upon the addition of lipid II, significant changes were monitored in the N-terminal part of nisin. An extremely low amide proton temperature coefficient (Delta delta/Delta T) was found for the amide proton of Ala3 (> -0.1 ppb/K) in the complex form. This suggests tight hydrogen bonding and/or isolation from the bulk solvent for this residue. Large chemical shift perturbations were also observed in the first two rings. In contrast, the C-terminal part of nisin was almost unaffected. This part of the molecule remains flexible and solvent-exposed. On the basis of our results, a multistep pore-forming mechanism is proposed. The N-terminal part of nisin first binds to lipid II, and a subsequent structural rearrangement takes place. The C-terminal part of nisin is possibly responsible for the activation of the pore formation. In light of the emerging antibiotic resistance problems, an understanding of the specific recognition mechanism of nisin with lipid II at the residue specific level may therefore aid in the development of novel antibiotics.


Subject(s)
Ion Channels/chemistry , Membrane Lipids/chemistry , Micelles , Nisin/analogs & derivatives , Nisin/chemistry , Nuclear Magnetic Resonance, Biomolecular/methods , Polyisoprenyl Phosphate Oligosaccharides/chemistry , Uridine Diphosphate N-Acetylmuramic Acid/analogs & derivatives , Uridine Diphosphate N-Acetylmuramic Acid/chemistry , Amides/chemistry , Amino Acid Sequence , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Binding Sites , Carbohydrate Sequence , Ion Channels/metabolism , Membrane Lipids/metabolism , Molecular Sequence Data , Nisin/metabolism , Peptidoglycan , Polyisoprenyl Phosphate Oligosaccharides/metabolism , Protein Conformation , Protons , Sodium Dodecyl Sulfate , Solutions , Solvents , Temperature , Thermodynamics , Titrimetry , Uridine Diphosphate N-Acetylmuramic Acid/metabolism
9.
Nature ; 415(6870): 447-50, 2002 Jan 24.
Article in English | MEDLINE | ID: mdl-11807558

ABSTRACT

N-linked glycosylation of proteins in eukaryotic cells follows a highly conserved pathway. The tetradecasaccharide substrate (Glc3Man9GlcNAc2) is first assembled at the membrane of the endoplasmic reticulum (ER) as a dolichylpyrophosphate (Dol-PP)-linked intermediate, and then transferred to nascent polypeptide chains in the lumen of the ER. The assembly of the oligosaccharide starts on the cytoplasmic side of the ER membrane with the synthesis of a Man5GlcNAc2-PP-Dol intermediate. This lipid-linked intermediate is then translocated across the membrane so that the oligosaccharides face the lumen of the ER, where the biosynthesis of Glc3Man9GlcNAc2-PP-Dol continues to completion. The fully assembled oligosaccharide is transferred to selected asparagine residues of target proteins. The transmembrane movement of lipid-linked Man5GlcNAc2 oligosaccharide is of fundamental importance in this biosynthetic pathway, and similar processes involving phospholipids and glycolipids are essential in all types of cells. The process is predicted to be catalysed by proteins, termed flippases, which to date have remained elusive. Here we provide evidence that yeast RFT1 encodes an evolutionarily conserved protein required for the translocation of Man5GlcNAc2-PP-Dol from the cytoplasmic to the lumenal leaflet of the ER membrane.


Subject(s)
Endoplasmic Reticulum/metabolism , Lipid Metabolism , Membrane Glycoproteins/metabolism , Oligosaccharides/metabolism , Phospholipid Transfer Proteins , Saccharomyces cerevisiae Proteins/metabolism , Asparagine/metabolism , Biological Transport , Carrier Proteins/metabolism , Conserved Sequence , Glycosylation , Intracellular Membranes/metabolism , Membrane Glycoproteins/genetics , Membrane Proteins/metabolism , Membrane Transport Proteins , Polyisoprenyl Phosphate Oligosaccharides/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics
10.
Biol Chem ; 382(2): 321-8, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11308030

ABSTRACT

The formation of N-glycosidic linkages of glycoproteins involves the ordered assembly of the common Glc3Man9GlcNAc2 core-oligosaccharide on the lipid carrier dolichyl pyrophosphate. Whereas early mannosylation steps occur on the cytoplasmic side of the endoplasmic reticulum with GDP-Man as donor, the final reactions from Man5GlcNAc2-PP-Dol to Man9GlcNAc2-PP-Dol on the lumenal side use Dol-P-Man. We have investigated these later stages in vitro using a detergent-solubilized enzyme extract from yeast membranes. Mannosyltransfer from Dol-P-Man to [3H]Man5GlcNAc2-PP-Dol with formation of all intermediates up to Man9GlcNAc2-PP-Dol occured in a rapid, time- and protein-dependent fashion. We find that the initial reaction from Man5GlcNAc2-PP-Dol to Man6GlcNAc2-PP-Dol is independent of metal ions, but further elongations need Mn2+ that can be partly replaced by Mg2+ or Ca2+. Zn2+ or Cd2+ ions were found to inhibit formation of Man(7-9)GlcNAc2-PP-Dol, but do not affect synthesis of Man6GlcNAc2-PP-Dol. Extension did not occur when the acceptor was added as a free Man5GlcNAc2 oligosaccharide or when GDP-Man was used as mannosyl donor. The alg3 mutant was described to accumulate Man5GlcNAc2-PP-Dol. We expressed a functional active HA-epitope tagged ALG3 fusion and succeeded to selectively immunoprecipitate the Dol-P-Man:Man5GlcNAc2-PP-Dol mannosyltransferase activity from the other enzymes of the detergent extract involved in the subsequent mannosylation reactions. This demonstrates that Alg3p represents the mannosyltransferase itself and not an accessory protein involved in the reaction.


Subject(s)
Fungal Proteins/metabolism , Lipopolysaccharides/biosynthesis , Mannosyltransferases/genetics , Mannosyltransferases/metabolism , Membrane Proteins/metabolism , Saccharomyces cerevisiae Proteins , Yeasts/metabolism , Chemical Precipitation , Fungal Proteins/genetics , Membrane Proteins/genetics , Metals , Polyisoprenyl Phosphate Oligosaccharides/metabolism , Solubility , Yeasts/genetics
11.
FEMS Microbiol Lett ; 191(2): 187-90, 2000 Oct 15.
Article in English | MEDLINE | ID: mdl-11024262

ABSTRACT

An in situ transglycosylase assay has been developed using endogenously synthesized lipid II. The assay involves the preferential synthesis and accumulation of lipid II in a reaction mixture containing the cell wall membrane material isolated from Escherichia coli, exogenously supplied UDP-MurNAc-pentapeptide, and radiolabeled UDP-GlcNAc. In the presence of Triton X-100, the radiolabeled product formed is almost exclusively lipid II, while the subsequent formation of peptidoglycan is inhibited. Removal of the detergent resulted in the synthesis of peptidoglycan (25% incorporation of radiolabeled material) from the accumulated lipid II. This reaction was inhibited by moenomycin, a known transglycosylase inhibitor. In addition, tunicamycin, which affects an earlier step of the pathway by inhibiting MraY, had no effect on the formation of peptidoglycan in this assay, as expected. Similarly, ampicillin and bacitracin did not inhibit the formation of peptidoglycan under the conditions established.


Subject(s)
Enzyme Inhibitors/pharmacology , Escherichia coli/enzymology , Glycosyltransferases/antagonists & inhibitors , Anti-Bacterial Agents/pharmacology , Bacteriological Techniques , Bambermycins/pharmacology , Glycosyltransferases/metabolism , Octoxynol/pharmacology , Peptidoglycan/metabolism , Polyisoprenyl Phosphate Oligosaccharides/metabolism , Tunicamycin/pharmacology
12.
Biochem Biophys Res Commun ; 272(1): 290-2, 2000 May 27.
Article in English | MEDLINE | ID: mdl-10872841

ABSTRACT

The biochemical characterization of bacterial glycosyltransferases involved in the assembly of cell-wall-associated polysaccharides is often hindered by the lack of the appropriate undecaprenyl-pyrophosphate-linked acceptor substrate. In order to find a suitable synthetic substrate for the alpha1,3-mannosyltransferase AceA from Acetobacter xylinum, phytanyl-pyrophosphate-linked cellobiose was prepared. In the presence of GDP-[14C]mannose and recombinant AceA, the phytanyl-pyrophosphate-linked cellobiose afforded a 14C-labeled trisaccharide that was sensitive to alpha-mannosidase degradation in a fashion analogous to the natural undecaprenyl-pyrophosphate-linked cellobiose substrate. These results suggest that phytanyl-pyrophosphate-linked oligosaccharides may be useful substrates for other important bacterial glycosyltransferases.


Subject(s)
Mannosyltransferases/metabolism , Acetobacter/enzymology , Cellobiose/chemistry , Cellobiose/metabolism , Molecular Structure , Polyisoprenyl Phosphate Oligosaccharides/chemistry , Polyisoprenyl Phosphate Oligosaccharides/metabolism , Recombinant Proteins/metabolism , Substrate Specificity
13.
J Biol Chem ; 275(6): 4267-77, 2000 Feb 11.
Article in English | MEDLINE | ID: mdl-10660594

ABSTRACT

N-Glycans in nearly all eukaryotes are derived by transfer of a precursor Glc(3)Man(9)GlcNAc(2) from dolichol (Dol) to consensus Asn residues in nascent proteins in the endoplasmic reticulum. The Saccharomyces cerevisiae alg (asparagine-linked glycosylation) mutants fail to synthesize oligosaccharide-lipid properly, and the alg9 mutant, accumulates Man(6)GlcNAc(2)-PP-Dol. High-field (1)H NMR and methylation analyses of Man(6)GlcNAc(2) released with peptide-N-glycosidase F from invertase secreted by Deltaalg9 yeast showed its structure to be Manalpha1,2Manalpha1,2Manalpha1, 3(Manalpha1,3Manalpha1,6)-Manbeta1,4GlcNAcbeta1, 4GlcNAcalpha/beta, confirming the addition of the alpha1,3-linked Man to Man(5)GlcNAc(2)-PP-Dol prior to the addition of the final upper-arm alpha1,6-linked Man. This Man(6)GlcNAc(2) is the endoglycosidase H-sensitive product of the Alg3p step. The Deltaalg9 Hex(7-10)GlcNAc(2) elongation intermediates were released from invertase and similarly analyzed. When compared with alg3 sec18 and wild-type core mannans, Deltaalg9 N-glycans reveal a regulatory role for the Alg3p-dependent alpha1,3-linked Man in subsequent oligosaccharide-lipid and glycoprotein glycan maturation. The presence of this Man appears to provide structural information potentiating the downstream action of the endoplasmic reticulum glucosyltransferases Alg6p, Alg8p and Alg10p, glucosidases Gls1p and Gls2p, and the Golgi Och1p outerchain alpha1,6-Man branch-initiating mannosyltransferase.


Subject(s)
Dolichols/analogs & derivatives , Fungal Proteins/metabolism , Mannans/metabolism , Mannosyltransferases , Membrane Proteins/metabolism , Polyisoprenyl Phosphate Oligosaccharides/metabolism , Polysaccharides/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , Amidohydrolases/metabolism , Carbohydrate Conformation , Carbohydrate Sequence , Chromatography, High Pressure Liquid , Dolichols/metabolism , Endoplasmic Reticulum/enzymology , Glycoproteins/chemistry , Glycoside Hydrolases/chemistry , Glycoside Hydrolases/metabolism , Glycosyltransferases/metabolism , Lipopolysaccharides , Magnetic Resonance Spectroscopy , Mannans/chemistry , Mannosides/chemistry , Molecular Sequence Data , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase , Saccharomyces cerevisiae/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , beta-Fructofuranosidase
14.
Science ; 286(5448): 2361-4, 1999 Dec 17.
Article in English | MEDLINE | ID: mdl-10600751

ABSTRACT

Resistance to antibiotics is increasing in some groups of clinically important pathogens. For instance, high vancomycin resistance has emerged in enterococci. Promising alternative antibiotics are the peptide antibiotics, abundant in host defense systems, which kill their targets by permeabilizing the plasma membrane. These peptides generally do not act via specific receptors and are active in the micromolar range. Here it is shown that vancomycin and the antibacterial peptide nisin Z use the same target: the membrane-anchored cell wall precursor Lipid II. Nisin combines high affinity for Lipid II with its pore-forming ability, thus causing the peptide to be highly active (in the nanomolar range).


Subject(s)
Anti-Bacterial Agents/pharmacology , Micrococcus/drug effects , Nisin/analogs & derivatives , Polyisoprenyl Phosphate Oligosaccharides/metabolism , Amino Acid Sequence , Anti-Bacterial Agents/metabolism , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Membrane Permeability/drug effects , Cell Wall/metabolism , Dose-Response Relationship, Drug , Membrane Lipids/metabolism , Microbial Sensitivity Tests , Micrococcus/metabolism , Molecular Sequence Data , Nisin/metabolism , Nisin/pharmacology , Peptides/pharmacology , Peptidoglycan , Vancomycin/pharmacology
15.
J Biol Chem ; 274(48): 34072-82, 1999 Nov 26.
Article in English | MEDLINE | ID: mdl-10567375

ABSTRACT

The assembly of the core oligosaccharide region of asparagine-linked glycoproteins proceeds by means of the dolichol pathway. The first step of this pathway, the reaction of dolichol phosphate with UDP-GlcNAc to form N-acetylglucosaminylpyrophosphoryldolichol (GlcNAc-P-P-dolichol), is under investigation as a possible site of metabolic regulation. This report describes feedback inhibition of this reaction by the second intermediate of the pathway, N-acetylglucosaminyl-N-acetylglucosaminylpyrophosphoryldolichol (GlcNAc-GlcNAc-P-P-dolichol), and product inhibition by GlcNAc-P-P-dolichol itself. These influences were revealed when the reactions were carried out in the presence of showdomycin, a nucleoside antibiotic, present at concentrations that block the de novo formation of GlcNAc-GlcNAc-P-P-dolichol but not that of GlcNAc-P-P-dolichol. The apparent K(i) values for GlcNAc-P-P-dolichol and GlcNAc-GlcNAc-P-P-dolichol under basal conditions were 4.4 and 2.8 microM, respectively. Inhibition was also observed under conditions where mannosyl-P-dolichol (Man-P-dol) stimulated the biosynthesis of GlcNAc-P-P-dolichol; the apparent K(i) values for GlcNAc-P-P-dolichol and GlcNAc-GlcNAc-P-P-dolichol were 2.2 and 11 microM, respectively. Kinetic analysis of the types of inhibition indicated competitive inhibition by GlcNAc-P-P-dolichol toward the substrate UDP-GlcNAc and non-competitive inhibition toward dolichol phosphate. Inhibition by GlcNAc-GlcNAc-P-P-dolichol was uncompetitive toward UDP-GlcNAc and competitive toward dolichol phosphate. A model is presented for the kinetic mechanism of the synthesis of GlcNAc-P-P-dolichol. GlcNAc-P-P-dolichol also exerts a stimulatory effect on the biosynthesis of Man-P-dol, i.e. a reciprocal relationship to that previously observed between these two intermediates of the dolichol pathway. This network of inhibitory and stimulatory influences may be aspects of metabolic control of the pathway and thus of glycoprotein biosynthesis in general.


Subject(s)
Polyisoprenyl Phosphate Monosaccharides/antagonists & inhibitors , Polyisoprenyl Phosphate Monosaccharides/metabolism , Acetylglucosamine/biosynthesis , Acids/pharmacology , Animals , Chick Embryo , Dolichol Phosphates/antagonists & inhibitors , Dolichol Phosphates/metabolism , Dolichols/analogs & derivatives , Dolichols/biosynthesis , Hydrolysis/drug effects , Kinetics , Lipids/biosynthesis , Microsomes/drug effects , Microsomes/metabolism , Polyisoprenyl Phosphate Monosaccharides/chemistry , Polyisoprenyl Phosphate Oligosaccharides/antagonists & inhibitors , Polyisoprenyl Phosphate Oligosaccharides/chemistry , Polyisoprenyl Phosphate Oligosaccharides/metabolism , Retina/drug effects , Retina/embryology , Retina/metabolism , Showdomycin/pharmacology , Transferases (Other Substituted Phosphate Groups)/metabolism , Tritium , Uridine Diphosphate N-Acetylglucosamine/antagonists & inhibitors , Uridine Diphosphate N-Acetylglucosamine/metabolism , Uridine Monophosphate/metabolism
17.
Bioorg Med Chem ; 7(3): 441-7, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10220030

ABSTRACT

Oligosaccharyltransferase (OST) catalyzes the transfer of a branched oligosaccharide from a dolichylpyrophosphate oligosaccharide (Dol-PP-OS) to the asparagine of a nascent polypeptide chain in vivo and peptide substrates in vitro. Here we report the isolation and purification of Dol-PP-OS from bovine pancreas and thyroid. Steady-state kinetic parameters comparing the two Dol-PP-OS to a shorter dolichylpyrophosphate disaccharide (DolPP-DS) previously synthesized in our laboratory are reported. These were determined for Dol-PP-OS, Dol-PP-DS, and the tripeptide Bz-Asn-Leu-Thr-NH2 with solubilized OST and, for the first time, saturation kinetics were observed for all substrates. The kinetic data provide a basis for analyzing quantitatively the individual contributions of oligosaccharide donor and peptide acceptor substrates to OST-catalyzed glycosylation.


Subject(s)
Polyisoprenyl Phosphate Oligosaccharides/isolation & purification , Animals , Cattle , Evaluation Studies as Topic , Kinetics , Pancreatin/chemistry , Polyisoprenyl Phosphate Oligosaccharides/metabolism , Substrate Specificity , Thyroid Gland/chemistry
19.
Mol Microbiol ; 30(2): 317-27, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9791177

ABSTRACT

It is generally assumed that type A lantibiotics primarily kill bacteria by permeabilization of the cytoplasmic membrane. As previous studies had demonstrated that nisin interacts with the membrane-bound peptidoglycan precursors lipid I and lipid II, we presumed that this interaction could play a role in the pore formation process of lantibiotics. Using a thin-layer chromatography system, we found that only nisin and epidermin, but not Pep5, can form a complex with [14C]-lipid II. Lipid II was then purified from Micrococcus luteus and incorporated into carboxyfluorescein-loaded liposomes made of phosphatidylcholine and cholesterol (1:1). Liposomes supplemented with 0.05 or 0.1 mol% of lipid II did not release any marker when treated with Pep5 or epilancin K7 (peptide concentrations of up to 5 mol% were tested). In contrast, as little as 0.01 mol% of epidermin and 0.1 mol% of nisin were sufficient to induce rapid marker release; phosphatidylglycerol-containing liposomes were even more susceptible. Controls with moenomycin-, undecaprenol- or dodecaprenolphosphate-doped liposomes demonstrated the specificity of the lantibiotics for lipid II. These results were correlated with intact cells in an in vivo model. M. luteus and Staphylococcus simulans were depleted of lipid II by preincubation with the lipopeptide ramoplanin and then tested for pore formation. When applied in concentrations below the minimal inhibitory concentration (MIC) and up to 5-10 times the MIC, the pore formation by nisin and epidermin was blocked; at higher concentrations of the lantibiotics the protective effect of ramoplanin disappeared. These results demonstrate that, in vitro and in vivo, lipid II serves as a docking molecule for nisin and epidermin, but not for Pep5 and epilancin K7, and thereby facilitates the formation of pores in the cytoplasmic membrane.


Subject(s)
Anti-Bacterial Agents/pharmacology , Depsipeptides , Nisin/pharmacology , Peptides, Cyclic , Peptides , Peptidoglycan/metabolism , Anti-Bacterial Agents/metabolism , Bacteriocins , Liposomes/metabolism , Micrococcus luteus/drug effects , Micrococcus luteus/metabolism , Nisin/metabolism , Peptidoglycan/drug effects , Polyisoprenyl Phosphate Oligosaccharides/metabolism , Staphylococcus/drug effects , Staphylococcus/metabolism
20.
Yeast ; 14(8): 773-81, 1998 Jun 15.
Article in English | MEDLINE | ID: mdl-9675821

ABSTRACT

Transfer of truncated oligosaccharides to yeast exoglucanase (Exg) in Saccharomyces cerevisiae alg1 has been investigated. When incubated at the non-permissive temperature, alg1 cells secreted into the culture medium, in addition to the exoglucanase glycoforms secreted by wild type, underglycosylated forms as well as material with ionic properties of the non-glycosylated enzyme. As expected, none of the latter had affinity towards concanavalin A, but part of it bound to wheat germ agglutinin (WGA), suggesting that it contained, in addition to non-glycosylated Exg, glycoforms carrying non-reducing terminal GlcNAc. Only the WGA-bound material could be labelled with galactosyltransferase; furthermore, the label could be released by treatment with peptide-N4-N-acetyl-beta-glucosamine asparagine amidase. These results unambiguously demonstrate that GlcNAc2 can be transferred from dolichol-PP-GlcNAc2 to one or both sequons of yeast Exg. Accordingly, they support previous observations suggesting that this early intermediate is able to translocate in vivo in order to make its sugar portion accessible to the oligosaccharyltransferase in the lumen of the endoplasmic reticulum.


Subject(s)
Polyisoprenyl Phosphate Oligosaccharides/metabolism , Saccharomyces cerevisiae/enzymology , beta-Glucosidase/chemistry , Amidohydrolases , Autoradiography , Blotting, Western , Carbohydrate Conformation , Chromatography, Affinity , Chromatography, Ion Exchange , Concanavalin A/metabolism , Galactose , Glucan 1,3-beta-Glucosidase , Glycosylation , Hexosaminidases , Mannosyltransferases/genetics , Mannosyltransferases/metabolism , Molecular Weight , Mutation , Saccharomyces cerevisiae/metabolism , beta-Glucosidase/isolation & purification , beta-Glucosidase/metabolism
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