Biosynthesis of dolichyl pyrophosphate N-acetylglucosaminyl intermediates in liver microsomes from hibernating ground squirrels (Citellus citellus L.)

Dolichyl pyrophosphate N-acetyl[14C]glucosamine was synthesized after incubation of liver microsomes from hibernating ground squirrels with UDP-N-acetyl[14C )glucosamine. The radioactivity of glycolipid formed by liver microsomes from hibernating ground squirrels was about 2-fold greater than by liver microsomes from active animals. Addition of exogenous dolichyl phosphate to the incubation mixture increased the formation of dolichyl pyrophosphate N-acetyl[14C]glucosamine by microsomes from both active and hibernating ground squirrels about 6 times. Liver microsomes from hibernating ground squirrels converted dolichyl pyrophosphate N-acetyl[14C]glucosamine into dolichyl pyrophosphate N,N'-diacetyl[14C]chitobiose in the presence of unlabelled UDP-N-acetylglucosamine. This conversion was maximal at 1.0 M concentration of unlabelled UDP-N-acetylglucosamine. The level of dolichyl phosphate assessed by the level of dolichyl pyrophosphate N-acetylglucosamine formation was nearly 2 times greater in liver microsomes from hibernating ground squirrels than from active animals.     

Biosynthesis of lipid-linked oligosaccharides in embryonic liver. Formation of mannose containing derivatives

In the presence of exogenous dolichyl phosphate mannosyl transferase activity towards dolichyl phosphate was nearly 3-fold higher in microsomes from pig embryonic liver compared to that from adult liver. After incubation of microsomes from embryonic liver with UDP-N-acetylglucosamine and GDP-[14C]mannose lipid-linked tri- to undecasaccharides were discovered in CHCl3-CH3OH (2:1, v/v) and CHCl3-CH3OH-H2O (1:1:0.3, by vol) extracts. The main proportion of the radioactivity was incorporated into penta-, sexta and undecasaccharides. Amphomycin at concentration 500 micrograms/ml inhibited almost completely dolichyl phosphate mannose synthesis in embryonic liver microsomes without inhibition the formation of lipid-linked penta- and sextasaccharides. It was suggested that mannose transferred to lipid-linked tetra- to heptasaccharides comes from GDP-mannose but not from dolichyl phosphate mannose.     

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.     

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.     

On the feasibility of electron transfer to singlet oxygen from mitochondrial components

The incorporation of [¹⁴C]mannose from GDP-[¹⁴C]mannose into dolichyl mannosyl phosphate in rat liver microsomes showed a biphasic time-course; an initial rapid incorporation of mannose which ceased within 2 min and a much slower incorporation which continued for 30 min. In the presence of 0.18 mM (250 μg/ml) bacitracin, the rapid incorporation proceeded normally whereas the slow incorporation was inhibited by about 70%. Upon addition of dolichyl pyrophosphate, the microsomes catalyzed the dephosphorylation of the added compound which was also inhibited by bacitracin. The results, coupled with several other observations, suggest that the rapid reaction represents the transfer of mannose to endogenous dolichyl phosphate whereas the bacitracin-sensitive, slow reaction represents a more complex process in which the enzymatic dephosphorylation of dolichyl pyrophosphate is involved as a rate-limiting step.     

Dietary effects on the formation of dolichyl monophosphate mannose by microsomal preparations of rat adipose tissue

Microsomal preparations from rat adipose tissue catalyse the transfer of [¹⁴C]mannose from GDP-[¹⁴C]mannose to an endogenous acceptor forming a [¹⁴C]mannosyl lipid. The mannosyl lipid co-chromatographs with hen oviduct dolichyl monophosphate β-mannose on three solvent systems. It is stable to mild alkaline hydrolysis, but strong alkaline treatment yields a compound that co-migrates with mannose 1-phosphate. The mannosyl lipid is labile to mild acid hydrolysis, yielding [¹⁴C]mannose. Formation of the compound is reversible by GDP, but not GMP, and is stimulated by exogenous dolichyl phosphate.

The kinetics of transfer of [¹⁴C]mannose from GDP-[¹⁴C]mannose to form dolichyl monophosphate mannose were studied by using preparations derived from rats fed on one of four diets: G (high glucose), L (high lard), F (fructose) or GC (high glucose, 0.9% cholesterol). The K_m and V_max. values for transfer from GDP-mannose were virtually indistinguishable in the four preparations.

In the absence of exogenous dolichyl phosphate, the largest amount of transfer of [¹⁴C]mannose into the mannosyl lipid was observed with preparations from fructose-fed animals. Preparations from glucose-fed animals showed about 60% as much transfer, whereas membranes from rats fed the other diets showed intermediate values between the fructose- and glucose-fed animals. The inclusion of cholesterol in the glucose diet elicited an increase in transfer of mannose.

Under conditions of saturating exogenous dolichyl phosphate, preparations from lard-fed animals have 1.5 times as much enzyme activity as do preparations from animals fed the other three diets.

     

Biosynthesis of dolichyl pentasaccharide diphosphate in calf pancreas microsomes

Incubation of synthetic dolichyl pyrophosphate tetrasaccharide and GDP-[14C]mannose with calf pancreas microsomes gave three lipid-linked oligosaccharides, which could be extracted with chloroform/methanol (2:1) and separated on silica gel plates. The fastest migrating product was characterized as dolichyl pyrophosphate pentasaccharide based on gel filtration and high pressure liquid chromatography. The formation of the pentasaccharide-lipid was greatly stimulated by addition of synthetic tetrasaccharide-lipid and required the presence of Triton X-100. Dolichyl phosphate mannose could not replace GDP-mannose as a sugar donor. The structure of the pentasaccharide was determined by degradation with endo-beta-N-acetylglucosaminidase D, acetolysis, alpha-D-mannosidase, and concanavalin A-Sepharose chromatography, showing that the following reaction was taking place: alpha-D-Manp-(1 leads to 3)-beta-D-Manp-(1 leads to 4)-beta-D-GlcpNAc-(1 leads to 4)-alpha-D-GlcpNAcPPDol + GDPMan leads to GDP + alpha-D-Manp-(1 leads to 3)-[alpha-D-Manp-(1 leads to 6)]-beta-D-Manp-(1 leads to 4)-beta-D-GlcpNAc-(1 leads to 4)-alpha-D-GlcpNAcPPDol. The mannosyltransferase was partially characterized.     

Tridecaptin stimulates the formation of lipid-linked saccharides in aorta.

The peptide antibiotic tridecaptin caused a 2--4-fold stimulation in the incorporation of mannose from GDP-[14C]mannose and glucose from UDP-[3H]glucose into lipid-linked monosaccharides by both the particulate and the soluble enzyme fractions from pig aorta. In both cases, the major products and the ones stimulated by antibiotic were dolichyl phosphate mannose and dolichyl phosphate glucose. The stimulation in activity was unaffected by increasing concentrations of dolichyl phosphate, GDP-mannose, UdP-glucose, Mn2+ or the detergent Nonidet P40. Tridecaptin stimulation was apparently not due to protection of sugar nucleotide substrate, since addition of various concentrations of sugar nucleotides did not alter the stimulation. Nor did the addition of tridecaptin result in any increase in the amount of radioactive sugar nucleotide recovered from incubation mixtures. Tridecaptin bound to the particulate enzyme and could not be removed by centrifugation of the particles.     

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 transfer of mannose from guanosine diphosphate mannose to dolichol phosphate and protein by pig liver endoplasmic reticulum

When pig liver microsomal preparations were incubated with GDP-[¹⁴C]mannose, 10–40% of the ¹⁴C was transferred to mannolipid and 1–3% to mannoprotein. The transfer to mannolipid was readily reversible and GDP was one of the products of the reaction. It was possible to reverse the reaction by adding excess of GDP and to show the incorporation of [¹⁴C]GDP into GDP-mannose. When excess of unlabelled GDP-mannose was added to a partially completed incubation there was a rapid transfer back of [¹⁴C]mannose from the mannolipid to GDP-mannose. The other product of the reaction, the mannolipid, had the properties of a prenol phosphate mannose. This was illustrated by its lability to dilute acid but stability to dilute alkali, and by its chromatographic properties. Dolichol phosphate stimulated the incorporation of [¹⁴C]mannose into both mannolipid and into protein, although the former effect was larger and more consistent than the latter. The incorporation of exogenous [³H]dolichol phosphate into the mannolipid, and its release, accompanied by mannose, on treatment of the mannolipid with dilute acid, confirmed that exogenous dolichol phosphate can act as an acceptor of mannose in this system. It was shown that other exogenous polyprenol phosphates (but not farnesol phosphate or cetyl phosphate) can substitute for dolichol phosphate in this respect but that they are much less efficient than dolichol phosphate in stimulating the transfer of mannose to protein. Since pig liver contained substances with the chromatographic properties of both dolichol phosphate and dolichol phosphate mannose, which caused an increase in transfer of [¹⁴C]mannose from GDP-[¹⁴C]mannose to mannolipid, it was concluded that endogenous dolichol phosphate acts as an acceptor of mannose in the microsomal preparation. The results indicate that the mannolipid is an intermediate in the transfer of mannose from GDP-mannose to protein. Some 4% of the mannose of a sample of mannolipid added to an incubation was transferred to protein. A scheme is proposed to explain the variations with time in the production of radioactive mannolipid, mannoprotein, mannose 1-phosphate and mannose from GDP-[¹⁴C]mannose that takes account of the above observations. ATP, ADP, UTP, GDP, ADP-glucose and UDP-glucose markedly inhibited the transfer of mannose to the mannolipid.     

Characterization of the reaction of GDP-mannose with dolichol phosphate in liver membranes.

The Mn-2+ dependent mannosyl transfer reaction between GDP-[14-C]mannose and dolichol phosphate, which is catalyzed by liver membranes, could not be followed accurately with the existing assay systems. Thus, GDP-[14-C]mannose is hydrolyzed rapidly by a pyrophosphatase present in microsomal and Golgi fractions from liver cells. The rate of the hydrolysis is rapid enough to limit the extent of incorporation of [14-c]mannose into endogenous acceptors. AMP was an effective inhibitor of the pyrophosphatase in Golgi membranes, and protected GDP-mannose from metabolism in alternative pathways. In the presence of AMP it was possible accurately to follow the time course of synthesis of dolichol phosphate [14-c]mannose over short time periods. Even though the time course of the reaction was measured over 2 s intervals, no linear portion could be detected in plots of product formed versus time. The kinetics of synthesis did, however, fit an equation for a first-order kinetic process. The basis for the first-order kinetics seems related to the very small amounts of dolichol phosphate in membranes. The values of the first-order rate constant is dependent on the concentrations of GDP-mannose and Mn-2+ added to the assays.     

Glycoprotein biosynthesis in Chlamydomonas. A mannolipid intermediate with the properties of a short-chain alpha-saturated polyprenyl monophosphate

A crude membrane preparation of the unicellular green alga Chlamydomonas reinhardii was found to catalyse the incorporation of D-[14C]mannose from GDP-D-[14C]-mannose into a chloroform/methanol-soluble compound and into a trichloroacetic acid-insoluble polymer fraction. The labelled lipid revealed the chemical and chromatographic properties of a short-chain (about C55-C65) alpha-saturated polyprenyl mannosyl monophosphate. In the presence of detergent both long-chain (C85-C105) dolichol phosphate and alpha-unsaturated undecaprenyl phosphate (C55) were found to be effective as exogenous acceptors of D-mannose from GDP-D-[14C]mannose to yield their corresponding labelled polyprenyl mannosyl phosphates. Exogenous dolichyl phosphate stimulated the incorporation of mannose from GDP-D-[14C]mannose into the polymer fraction 5-7-fold, whereas the mannose moiety from undecaprenyl mannosyl phosphate was not further transferred. Authentic dolichyl phosphate [3H]mannose and partially purified mannolipid formed from GDP-[14C]mannose and exogenous dolichyl phosphate were found to function as direct mannosyl donors for the synthesis of labelled mannoproteins. These results clearly indicate the existence of dolichol-type glycolipids and their role as intermediates in transglycosylation reactions of this algal system. Both the saturation of the alpha-isoprene unit and the length of the polyprenyl chain may be regarded as evolutionary markers.     

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.     

Lipid-bound sugars in malignant human breast tumors

Microsomal preparations from malignant human breast tumors catalyzed the transfer of mannose and glucose from GDP-[14C]-Man and UDP-[14C]-Glc into lipid-linked sugars and glycoprotein-like substances. As judged by several criteria the obtained lipid-linked monosaccharides behaved as dolichyl phosphate mannose and dolichyl phosphate glucose whereas lipid-linked oligosaccharides behaved as polyprenyl diphosphate derivatives. The optimum conditions for mannosyl- and glucosyl-transfer reactions and the effect of dolichyl phosphate, detergent and EDTA on incubation mixture were described.     

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.     

Anomeric configuration of the dolichyl D-mannosyl phosphate formed in calf pancreas microsomes.

Dolichyl D-[14C]mannosyl phosphate formed in calf pancreas microsomes was compared to dolichyl alpha-D-[14C]mannopyranosyl phosphate, a chemical synthesis of which is described. Jack bean alpha-mannosidase, which converted citronellyl alpha-D-mannopyranosyl phosphate, but not its beta anomer, to citronellyl phosphate and D-mannose, was effective in releasing D-[14C]mannose from dolichyl alpha-D-[14C]manopyranosyl phosphate in the presence of detergent. In contrast, alpha-mannosidase did not cause any significant release from the pancreatic dolichyl D-[14C]mannosyl phosphate. Alkali treatment (0.1 M NaOH in propanol at 65 and 90 degrees) degraded both dolichyl D-[14C]mannosyl phosphates with the formation of water-soluble 14C-labeled products. The pattern of 14C-labeled breakdown products formed from the synthetic dolichyl alpha-D-[14C]mannopyranosyl phosphate differed from that obtained from the pancreatic dolichyl D-[14C]mannosyl phosphate. Dolichyl alpha-D-[14C]mannopyranosyl phosphate yielded several 14C-labeled products, including a trace of D-[14C]mannosyl phosphate, and an acidic fraction which appeared to result from degradation of D-[14C]mannose. The pancreatic dolichyl D-[14C]mannosyl phosphate gave various products, depending on the temperature of the reaction: at 90 degrees, 20 to 30% of the radioactivity was found in D-[14C]mannosyl phosphate and the rest in acidic breakdown products; at 65 degrees, about two-thirds of the radioactivity was recovered in a compound which behaved as D-MANNOSE 2-PHOSPHATE, A Product characteristic of a beta-linked D-mannosyl residue. It is concluded that the pancreatic compound is dolichyl beta-D-[14C]mannosyl phosphate.     

alpha-Dihydrodecaprenyl phosphate as a sugar carrier in the membrane of AH 70Btc hepatoma cells

The effect of alpha-dihydrodecaprenyl phosphate, dolichyl phosphate and solanesyl phosphate on the lipid intermediate pathway for protein glycosylation was studied with crude membrane fraction prepared from AH 70Btc hepatoma cells. alpha-Dihydrodecaprenyl phosphate increased the incorporations of [14C]mannose from GDP-[14C]mannose into CHCl3-CH3OH (2:1, v/v) extract, oligosaccharide-lipid and proteins. The above and the other data showed that alpha-dihydrodecaprenyl phosphate may function as a mannose carrier in the lipid intermediate pathway.     

Formation of polyprenol-linked mono- and oligosaccharides in Phaseolus aureus

A crude membrane preparation from Phaseolus aureus hypocotyls catalyzes the incorporation of mannose from GDP-[¹⁴C]mannose into a acid labile glycolipid and a methanol insoluble fraction. Addition of dolichyl monophosphate to the incubation mixture stimulated the formation of both the mannolipid and the methanol insoluble endproduct. Thin-layer chromatography of endogenous lipid and of the stimulated lipid fraction revealed that both compounds run identical. Ficaprenyl monophosphate also stimulates the incorporation of mannose; however, the ficaprenyl monophosphate mannose formed is not identical to the endogenous mannolipid. This suggests that the endogenous acceptor has the properties of an α-saturated polyprenyl monophosphate rather than those of the ficaprenyl phosphate type. The same membrane preparation also incorporates N-acetylglucosamine into an acid labile glyolipid as well as into a polymer fraction. Evidence is presented that the N-acetylglucosamine containing lipid consists of a mixture of dolichyl pyrophosphate N-acetylglucosamine and dolichyl pyrophosphate di-N-acetylchitobiose. It seems likely that the two compounds have a precursor-product relationship. Incubation of dolichyl pyrophosphate di-N-acetylchitobiose together with GDP-mannose gives rise to lipid-bound mannosyl-di-N-acetylchitobiose. Radioactivity from either the [¹⁴C]mannolipid or the N-acetyl[¹⁴C]glucosamine containing lipid is incorporated into a methanol insoluble product to 3.4 and 6.3%, respectively; it seems, at least in part, to be a glycoprotein.     

Solubilization and properties of mannose and N-acetylglucosamine transferases involved in formation of polyprenyl-sugar intermediates.

A particulate fraction from porcine aorta catalyzed the incorporation of N-acetylglucosamine (GlcNAc) from UDP-[3H]GlcNAc into both GlcNAc-pyrophosphorylpolyprenol and GlcNAc-GlcNAc-pyrophosphorylpolyprenol. This transfer utilized endogenous lipid and required a divalent cation. Mn2+ was the best metal ion and was optimum at 2.3 mM. This same particulate fraction was previously shown to transfer mannose from GDP-[14C]mannose to endogenous lipid to form mannosylphosphorylpolyprenol (Chambers, J., and Elbein, A.D. (1975) J. Biol. Chem. 250, 6904-6915). Both the GlcNAc activities and the mannose activity were solubilized by treatment of the particulate fraction with the detergent Nonidet P-40. The enzymes were partially purified by chromatography on DEAE-cellulose and on Sephadex G-200. These soluble enzymes required the addition of acceptor lipid for activity. An acidic lipid fraction, isolated from pig liver and having the properties of dolichyl phosphate, was active with either the GlcNAc or the mannose transferase. Chemically synthesized dolichyl phosphate was also active with either of these enzymes. The products formed from either GlcNAc or mannose by the soluble transferases were similar to those formed by the particulate enzyme. Thus the major product formed from UDP-[3H]GlcNAc was GlcNAc-pyrophosphoryldolichol with small amounts of the disaccharide-lipid while the product formed from GDP-[14C]mannose was mannosylphosphoryldolichol.     

Glycoprotein biosynthesis in intestinal epithelial cells during differentiation. Incorporation of [14C]mannose from GDP-[14C]mannose into dolichol derivatives

Epithelial cells of the rat small intestine were collected as a gradient of villus to crypt cells. Homogenates of these cells incubated with GDP-D-[14C]mannose in the presence of MnCl2 incorporated radioactivity into dolichyl mannosyl phosphate and a mixutre of dolichyl pyrophosphate oligosaccharides varying in the size of their oligosaccharide moiety. The labeled oligosaccharides formed in villus cell homogenates appeared shorter than those formed in crypt cell homogenates. The addition of dolichyl phosphate greatly stimulated the synthesis of dolichyl mannosyl phosphate. The initial rate of synthesis of dolichyl mannosyl phosphate from GDP-D-[14C]mannose and exogenous dolichyl phosphate was highest in an intermediate cell fraction having a low specific activity of sucrase and alkaline phosphatase and an intermediate specific activity of thymidine kinase. To compare the rates of dolichyl mannosyl phosphate synthesis in the different cell fractions, it was essential to control degradation of GDP-D-[14]mannose by the addition of AMP to the incubation, since villus cells degraded GDP-D-[14C]mannose much faster than crypt cells.