dolichol-monophosphate and mannosylretinylphosphate

Microsomal membrane preparations from rat livers, when incubated with labelled sugar-nucleotides, were shown to synthesize labelled oligosaccharide-lipids in the presence of excess exogenous dolichyl phosphate. Under the incubation conditions defined in the present study, dolichyl pyrophosphoryl(DolPP)GlcNAc2-Man5, DolPPGlcNAc2Man9 and DolPPGlcNAc2Man9Glc3 were the principal oligosaccharide-lipids formed by both control and vitamin A-deficient membranes. However, deficient membranes synthesized 3.2 +/- 0.8 times as much oligosaccharide-lipids and 2.6 +/- 0.7 times as much dolichyl phosphate mannose (DolPMan) and dolichyl phosphate glucose (DolPGlc) as the controls. The transfer of the oligosaccharide chain from the dolichol carrier to the endogenous protein acceptors in vitamin A-deficient microsomes (microsomal fractions) was only 57.5 +/- 9.5% of that of controls. After endo-beta-N-acetylglucosaminidase treatment, only one oligosaccharide species was isolated from both control and vitamin A-deficient microsomal glycoproteins, and was characterized as GlcNAcMan9Glc3. We conclude that the decreased incorporation of labelled mannose and glucose from sugar-nucleotides into the glycoproteins must be due to decreased transfer of GlcNAc2Man9Glc3 from the dolichol carrier to the protein acceptors. This conclusion was further substantiated by the finding that control membranes transferred 4-6 times as much labelled oligosaccharides from exogenously added dolichol-linked substrate (DolPPGlcNAc2Man9Glc3) to endogenous microsomal protein acceptors as compared with the vitamin A-deficient membranes. Attempts to reverse this defect by addition of retinol or retinyl phosphate (a source of retinyl phosphate mannose) to the incubations were unsuccessful.

Topics: Animals; Chromatography, High Pressure Liquid; Diterpenes; Dolichol Phosphates; Glycoproteins; Glycosylation; Intracellular Membranes; Male; Microsomes, Liver; Oligosaccharides; Polyisoprenyl Phosphate Monosaccharides; Rats; Vitamin A

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

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

Rat liver microsomal fraction synthesized Ret-P-Man (retinyl phosphate mannose) and Dol-P-Man (dolichyl phosphate mannose) from endogenous Ret-P (retinyl phosphate) and Dol-P (dolichyl phosphate). Ret-P-Man synthesis displayed an absolute requirement for a bivalent cation, and also Dol-P-Man synthesis was stimulated by bivalent metal ions. Mn2+ and Co2+ were the most active, with maximum synthesis of Ret-P-Man occurring at 5-10 mM: Mg2+ was also active, but at higher concentrations. At 5mM-Mn2+ the amount of endogenous Ret-P mannosylated in incubation mixtures containing 5 microM-GDP-mannose in 15 min at 37 degrees C was approx. 3 pmol/mg of protein. In the same assays about 7-10 pmol of endogenous Dol-P was mannosylated. Bivalentcation requirement for Ret-P-Man synthesis from exogenous Ret-P showed maximum synthesis at 2.5 mM-Mn2+ or -Co2+. In addition to Ret-P-Man and Dol-P-Man, a mannolipid co-chromatographing with undecaprenyl phosphate mannose was detected. Triton X-100 (0.5%) abolished Ret-P-Man synthesis from endogenous Ret-P and caused a 99% inhibition of Ret-P-Man synthesis from exogenous Ret-P. The presence of detergent (0.5%) also inhibited Dol-P-Man synthesis from endogenous Dol-P and altered the requirement for Mn2+. Microsomal fraction from Syrian golden hamsters was also active in Ret-P-Man and Dol-P-Man synthesis from endogenous Ret-P and Dol-P. At 5 mM-Mn2+ about 2.5 pmol of endogenous Ret-P and 3.7 pmol of endogenous Dol-P were mannosylated from GDP-mannose per mg of protein in 15 min at 37 degrees C. On the other hand, microsomal fraction from vitamin A-deficient hamsters contained 1.2 pmol of Ret-P and 14.1 pmol of Dol-P available for mannosylation. Since GDP-mannose: Ret-P and GDP-mannose: Dol-P mannosyltransferase activities were not affected, depletion of vitamin A must affect Ret-P and Dol-P pools in opposite ways.

Topics: Animals; Cations, Divalent; Chromatography, Thin Layer; Cricetinae; Detergents; Diterpenes; Dolichol Monophosphate Mannose; Dolichol Phosphates; In Vitro Techniques; Kinetics; Male; Mesocricetus; Microsomes, Liver; Octoxynol; Polyethylene Glycols; Polyisoprenyl Phosphate Monosaccharides; Polyisoprenyl Phosphate Sugars; Polyisoprenyl Phosphates; Rats; Serum Albumin, Bovine; Vitamin A; Vitamin A Deficiency