dolichol-monophosphate-mannose and mannosylretinylphosphate

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

Topics: Animals; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Cricetinae; Diterpenes; Dolichol Monophosphate Mannose; Guanosine Diphosphate; Intracellular Membranes; Liver; Mannose; Mannosyltransferases; Mesocricetus; Polyisoprenyl Phosphate Monosaccharides; Polyisoprenyl Phosphate Sugars; Rats; Vitamin A

We have shown earlier that in HeLa S3G cells, glucocorticoids stimulate the synthesis of dolichyl phosphorylmannose (Dol-P-Man) with a concomitant increase in the glycosylation of proteins (Ramachandran, C.K., Gray, S.L. and Melnykovych G. (1982) Biochem. J. 208, 47-52). Although controversial, there have been several lines of evidence suggesting that the synthesis of retinyl phosphorylmannose (Ret-P-Man) and Dol-P-Man may be carried out by the same enzyme. We examined this possibility and conclude that in HeLa S3G cells the syntheses of Dol-P-Man and Ret-P-Man are catalyzed by two different enzymes located in the same microenvironment. Our conclusion is based on the following observations: exogenously added dolichyl phosphate and retinyl phosphate did not compete with each other; when the cells were grown in the presence of 1 microM dexamethasone, the microsomal synthesis of Dol-P-Man was stimulated, without affecting the Ret-P-Man synthesis; Arrhenius plots on Ret-P-Man and Dol-P-Man synthesis showed breaks at 22 and 37.7 degrees C.

Topics: Animals; Dexamethasone; Diterpenes; Dolichol Monophosphate Mannose; HeLa Cells; Humans; Hydroxymethylglutaryl CoA Reductases; Liposomes; Mannosyltransferases; Polyisoprenyl Phosphate Monosaccharides; Polyisoprenyl Phosphate Sugars; Rats; Temperature

It is well established that mannosylphosphoryldolichol participates in the synthesis of N-linked glycoproteins by donating mannosyl residues to oligosaccharide-lipid intermediates. It has been suggested that mannosylphosphorylretinol also is involved in glycoprotein biosynthesis. We conclude that one synthase catalyzes the synthesis of both mannosylphosphoryldolichol and mannosylphosphorylretinol in rat liver tissue and Chinese hamster ovary cells, based on the following results. 1) The enzyme in rat liver microsomes that synthesizes mannosylphosphoryldolichol and mannosylphosphorylretinol is inactivated at the same rate at 55 degrees C. 2) In membranes of both rat liver and Chinese hamster ovary cells, exogenous dolichyl phosphate and retinyl phosphate compete with each other for mannosyl-lipid synthesis. However, in both systems adding exogenous retinyl phosphate has no effect on the synthesis of mannosylphosphoryldolichol from endogenous dolichyl phosphate in the membranes. 3) Membranes prepared from a mutant of Chinese hamster ovary cells which is devoid of mannosylphosphoryldolichol synthase lack the ability to synthesize mannosylphosphorylretinol.

Topics: Animals; Cell Line; Cell Membrane; Cricetinae; Cricetulus; Diterpenes; Dolichol Monophosphate Mannose; Female; Hexosyltransferases; In Vitro Techniques; Kinetics; Liver; Male; Mannosyltransferases; Microsomes, Liver; Ovary; Polyisoprenyl Phosphate Monosaccharides; Polyisoprenyl Phosphate Sugars; Rats; Rats, Inbred Strains

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

A remarkable and immediate decrease in GDP-mannose:retinyl phosphate mannosyltransferase activity was found on pre-incubation of rat liver postnuclear membranes with phospholipase A2 or phospholipase C. Under the same conditions of pre-incubation (1 min at 37 degrees C) trypsin did not affect the enzyme activity, whereas pre-incubation for 30 min with trypsin and Pronase abolished enzyme activity. The lipid extract of untreated rat liver membranes partially restored enzyme activity after phospholipase treatment. Sphingomyelin was as active as the endogenous lipids. Other phospholipids were less active in the following order: phosphatidylcholine greater than phosphatidylethanolamine greater than phosphatidylinositol = phosphatidylserine. Dolichyl phosphate mannose synthesis was inhibited less (33%) by phospholipase C than was Ret-P-Man synthesis (98.5%) under identical conditions of incubation, which included 0.025% Triton. However, retinyl phosphate mannose synthesis by purified endoplasmic reticulum was found to be resistant to phospholipase C. Mixing experiments failed to demonstrate an inhibitory effect of the phospholipase-treated postnuclear membrane fraction on the synthetic activity of the endoplasmic reticulum, thus excluding the release of an inhibitory factor from the postnuclear membranes.

Topics: Animals; Cell Membrane; Diterpenes; Dolichol Monophosphate Mannose; Endoplasmic Reticulum; Guanosine Diphosphate Mannose; In Vitro Techniques; Liver; Male; Phospholipases; Phospholipids; Polyisoprenyl Phosphate Monosaccharides; Polyisoprenyl Phosphate Sugars; Rats; Sphingomyelins; Trypsin

Topics: Animals; Cricetinae; Diterpenes; Dolichol Monophosphate Mannose; Male; Mesocricetus; Polyisoprenyl Phosphate Monosaccharides; Polyisoprenyl Phosphate Sugars; Rats; Vitamin A Deficiency

The mannosyl derivative of phosphorylated vitamin A, mannosylretinylphosphate, is synthesized in vivo and in vitro by most epithelial tissues, including mouse epidermal cells. This compound is but one component of the complex biosynthetic machinery responsible for the assembly of glycoproteins in mammalian membranes; its specific role remains to be understood, even though it has been established that it functions as a carrier of mannose mostly in a direct transfer to protein. The system of five conjugated double bonds appears necessary for this function, since upon saturation the resulting perhydroretinylphosphate is incapable of acting in mannosyl transfer to endogenous microsomal proteins. Using vitamin A - deficient hamsters and rats, retinoic acid was shown to be as active as retinol in restoring the in vivo in corporation of mannose into glycoproteins to normal levels within a short time, in liver and tracheal tissues. Retinoic acid was also active in increasing adhesive properties of spontaneously transformed mouse dermal fibroblasts (3T12 cells) in a reversible manner. The free carboxyl group appeared to be a requirement for this activity inasmuch as the lactonized form of 11-hydroxyretinoic acid was inactive. Even though retinoic acid, as well as retinol, was able to enhance adhesion of 3T12 cells, only the cellular retinoic acid-binding protein was detected, suggesting that the cellular retinol-binding protein is not a requirement for this activity. A metabolite of retinoic acid, compound X, different from retinol, was found incorporated into mannosylretinoidphosphate (MXP) by 3T12 cells. This derivative constitutes up to 40% of the total radioactive pool of derivatives of retinoic acid after 48 hours of labeling. These data suggest that retinol, as mannosylretinylphosphate, and retinoic acid, as mannosylretinoidphosphate, function as mannosyl carriers in biologic membranes.

Topics: Animals; Cell Line; Diterpenes; Dolichol Monophosphate Mannose; Epithelium; Fibroblasts; Hexosephosphates; Mannose; Mannosephosphates; Mice; Mice, Inbred BALB C; Microsomes, Liver; Polyisoprenyl Phosphate Monosaccharides; Polyisoprenyl Phosphate Sugars; Rats; Skin; Tretinoin; Vitamin A; Vitamin A Deficiency

Topics: Animals; Chromatography, High Pressure Liquid; Diterpenes; Dolichol Monophosphate Mannose; Dolichol Phosphates; Dolichols; Mannose; Mannosephosphates; Polyisoprenyl Phosphate Monosaccharides; Rats; Tretinoin; 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

Rat liver microsomes synthesized [14C]mannosylretinylphosphate and dolichyl [14C]mannosylphosphate from guanosinedisphosphate [14C]mannose, retinylphosphate and dolichylphosphate. Two distinct enzyme activities were shown to be responsible for the biosynthesis of the two mannolipids. A higher affinity mannosyl transferase (EA I), responsible for dolichylmannosylphosphate synthesis, displayed a Km for GDP-mannose of 1.7 microM; while a lower affinity enzyme (EA II), responsible for mannosylretinylphosphate synthesis, displayed a Km for GDP-mannose of 12.5 microM. These Km values were unaffected by the addition of either dolichylphosphate for EA II, or retinylphosphate for EA I. The same Km values were found before and after solubilization of the enzyme activity with 1% Triton X-100. Differential solubilization of EA I and EA II was demonstrated, utilizing different concentrations of Triton X-100. Triple-labeled mannosylretinylphosphate was prepared from [3H]retinylphosphate, retinyl[32P]phosphate and GDP-[14C]mannose from incubations containing rat liver microsomes. This compound was shown to donate [14C]mannose to endogenous acceptors of rat liver microsomes.

Topics: Animals; Diterpenes; Dolichol Monophosphate Mannose; Guanosine Diphosphate Mannose; Hexosephosphates; Hexosyltransferases; Kinetics; Mannosephosphates; Mannosyltransferases; Membranes; Microsomes, Liver; Polyisoprenyl Phosphate Monosaccharides; Polyisoprenyl Phosphate Sugars; Polyisoprenyl Phosphates; Rats; Vitamin A

In the absence of detergent, the transfer of mannose from GDP-mannose to rat liver microsomal vesicles was highly stimulated by exogenous retinyl phosphate in incubations containing bovine serum albumin, as measured in a filter binding assay. Under these conditions 65% of mannose 6-phosphatase activity was latent. The transfer process was linear with time up to 5min and with protein concentration up to 1.5mg/0.2ml. It was also temperature-dependent. The microsomal uptake of mannose was highly dependent on retinyl phosphate and was saturable against increasing amounts of retinyl phosphate, a concentration of 15mum giving half-maximal transfer. The uptake system was also saturated by increasing concentrations of GDP-mannose, with an apparent K(m) of 18mum. Neither exogenous dolichyl phosphate nor non-phosphorylated retinoids were active in this process in the absence of detergent. Phosphatidylethanolamine and synthetic dipalmitoylglycerophosphocholine were also without activity. Several water-soluble organic phosphates (1.5mm), such as phenyl phosphate, 4-nitrophenyl phosphate, phosphoserine and phosphocholine, did not inhibit the retinyl phosphate-stimulated mannosyl transfer to microsomes. This mannosyl-transfer activity was highest in microsomes and marginal in mitochondria, plasma and nuclear membranes. It was specific for mannose residues from GDP-mannose and did not occur with UDP-[(3)H]galactose, UDP- or GDP-[(14)C]glucose, UDP-N-acetyl[(14)C]-glucosamine and UDP-N-acetyl[(14)C]galactosamine, all at 24mum. The mannosyl transfer was inhibited 85% by 3mm-EDTA and 93% by 0.8mm-amphomycin. At 2min, 90% of the radioactivity retained on the filter could be extracted with chloroform/methanol (2:1, v/v) and mainly co-migrated with retinyl phosphate mannose by t.l.c. This mannolipid was shown to bind to immunoglobulin G fraction of anti-(vitamin A) serum and was displaced by a large excess of retinoic acid, thus confirming the presence of the beta-ionone ring in the mannolipid. The amount of retinyl phosphate mannose formed in the bovine serum albumin/retinyl phosphate incubation is about 100-fold greater than in incubations containing 0.5% Triton X-100. In contrast with the lack of activity as a mannosyl acceptor for exogenous dolichyl phosphate in the present assay system, endogenous dolichyl phosphate clearly functions as an acceptor. Moreover in the same incubations a mannolipid with chromatographic properties of retinyl phosphate mannose was also synthes

Topics: Animals; Detergents; Diterpenes; Dolichol Monophosphate Mannose; Guanosine Diphosphate Mannose; Immune Sera; In Vitro Techniques; Lipid Metabolism; Male; Microsomes, Liver; Nucleoside Diphosphate Sugars; Octoxynol; Polyethylene Glycols; Polyisoprenyl Phosphate Monosaccharides; Proteins; Rats; Rats, Inbred Strains; Vitamin A

Topics: Animals; Cell Membrane; Diterpenes; Dolichol Monophosphate Mannose; Glycoproteins; Hexosephosphates; Kinetics; Liver; Male; Mannosephosphates; Membrane Proteins; Polyisoprenyl Phosphate Monosaccharides; Polyisoprenyl Phosphate Sugars; Rats; Spectrophotometry, Ultraviolet

Topics: Animals; Chromatography, DEAE-Cellulose; Diterpenes; Dolichol Monophosphate Mannose; Liver; Male; Mannosephosphates; Polyisoprenyl Phosphate Monosaccharides; Polyisoprenyl Phosphate Sugars; Rats; Subcellular Fractions

Excess vitamin A stimulated the incorporation of mannose into rat liver mannosylretinylphosphate (MRP), dolichylmannosylphosphate (DMP), and into glycoproteins by over 200% during a 20-min labeling period. The glycoproteins were digested with pronase and separated into three components by molecular sieve chromatography. The stimulation of mannose incorporation was greatest in the Peak II glycopeptide (Mr = 6500). In contrast, the incorporation of galactose into glycolipids or glycopeptides was not altered by vitamin A treatment. Analysis of the glycopeptide stimulated by vitamin A treatment showed it to contain mannose, glucose, galactose, and glucosamine in the respective molar ratios of 7:10:17:1 and to be rich in glutamic acid, serine, glycine, aspartic acid, and threonine. The results suggest that excess vitamin A stimulates the incorporation of mannose into glycoproteins by enhancing the synthesis of lipid intermediates involved in specific mannosyl transfer reactions.

Topics: Animals; Diterpenes; Dolichol Monophosphate Mannose; Dose-Response Relationship, Drug; Female; Glycopeptides; Glycoproteins; Liver; Mannose; Polyisoprenyl Phosphate Monosaccharides; Polyisoprenyl Phosphate Sugars; Rats; Vitamin A