guanosine-diphosphate-mannose and dolichol-monophosphate

Dolichyl mannosyl phosphate synthase is associated with membranes of the rough endoplasmic reticulum and catalyzes mannosyl transfer from GDP-mannose to the hydrophobic long-chain acceptor dolichyl-phosphate. The gene for the yeast enzyme encodes a protein with a molecular mass of 30.36 kDa containing three cysteine residues, at positions 93, 172 and 259 [Orlean, P., Albright, C. & Robbins, P. W. (1988) J. Biol. Chem. 263, 17499-17507]. Inhibition of the synthase by thiol-specific reagents, including N-ethylmaleimide, p-hydroxymercuribenzoate, 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2), and lucifer yellow iodoacetamide (LYI), suggests that sulfhydryl groups might play a role in the catalytic mechanism of the enzyme. Titration of the synthase with Nbs2 or LYI indicated that 1 mol sulfhydryl/mol protein was accessible to these reagents, and that saturation of this site completely inhibited enzyme activity. To ascertain the reactive group and its possible function in enzyme catalysis, each of the cysteine residues was replaced individually by site-directed mutagenesis. The mutant enzymes had specific activities comparable to that of the wild-type enzyme, demonstrating that none of the cysteine residues were essential for catalytic activity. All of the mutant proteins except those containing a substitution at Cys93 were inhibited by thiol-blocking reagents, indicating that Cys93 might be physically located near the catalytic site of the enzyme. GDP-mannose, dolichyl phosphate and substrate analogs were found to protect against Nbs2 inactivation, further suggesting that Cys93 was physically near, or within, the substrate-binding site of the enzyme.

Topics: Binding Sites; Cysteine; Dolichol Phosphates; Enzyme Inhibitors; Genes, Fungal; Guanosine Diphosphate Mannose; Mannosyltransferases; Mutagenesis, Site-Directed; Organophosphates; Phospholipids; Recombinant Proteins; Saccharomyces cerevisiae; Sulfhydryl Reagents

The glycosylphosphatidylinositol membrane anchor of variant surface glycoprotein of the African trypanosome Trypanosoma brucei contains several mannosyl residues for which dolichol phosphoryl mannose is supposed to be the precursor; this itself is probably synthesised by a dolichol-dependent mannosyltransferase. We have characterised and localised a mannosyltransferase activity of T. brucei which transfers mannose from GDP-[14C]mannose to exogenously added dolichyl phosphate. The enzyme was saturable for both its substrates and had a Km of 7.8 microM and 3.3 microM, respectively, for dolichyl phosphate and GDP-mannose. Mannosyltransferase was labile at 37 degrees C in the presence of Triton X-100, but its activity remained constant for at least 60 min at temperatures between 10-15 degrees C. The enzyme was inhibited by amphomycin and this inhibition was potentiated by the presence of 10 mM CaCl2. After subcellular fractionation of cell homogenates by differential centrifugation, mannosyltransferase was recovered mainly in the microsomal fraction and its distribution was very similar to that of RNA, a marker for the rough endoplasmic reticulum. After isopycnic centrifugation in a linear sucrose gradient the distribution of mannosyltransferase also resembled that of RNA. Both constituents exhibited a shift towards lower densities after pre-treatment of microsomal membranes with inorganic pyrophosphate, while other membrane markers such as acid phosphatase and nucleoside diphosphatase did not. It is concluded that the formation of dolichol phosphoryl mannose from GDP-mannose and dolichyl phosphate in T. brucei occurs mainly in the rough endoplasmic reticulum.

Topics: Animals; Anti-Bacterial Agents; Cell Compartmentation; Cell Fractionation; Cytoplasm; Detergents; Diphosphates; Dolichol Phosphates; Endoplasmic Reticulum; Enzyme Activation; Guanosine Diphosphate Mannose; Lipopeptides; Mannose; Mannosyltransferases; Membrane Proteins; Microsomes; Oligopeptides; Subcellular Fractions; Trypanosoma brucei brucei

Synthetic truncated dolichyl phosphates of chain lengths from four to thirteen isoprene units (Jaenicke L. and Siegmund H.-U., Chem. Phys. Lipids 51 (1989) 159-170) were assayed for their cofactor activity in the enzymatic transfer of hexoses and hexosamines. The enzymes were microsomal preparations from the green alga Volvox carteri, baker's yeast, and mammalian liver cells. Under saturating conditions, the acceptor activities of the truncated dolichyl phosphates increased from zero to full strength as compared to the mixture of long-chain dolichyl phosphates from natural sources with growing chain length from five to nine isoprene units. Km determinations confirmed the results. Of the geometric isomers of dolichyl 7-phosphate (35 carbon atoms), the 14-trans compound has unchanged acceptor activity; all-trans dolichyl 7-phosphate, however, was almost inactive. The data suggest that hydrophobicity may be an important, but not the only criterion for the binding of the isoprene moiety to the active sites of the transferase enzyme(s) and that the geometry of more than only one double bond in the dolichols is recognized.

Topics: Animals; Cattle; Chlorophyta; Coenzymes; Dolichol Phosphates; Glycosyltransferases; Guanosine Diphosphate Mannose; In Vitro Techniques; Isomerism; Kinetics; Microsomes, Liver; Phosphorylation; Saccharomyces cerevisiae; Uridine Diphosphate

Hamster liver post-nuclear membranes catalyze the transfer of mannose from GDP-mannose to endogenous dolichyl phosphate and to a second major endogenous acidic lipid. This mannolipid was believed to be synthesized from endogenous retinyl phosphate and was tentatively identified as retinyl phosphate mannose (Ret-P-Man) (De Luca, L. M., Brugh, M. R. Silverman-Jones, C. S. and Shidoji, Y. (1982) Biochem. J. 208, 159-170). To characterize this endogenous mannolipid in more detail, we isolated and purified the mannolipid from incubations containing hamster liver membranes and GDP-[14C]mannose and compared its properties to those of authentic Ret-P-Man. We found that the endogenous mannolipid was separable from authentic Ret-P-Man on a Mono Q anion exchange column, did not exhibit the absorbance spectrum characteristic of a retinol moiety, and was stable to mild acid under conditions which cleave authentic Ret-P-Man. The endogenous mannolipid was sensitive to mild base hydrolysis and mannose was released from the mannolipid by snake venom phosphodiesterase digestion. These properties were consistent with the endogenous acceptor being phosphatidic acid. Addition of exogenous phosphatidic acid, but not phospholipids with a head group blocking the phosphate moiety, to incubations containing hamster liver membranes and GDP-[14C]mannose resulted in the synthesis of a mannolipid with chromatographic and physical properties identical to the endogenous mannolipid. A double-labeled mannolipid was synthesized in incubations containing hamster liver membranes, GDP-[14C]mannose, and [3H]phosphatidic acid. Mannosyl transfer to exogenous phosphatidic acid was saturable with increasing concentrations of phosphatidic acid and GDP-mannose and specific for glycosyl transfer from GDP-mannose. Class E Thy-1-negative mutant mouse lymphoma cell membranes, which are defective in dolichyl phosphate mannose synthesis, also fail to transfer mannose from GDP-mannose to exogenous phosphatidic acid or retinyl phosphate. Amphomycin, an inhibitor of dolichyl phosphate mannose synthesis, blocked mannosyl transfer to the endogenous lipid, and to exogenous retinyl phosphate and phosphatidic acid. We conclude that the same mannosyltransferase responsible for dolichyl phosphate mannose synthesis can also utilize in vitro exogenous retinyl phosphate and phosphatidic acid as well as endogenous phosphatidic acid as mannosyl acceptors.

Topics: Animals; Carbon Radioisotopes; Cricetinae; Diterpenes; Dolichol Monophosphate Mannose; Dolichol Phosphates; Glycolipids; Guanosine Diphosphate Mannose; In Vitro Techniques; Male; Mannose; Mannosyltransferases; Mesocricetus; Mice; Microsomes, Liver; Phosphatidic Acids; Polyisoprenyl Phosphate Monosaccharides

The enzymatic transfer of GlcNAc from UDP-GlcNAc and Man from GDP-Man to Dol-P has been characterized in human liver preparations. The presence of low concentrations of detergent, divalent cation and exogenous Dol-P are required for both enzymatic activities. The pH optimum of both reactions is broad with maximal activity near pH 7.8. The majority of N-acetylglucosaminyltransferase (90%) and mannosyltransferase (85%) activities is particulate but approximately 90% of both activities can be released into supernatant fluids by using Triton X-100 in the homogenizing buffer. The supernatant fluid enzymes have properties similar to those of the particulate enzymes although their activities are considerably less stable. Preliminary characterization of the enzymatic reaction products gave the following evidence for formation of GlcNAc and Man derivatives of Dol-P: (1) radiolabelled products are soluble in organic solvents; (2) for each reaction no detectable product is found without addition of exogenous Dol-P and increasing amounts of product are found with increasing amounts of this lipid; (3) acid and base hydrolysis of the glycolipid product (from the N-acetylglucosaminyltransferase reaction) result in radioactive, water-soluble compounds which comigrate with authentic GlcNAc and GlcNAc-1-P, respectively; (4) acid and base hydrolysis of the glycolipid product (from the mannosyltransferase reaction) result in radioactive, water-soluble compounds which comigrate with authentic Man and Man-1-P, respectively.

Topics: Dolichol Phosphates; Glucosyltransferases; Glycoproteins; Guanosine Diphosphate Mannose; Humans; In Vitro Techniques; Lipid Metabolism; Liver; Mannosyltransferases; N-Acetylglucosaminyltransferases; Uridine Diphosphate N-Acetylglucosamine

A membrane fraction from Saccharomyces cerevisiae as well as a mannosyltransferase purified therefrom was shown to catalyze the transfer of mannose from GDPmannose to retinyl phosphate. The product formed has chromatographic and chemical properties characteristic for retinylphosphate mannose. The enzyme requires divalent cations. Mg2+ is more effective than Mn2+ with an optimum concentration around 25 mM. Amphomycin at a concentration of 0.1 mg/ml inhibits the reaction to 50%. Glycosyl transfer was specific for mannose residues from GDPmannose and did not occur with dolichylphosphate mannose nor with UDP galactose; UDPglucose is a poor donor. Formation of retinylphosphate mannose is inhibited by dolichyl phosphate. This observation as well as similarities between retinylphosphate mannose and dolichylphosphate mannose synthesis in respect to ion requirement, inhibition by amphomycin are suggestive that both reactions are catalyzed by one and the same enzyme. In experiments studying the glycosyl donor specificity in the assembly of lipid-linked oligosaccharide intermediates involved in N-glycosylation of proteins, it could be demonstrated that retinylphosphate mannose can replace dolichylphosphate mannose in the final steps of mannosylation.

Topics: Catalysis; Diterpenes; Dolichol Phosphates; Guanosine Diphosphate Mannose; Lipid Metabolism; Oligosaccharides; Polyisoprenyl Phosphate Monosaccharides; Polyisoprenyl Phosphate Sugars; Saccharomyces cerevisiae; Substrate Specificity

Of the subcellular fractions of rat liver the endoplasmic reticulum was the most active in GDP-mannose: retinyl phosphate mannosyl-transfer activity. The synthesis of retinyl phosphate mannose reached a maximum at 20-30 min of incubation and declined at later times. Retinyl phosphate mannose and dolichyl phosphate mannose from endogenous retinyl phosphate and dolichyl phosphate could also be assayed in the endoplasmic reticulum. About 1.8 ng (5 pmol) of endogenous retinyl phosphate was mannosylated per mg of endoplasmic reticulum protein (15 min at 37 degrees C, in the presence of 5 mM-MnCl2), and about 0.15 ng (0.41 pmol) of endogenous retinyl phosphate was mannosylated with Golgi-apparatus membranes. About 20 ng (13.4 pmol) of endogenous dolichyl phosphate was mannosylated in endoplasmic reticulum and 4.5 ng (3 pmol) in Golgi apparatus under these conditions. Endoplasmic reticulum, but not Golgi-apparatus membranes, catalysed significant transfer of [14C]mannose to endogenous acceptor proteins in the presence of exogenous retinyl phosphate. Mannosylation of endogenous acceptors in the presence of exogenous dolichyl phosphate required the presence of Triton X-100 and could not be detected when dolichyl phosphate was solubilized in liposomes. Dolichyl phosphate mainly stimulated the incorporation of mannose into the lipid-oligosaccharide-containing fraction, whereas retinyl phosphate transferred mannose directly to protein.

Topics: Animals; Chlorides; Diterpenes; Dolichol Monophosphate Mannose; Dolichol Phosphates; Endoplasmic Reticulum; Golgi Apparatus; Guanosine Diphosphate Mannose; In Vitro Techniques; Kinetics; Lipopolysaccharides; Liver; Male; Manganese; Manganese Compounds; Nucleoside Diphosphate Sugars; Polyisoprenyl Phosphate Monosaccharides; Polyisoprenyl Phosphates; Rats; Vitamin A

Regulation of dolichyl phosphate-linked oligosaccharide assembly has been studied during the course of diethylstilbestrol-induced chick oviduct differentiation. Oviduct membranes from treated chicks form 4.6 times as much GlcNAc-P-P-Dol and GlcNAc2-P-P-Dol upon incubation with UDP-[14C]GlcNAc and MgCl2 than do membranes from untreated chicks. Assembly of oligosaccharide-lipid was studied by incubation of membranes with purified exogenous [14C]GlcNAc2-P-P-Dol and GDP-Man. Man transfer required a divalent cation (10 mM Mg2+) and detergent (0.5% Nonidet P-40 is optimal) and occurs in the presence of amphomycin (500 micrograms/ml). The apparent Km for GDP-Man is 1 microM and for [14C]GlcNAc2-P-P-Dol is 0.45 microM. The products are a series of sequentially formed dolichyl pyrophosphate-linked saccharides up to Man5GlcNAc2, the first of which is Man beta 1,4GlcNAc2. The same products are formed either in the presence or absence of amphomycin. Conversion of GlcNAc2-P-P-Dol to higher oligosaccharides is stimulated 3-fold by estrogen treatment of chicks. Similarly, the conversion of partially purified exogenously added Man beta-[14C]GlcNAc2-P-P-Dol is 4.6-fold higher after diethylstilbestrol treatment.

Topics: Animals; Cell Differentiation; Chickens; Diethylstilbestrol; Dolichol Phosphates; Female; Guanosine Diphosphate Mannose; Kinetics; Lipid Metabolism; Mannose; Oviducts; Polyisoprenyl Phosphate Oligosaccharides; Polyisoprenyl Phosphate Sugars