Mannosyl carrier functions of retinyl phosphate and dolichyl phosphate in rat liver endoplasmic reticulum

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.     

The conversion of exogenous retinol and related compounds into retinyl phosphate mannose by adult Brugia pahangi in vitro.

Adult Brugia pahangi took up and incorporated beta-carotene and free retinol in vitro. The uptake of retinol was 50 times greater than that of beta-carotene under similar incubation conditions. beta-Carotene was almost entirely metabolized, primarily to retinol. The metabolism of retinol by B. pahangi in vitro was less extensive, with a variety of retinoids tentatively identified, including retinyl phosphate (Ret-P), retinyl phosphate mannose (Ret-P-Man) and anhydroretinol as minor metabolites. B. pahangi microsomes were also shown to biosynthesize Ret-P-Man from exogenous Ret-P and GDP-mannose, but not from endogenous lipid acceptors alone. In this circumstance an unidentified lipid appeared to be mannosylated by B. pahangi. The rate of mannose transfer to exogenous Ret-P by B. pahangi microsomes was 150 pmol X min -1. (mg of protein) -1. Ret-P-Man synthetase activity from both B. pahangi and rat liver microsomes had an absolute requirement for bovine serum albumin and MnCl2, and occurred in the absence of detergent. The results suggest a biochemical role for vitamin A in B. pahangi, possibly in filarial glycoprotein synthesis.     

Mannosylation of endogenous and exogenous phosphatidic acid by liver microsomal membranes. Formation of phosphatidylmannose

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.     

Interactions between retinyl phosphate and bivalent cations.

In the presence of Mn(II) ions, the u.v. absorption spectrum of retinyl phosphate (Ret-P) solubilized in Triton X-100 micelles, phosphatidylcholine liposomes or rat liver microsomes exhibited a shift from the maximum of 330 nm to 287 nm. The effect of Mn(II) was reversed by adding EDTA or phosphate buffer. The same spectral change was found in the presence of poly-L-lysine in place of Mn(II) ions. The e.s.r. spectrum of Mn(II) in the presence or in the absence of Ret-P clearly showed that approx. 75% of the initial concentration of Mn(II) ions is bound to Ret-P when the molar ratio of Ret-P to Mn(II) ions is 4:1; no such binding occurred in the presence of retinol or retinoic acid. The appearance of two isosbestic points at 303 and 368 nm, in the presence of Mn(II) ions, suggests the existence of an equilibrium between an Mn(II)-bound monomer and an Mn(II)-bound dimer of Ret-P in Triton X-100 micelles. The same effect on the u.v.-absorption spectrum of Ret-P was also induced by Co(II), Cr(II), Zn(II) and Fe(II), but not by Mg2+ or Cu(II). The formation of the 'metachromatic complex' between Ret-P and Mn(II) or Co(II) inhibited the synthesis of retinyl phosphate mannose (Ret-P-Man) from exogenous and endogenous Ret-P and guanosine diphosphate [14C]mannose when bovine serum albumin was added after the metal ion. However, the order of addition did not influence Ret-P-Man synthesis in incubations containing MgCl2, which does not form the metachromatic complex with Ret-P. These results suggest that the bioavailability of proteins, polyamines and metal ions may control the extent to which Ret-P can be mannosylated in the intact membrane.     

Characterization of three kinetically distinct forms of glutamate decarboxylase from pig brain.

Hamster liver microsomal membranes catalyse the synthesis of retinyl phosphate mannose (Ret-P-Man) from GDP-mannose and exogenous retinyl phosphate (Ret-P). We have previously shown that maximal Ret-P-Man synthesis occurs in vitro at 20-30 min, followed by a subsequent loss of mannose from Ret-P-Man, suggestive of an intermediary function of Ret-P-Man and/or Ret-P-Man breakdown [Shidoji, Silverman-Jones & De Luca (1982) Biochem. J. 208, 865-868; Creek, Morre, Silverman-Jones, Shidoji & De Luca (1983) Biochem. J. 210, 541-547). To monitor Ret-P-Man synthesis and breakdown carefully, we developed a chromatographic system in which mannose, Ret-P-Man, mannose phosphate and GDP-mannose are separated in a single analysis on a Mono Q column eluted with a gradient of NaCl. Using this chromatographic system, we have determined that 80-90% of the Ret-P-Man made in vitro by hamster liver membranes in 30 min is recovered with the membranes upon centrifugation. Subsequent incubation of Ret-P-Man-loaded membranes at 37 degrees C results in a non-enzymic breakdown of Ret-P-Man to beta-mannopyranosyl phosphate and anhydroretinol. However, incubation of the Ret-P-Man-loaded hamster liver membranes with GDP, but not GMP, ADP, CDP or UDP, results in a loss of mannose from Ret-P-Man and the formation of GDP-mannose and Ret-P. These results demonstrate that Ret-P-Man synthesized in vitro is subject to non-enzymic breakdown to beta-mannopyranosyl phosphate and anhydroretinol and that the GDP-mannose:retinyl phosphate mannosyltransferase reaction is reversible.     

Rat liver microsomes catalyse mannosyl transfer from GDP-d-mannose to retinyl phosphate with high efficiency in the absence of detergents

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 synthesized from endogenous lipid acceptor. The biosynthesis of this mannolipid (retinyl phosphate mannose) was optimal at MnCl(2) concentrations between 5 and 10mm and could not be detected below 0.6mm-MnCl(2), when synthesis of dolichyl phosphate mannose from endogenous dolichyl phosphate was about 80% of optimal synthesis. Under optimal conditions (5mm-MnCl(2)) endogenous retinyl phosphate mannose represented about 20% of dolichyl phosphate mannose at 15min of incubation at 37 degrees C.     

Fast atom bombardment and collisional activation mass spectrometry of retinyl phosphate mannose synthesized by liver membranes

Fast atom bombardment (FAB) and collisional activation dissociation (CAD) mass-analysed ion kinetic energy (MIKE) spectra have confirmed the structures of retinyl phosphate (Ret-P), retinyl phosphate mannose (Ret-P-Man) and guanosine 5'-diphospho-D-mannose (GDP-Man). Ret-P-Man was made in vitro while Ret-P and GDP-Man were chemically synthesized. Positive ion FAB mass spectrometry of Ret-P showed an observable short-lived spectrum with a mass ion at m/z 367 [M + H]+, and a major fragment ion at m/z 269 [M + H - H3PO4]+. Negative ion FAB mass spectrometry of Ret-P showed a strong stable spectrum with a parent ion at m/z 365 [M - H]-, a glycerol (G) adduct ion at m/z 457 [M - H + G]- and a dimer ion at m/z 731 [2M - H]-. GDP-Man showed an intense spectrum with parent ion at m/z 604 [M - H]- and cationized species at m/z 626 [M + Na - 2H]- and 648 [M + 2Na - 3H]-. Negative ion FAB mass spectrometry of Ret-P-Man showed a parent ion at m/z 527 [M - H]- and a fragment ion at m/z 259 [C6H12PO9]-. The CAD-MIKE spectra showed structurally significant fragment ions at m/z 442 and 361 for the [M - H]- ion of GDP-Man, and at m/z 509, 406, 364 and 241 for the [M - H]- ion of Ret-P-Man. FAB and CAD-MIKE spectra have been applied successfully to confirm the structure of Ret-P-Man made in vitro from Ret-P and GDP-Man.     

Reduced mannose incorporation into GDP-mannose and dolichol-linked intermediates of N-glycosylation in hamster liver during vitamin A deficiency

Summary The molecular mechanism of reduced incorporation of radioactively labeled mannose into hamster liver glycoconjugates during the progression of vitamin A deficiency was investigated. In particular the in vivo incorporation of [2-³H]mannose into GDP-mannose, dolichyl phosphate mannose (Dol-P-Man), lipid-linked oligosaccharides, and glycopeptides of hamster liver was examined. Hamsters maintained on a vitamin A-free diet showed a reduction in the incorporation of mannose into GDP-mannose about 10 days before clinical signs of vitamin A deficiency could be observed. The decrease in [2-³H]mannose incorporated into GDP-mannose was accompanied by a reduction in label incorporated into Dol-P-Man, lipid linked oligosaccharides and glycopeptides, which became more severe with the progression of vitamin A deficiency. By the time they reached a plateau stage of growth, hamsters fed the vitamin A-free diet showed a 50% reduction in the amount of [2-³H]mannose converted to GDP-mannose, and the radioactivity associated with Dol-P-Man and glycopeptides was reduced by approximately 60% as compared to retinoic acid-supplemented controls. These results strongly indicate that the reduced incorporation of mannose into lipidic intermediates and glycoproteins observed during vitamin A deficiency is due to impaired GDP-mannose synthesis.Abbreviations Dol-P-Man Dolichyl Phosphate Mannose - Dol-P Dolichyl Phosphate     

Differential regulation of the synthesis of mannosylphosphoryl derivatives of dolichol and retinol in HeLa cells

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.     

Enzymatic synthesis of mannosyl retinyl phosphate from retinyl phosphate and guanosine diphosphate mannose.

A study was conducted to determine whether retinyl phosphate would act as substrate for the enzymatic synthesis of mannosyl retinyl phosphate. Retinyl phosphate, prepared chemically, supported the growth of vitamin A-deficient rats at the same rate as retinol. It also stimulated the uptake of [14C]mannose from GDP-[14C]mannose into total chloroform-methanol extractable lipid. This reaction occurred in the presence of ATP, Mn2+, detergent (Zonyl A), and a membrane-rich enzyme preparation from the livers of vitamin A-deficient rats, provided that a lipid extract of the membrane preparation of alpha-L-lecithin was also added. Total chloroform-methanol-extractable, labeled mannolipid was separated into two principal labeled mannolipids by thin-layer or column chromatography or by differential solvent extraction. The properties of these mannolipids identified them as glycophospholipids: one was identical with authentic synthetic dolichyl mannosyl phosphate, and the other was concluded to be mannosyl retinyl phosphate because of its incorporation of radioactivity from [3H]retinyl phosphate, its rapid hydrolysis by dilute acid, and the formation of substance that cochromatographed with retinol upon its acid hydrolysis. The presence of ATP or GTP was essential for the stimulation of mannolipid synthesis, probably because of their protective action on the substrates against phosphatases present in the crude enzyme fraction. A pH of 6.0-6.2 favored the formation of dolichyl mannosyl phosphate; a higher pH (6.7-7.0) that of mannosyl retinyl phosphate.     

Retinyl phosphate mannose synthesis in rat liver membranes. Phospholipase sensitivity and phospholipid requirement.

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.     

Inhibition of lipid-linked oligosaccharide synthesis by aryl phosphates.

Our previous work has shown that phenyl phosphate acts as an exogenous substrate for GDP-mannose:dolichyl phosphate mannosyltransferase in rat liver microsomal fractions to give rise to phenyl phosphate beta-D-mannose, a compound which, unlike Dol-P-Man (dolichyl phosphate beta-D-mannose), cannot act as mannose donor for further mannose-adding reactions in microsomal fractions. The study has now been extended to the action of various aryl phosphates and structurally related compounds on several other glycosyltransferase systems in the microsomal fractions. (1) Examination of the ability of these compounds to accept sugars from various sugar nucleotides indicated that the individual compounds have specificity as sugar acceptors. Thus phenyl phosphate acted as an effective acceptor for both mannose and glucose, whereas benzenephosphonic acid was active only in accepting mannose. p-Nitrophenyl phosphate was a more effective glucose acceptor than phenyl phosphate, but had only 8% of the mannose-accepting activity of phenyl phosphate. (2) Phenyl phosphate had an inhibitory effect on the transfer of mannose form GDP-mannose to lipid-linked oligosaccharides and to glycoproteins in rat liver microsomal fractions. The inhibition depended on the concentration of phenyl phosphate and on the extent of inhibition of Dol-P-Man synthesis. It is proposed that phenyl phosphate has a direct effect on the synthesis of Dol-P-Man and that its inhibition of synthesis of lipid-linked oligosaccharides and glycoproteins could be a consequence of this effect.     

A developmental change in dolichyl phosphate mannose synthase activity in pig brain

The initial rate of dolichyl phosphate mannose biosynthesis was measured in white-matter membranes from pig brain at various ages from before birth throughout the period of most rapid brain development. Dolichyl phosphate mannose synthase activity increased from prenatal values to a maximum in 3 week-old animals, and gradually decreased to adult values after 8 weeks of age. The nature of the developmental change was investigated by enzymic and biochemical comparisons of the membrane preparations from the most active age (3 weeks) and adult controls. The specific activity of dolichyl phosphate mannose synthase in preparations from actively myelinating animals was approx. 3-fold higher than adults when mannolipid formation was assayed with saturating concentrations of GDP-[¹⁴C]mannose and utilizing only endogenous acceptor lipid. No major variations were found in the apparent K_m values for GDP-mannose or exogenous dolichyl monophosphate. However, the ratio of dolichyl phosphate mannose synthase activity for myelinating animals/adult animals decreased significantly when large amounts of exogenous dolichyl monophosphate were added to the incubation mixtures. Dolichyl phosphate mannose synthase activity was also compared in white-matter membranes depleted of endogenous dolichyl monophosphate by enzymic mannosylation or treatment with butanol. When these preparations were assayed with identical amounts of exogenous dolichyl monophosphate, the dolichyl monophosphate-depleted membranes from actively myelinating animals contained only 20–30% more dolichyl phosphate mannose synthase activity. Overall, these studies strongly suggest that the developmental change in dolichyl phosphate mannose synthase activity is due primarily to the presence of a relatively lower amount of endogenous dolichyl monophosphate being accessible to the mannosyltransferase in the white-matter membranes from adult animals.     

The submicrosomal distribution of dolichyl phosphate and dolichyl phosphate phosphatase in rat liver

Rat liver microsomes were isolated and fractionated into Golgi, smooth endoplasmic reticulum (SER), and rough endoplasmic reticulum (RER), and the purity of these preparations was determined. The dolichyl phosphate (Dol-P) content of whole microsomes and of each of the submicrosomal fractions was estimated using high pressure liquid chromatography. Dol-P accounts for 4 and 40% of the sum of the alcohol, the fatty acyl esters of dolichol, and monophosphate forms present in whole liver and in purified microsomes, respectively. Concentrations equal to 58, 77, and 108 ng of Dol-P/mg of protein were found in Golgi, SER, and RER, respectively. These values represent 3, 36, and 54% of the sum of the alcohol, the fatty acyl esters of dolichol, and monophosphate forms present in each of these same fractions, respectively. Increases in the Dol-P content of rat liver were observed as early as 12 h after turpentine-induced inflammation and increased 2-fold over 36 h. In this system, Dol-P accounts for no more than 50% of the sum of all phosphorylated and pyrophosphorylated dolichol intermediates present. The specific activity for dolichyl phosphate phosphatase was highest by more than a factor of 2 in Golgi membrane. Specific activities obtained for SER and RER were 42 and 11% of those present in Golgi. The major requirement for Dol-P is thought to be for the saccharide and oligosaccharide transferase reactions which are presumed to take place in RER. The discovery of significant quantities of Dol-P in Golgi and SER is consistent with a possible role of Dol-P in the transport of sugars required for glycoprotein synthesis and processing from a cytosolic to luminal orientation.     

Glycoprotein biosynthesis: studies on thyroid mannosyltransferases. II. Characterization of a polyisoprenyl mannosyl phosphate and evaluation of its intermediary role in the glycosylation of exogenous acceptors

The transfer of mannose from GDP-mannonse to exogenous glycopeptides and simple glycosides has been shown to be carried out by calf thyroid particles (Adamany, A. M., and Spiro, R. G. (1975) J. Biol. Chem. 250, 2830-2841). The present investigation indicates that this mannosylation process is accomplished through two sequential enzymatic reactions. The first involves the transfer of mannose from the sugar nucleotide to an endogenous acceptor to form a compound which has the properties of dolichyl mannosyl phosphate, while in the properties of dolichyl mannosyl phosphate, while in the second reaction this mannolipid serves as the glycosyl donor to exogenous acceptors. The particle-bound enzyme which catalyzed the first reaction utilized GDP-mannose (Km = 0.29 microM) as the most effective mannosyl donor, required a divalent cation, preferably manganese or calcium, and acted optimally at pH 6.3. Mannolipid synthesis was reversed by addition of GDP and a ready exchange of the mannose moiety was observed between [14C]mannolipid and unlabeled GDP-mannose. Exogenously supplied dolichyl phosphate, and to a lesser extent ficaprenyl phosphate, served as acceptors for the transfer reaction. The 14C-labeled endogenous lipid had the same chromatographic behavior as synthetic dolichyl mannosyl phosphate and enzymatically mannosylated dolichyl phosphate. The mannose component in the endogenous lipid was not susceptible to reduction with sodium borohydride and was released by mild acid hydrolysis. Alkaline treatment of the mannolipid released a phosphorylated mannose with properties consistent with that of mannose 2-phosphate. The formation of this compound which can arise from a cyclic 1,2-phosphate indicated, on the basis of steric considerations, that the mannose is present in beta linkage to the phosphate of the lipid. An intermediate role of the mannolipid in the glycosylation of exogenous acceptors was suggested by the observation that addition of dolichyl phosphate to thyroid particles resulted in a marked enhancement of mannose transfer from GDP-mannose to methyl-alpha-D-mannopyranoside acceptor while the presence of the glycoside caused a decrease in the mannolipid level. The glycosyl donor function of the polyisoprenyl mannosyl phosphate in the second reaction of the mannosylation sequence could be directly demonstrated by the transfer of [14C]mannose from purified endogenous mannolipid to either methyl-alpha-D-mannoside or dinitrophenyl unit A glycopeptides by thyroid enzyme in the presence of Triton X-100. The mannosylation of the glycoside was not inhibited by EDTA whereas the transfer of mannose to glycopeptide was cation-dependent. While dolichyl [14C]mannosyl phosphate, prepared from exogenous dolichyl phosphate, served as a donor of mannose to exogenous acceptor, this function could not be fulfilled by ficaprenyl [14C]mannosyl phosphate. The two-step reaction sequence carried out by thyroid enzymes which leads to the formation of an alpha-D-manno-pyranosyl-D-mannose linkage in exogenous acceptors by transfer of mannose from GDP-mannose through a beta-linked intermediate appears to involve a double inversion of anomeric configuration of this sugar.     

Control of N-linked carbohydrate unit synthesis in thyroid endoplasmic reticulum by membrane organization and dolichyl phosphate availability

Thyroid rough endoplasmic reticulum (ER) has been shown to contain a highly organized multienzyme system capable of carrying out the N-glycosylation of newly synthesized proteins. These reactions were studied in isolated ER vesicles and found to be controlled to a large extent by the availability of a key substrate, dolichyl phosphate (Dol-P), as well as by the amount of endogenous polypeptide acceptor present. Although in intact vesicles UDP-Glc was utilized in an efficient manner to form Dol-P-Glc and glucosylated oligosaccharide-lipid, after disruption of vesicle integrity, even with low concentrations of Triton X-100, the coupling of Dol-P-Glc formation to lipid-linked oligosaccharide assembly and subsequent N-glycosylation was substantially impaired. Increased incubation temperatures also resulted in a decreased effectiveness of glucose transfer from Dol-P-Glc to lipid-oligosaccharide, presumably because of a decline in the extent of structural organization of the ER membranes. The limited availability of endogenous Dol-P was demonstrated by the pronounced stimulation in Dol-P-Glc formation resulting from the addition of this lipid acceptor to Triton-disrupted ER membranes as well as by its generation in intact vesicles. The latter was accomplished by stimulating recycling of endogenous Dol-P through the addition of a peptide (Tyr-Asn-Leu-Thr-Ser-Val) which is an N-glycosylation substrate. The inhibition of Dol-P-Glc synthesis from UDP-Glc observed in the presence of elevated levels of GDP-Man which could be relieved in Triton-disrupted or intact ER vesicles by the addition or generation, respectively, of Dol-P, is considered to be the result of a competing requirement for Dol-P by the mannosyltransferase. Moreover GTP, by selectively inhibiting the mannosyltransferase, prevented the decrease of Dol-P-Glc formation caused by GDP-Man. Since addition of the acceptor peptide to intact vesicles stimulated Dol-P-P-GlcNAc as well as Dol-P-Glc and Dol-P-Man synthesis it would appear that a pool of Dol-P available in common to all three enzymes responsible for dolichol-linked monosaccharide synthesis exists in the ER membranes.     

Enzymatic activities in cultured human lymphocytes that dephosphorylate dolichyl pyrophosphate and dolichyl phosphate

Enzymatic activities which dephosphorylate dolichyl phosphate (Dol-P) and dolichyl pyrophosphate (Dol-P-P) have been observed in membranes from cultured human lymphocytes. Neither activity requires divalent metals. Dol-P phosphatase is inhibited by inorganic phosphate but not by other phosphate-containing compounds. Dol-P-P phosphatase is inhibited by bacitracin but not by phosphate-containing compounds including the methylene analogue of pyrophosphate. These reactions are similar to those previously found in the cycle of bacterial wall peptidoglycan biosynthesis. A chemical synthesis of [32P]Dol-P and [32P]Dol-P-P is reported.     

Formation of alpha-1,2- and alpha-1,3-linked mannose disaccharides from dolichyl mannosyl phosphate by rat liver membrane enzymes

Dolichyl mannosyl phosphate and GDPmannose were active substrates for the transfer of mannose to methyl-alpha-D-mannose, p-nitrophenyl-alpha-D-mannose, and free mannose with rat liver microsomal membranes. The products formed during dolichyl mannosyl phosphate incubation with methyl-alpha-D-mannose or with mannose were alpha-linked. The disaccharides formed by incubation of dolichyl mannosyl phosphate or GDPmannose with mannose were identified by paper chromatography and electrophoresis as mannose-alpha-1,2-mannose and mannose-alpha-1,3-mannose. synthesis of each product was dependent on the assay conditions used and was most markedly affected by the presence of detergent. Transfer of mannose from either substrate to form mannose-alpha-1,3-mannose was severely inhibited by Triton X-100.     

Subcellular localization of the enzyme that forms mannosyl retinyl phosphate from guanosine diphosphate [14C]mannose and retinyl phosphate.

The subcellular distribution of the enzyme catalysing the conversion of retinyl phosphate and GDP-[14C]mannose into [14C]mannosyl retinyl phosphate was determined by using subcellular fractions of rat liver. Purity of fractions, as determined by marker enzymes, was 80% or better. The amount of mannosyl retinyl phosphate formed (pmol/min per mg of protein) for each fraction was: rough endoplasmic reticulum 0.48 +/- 0.09 (mean +/- S.D.); smooth membranes (consisting of 60% smooth endoplasmic reticulum and 40% Golgi apparatus), 0.18 +/- 0.03; Golgi apparatus, 0.13 +/- 0.03; and plasma membrane 0.02.     

Conversion of dolichyl pyrophosphate N,N'-diacetylchitobiose to lipid-tri- to heptasaccharides by liver microsomes from hibernating ground squirrels (Citellus citellus L.)

Incubation of liver microsomes from hibernating ground squirrel with GDP-[14C]mannose and exogenous dolichyl phosphate resulted in the synthesis of dolichyl phosphate [14C]mannose. The mannosyltransferase activity was about 3-fold higher in microsomes from hibernating ground squirrels than in those from active animals. Incubation for 30 min of liver microsomes from hibernating animals with dolichyl pyrophosphate N,N'-diacetyl-[14C]chitobiose and GDP-[14C]mannose led to the synthesis of lipid-[14C]trisaccharide. When liver microsomes were incubated with lipid-[14C]trisaccharide and unlabelled GDP-mannose, lipid-tetra- to heptasaccharides were discovered in the chloroform-methanol (2:1) extract. Since, under the experimental conditions, negligible synthesis of dolichyl phosphate mannose was observed, it was assumed that GDP-mannose was a donor of mannose in the conversion of lipid-trisaccharide into lipid-oligosaccharides containing 2-5 mannose residues.