The subcellular localization of enzymes of dolichol metabolism in rat liver

Dolichyl phosphate is an intermediate in the glycosylation of N-glycosamidic linked glycoproteins in mammalian systems, and its availability may be a limiting factor in glycoprotein biosynthesis. The basic kinetics and subcellular distribution of enzymes which may influence the concentration of dolichyl phosphate in rat liver have therefore been investigated. These include dolichyl phosphate phosphatase, dolichol phosphokinase, dolichyl fatty acyl ester synthetase, GDP-mannose dolichyl phosphate mannosyl transferase, and UDP-glucose dolichyl phosphate glucosyl transferase. The specific activity of the enzymes was highest in the microsomes, except for dolichyl phosphate phosphatase and dolichyl fatty acyl ester synthetase, which were most concentrated in the plasma membrane and the cytosol fraction, respectively. The nuclei contained all of the enzyme activities while the mitochondria and cytoplasm were generally less active. The presence of both dolichol phosphokinase and dolichyl phosphate phosphatase in microsomes and nuclei, which contain the highest glycosyl transferase activities, may provide a means for direct enzymatic control of levels of dolichyl phosphate.     

Dolichyl sulphate and H-phosphonate: enzymatic reactions with activated sugars.

Two phosphate-modified analogues of dolichyl phosphate were evaluated as substrates or inhibitors of the reactions catalyzed by mammalian microsomal enzymes. Dolichyl H-phosphonate could serve as an efficient acceptor for mannosyl and glucosyl transfer. The reaction products were chromatographically different from those formed from dolichyl phosphate. Lower activity of the H-phosphonate was observed for the reaction of N-acetylglucosaminyl phosphate transfer from UDP-GlcNAc. Dolichyl sulphate was shown not to serve as a substrate for the transfer of mannosyl (from GDP-Man), glucosyl (from UDP-Glc) or N-acetylglucosaminyl phosphate (from UDP-GlcNAc) residues in the presence of rat liver microsomes. Weak inhibitory properties of this analogue were demonstrated.     

Characterization of mannosyl-transfer reactions catalyzed by dolichyl-mannosyl-phosphate-synthase

Evidence suggesting that a single enzyme catalyzes mannosyl transfer from GDP-mannose to both dolichyl phosphate and to phenyl phosphate was obtained as follows: (a) The two activities were coeluted from columns of DEAE-cellulose and Sepharose CL-6B, (b) both reactions demonstrated similar kinetic constants for the glycosyl donor and for guanosine nucleoside inhibitors, (c) both reactions were sensitive to inhibition by low concentrations of nonionic detergents, and (d) both activities were found to be thermally inactivated at similar rates upon incubation at 55 degrees. The reaction conditions required for optimal mannosyl transfer by the purified enzyme preparation to the hydrophobic and water soluble acceptors, however, were found to be quite different. Whereas mannosyl transfer from GDP-mannose to dolichyl phosphate occurred at maximal rates only in the presence of specific phospholipids, the rate of mannosyl transfer to phenyl phosphate was essentially unaffected by the addition of phospholipid. These results indicate that dolichyl-mannosyl-phosphate-synthase, which has some of the properties of an intrinsic membrane protein, does not have an absolute requirement for phospholipid for catalytic activity, but rather that phospholipid is required for interaction of the enzyme with the long chain polyisoprenol substrate dolichyl phosphate.     

Inhibition of beta-1,4-Glucan Biosynthesis by Deoxyglucose : The Effect on the Glucosylation of Lipid Intermediates

GDP- and UDP-deoxyglucose inhibit the incorporation of glucose from UDP-glucose into dolichyl phosphate glucose and dolichyl pyrophosphate oligosaccharides. GDP-deoxyglucose inhibits by competing with the physiological nucleotide sugars for dolichyl phosphate, and dolichyl phosphate deoxyglucose is formed. This inhibition is reversed by excess of dolichyl phosphate. UDP-deoxyglucose does not give rise to a lipid-linked derivative, and inhibition by this analog is not reversed by dolichyl phosphate. The UDP- and GDP-derivatives of deoxyglucose inhibit the incorporation of glucose into glucose-containing glycoproteins. This effect seems to be the result of the inhibition of lipid intermediates glucosylation and is comparable to the effect produced by coumarin. Cellulose synthetase activity is not affected by UDP- or GDP-deoxyglucose. On the other hand, deoxyglucose inhibits the formation of β-1,4-glucans in vivo.     

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.     

Inhibition of lipid-linked mannose and mannoprotein synthesis in yeast by diumycin in vitro

Diumycin, a phosphoglycolipid antibiotic, inhibits different mannosyl transfer reactions in yeast. Using membrane preparations, the drug effectively inhibited the formation of dolichyl phosphate mannose (DolP-Man); 50% inhibition was observed at approximately 10 microgram/ml. To a lesser extent also mannosyl transfer from DolP-Man to protein decreased in presence of diumycin. Both mannosyl transfer to protein-serine/threonine acceptor sites as well as into positions within the asparagine-linked polymannose part of the yeast mannoprotein are inhibited to about 60% under conditions where DolP-Man formation is blocked. DolP-Man synthesis as well as mannosyl transfer from DolP-Man to protein are also inhibited by diumycin using solubilized enzymes and exogenous acceptor substrates. Glycosyltransfer reactions from GDP-mannose either to protein-serine/threonine-linked mannose (formation of short manno-oligosaccharides) or to dolichyl-diphosphate-linked chitobiose (formation of lipid-linked trisaccharide) are not inhibited by diumycin under conditions where DolP-Man synthesis is blocked by the antibiotic. The inhibitory action of diumycin on DolP-Man formation does not seem to be competitive with respect to dolichyl phosphate, since it cannot be overcome by higher concentrations of dolichyl phosphate.     

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.

     

Dolichol pathway in lymphocytes from rat spleen. Influence of the glucosylation on the cleavage of dolichyl diphosphate oligosaccharides into phosphooligosaccharides

Incubation of rat-spleen lymphocytes with UDP-glucose together with GDP-mannose and UDP-N-acetylglucosamine leads to the formation of glucosylated lipid intermediates characterized as dolichyl phosphate glucose and dolichyl diphosphate oligosaccharides. This latter can be either transferred onto endogenous protein acceptors or cleaved into phosphooligosaccharides. The striking fact is that phosphooligosaccharide populations contain far less glucosylated products than the dolichyl diphosphate oligosaccharide ones from which they are derived. Two hypotheses have been investigated: either a rapid action of glucosidases on the liberated phosphooligosaccharides or a preferential splitting of the non-glucosylated population of dolichyl diphosphate oligosaccharides. Addition of p-nitrophenyl-alpha-D-glucoside inhibits glucosidase activities and allows the production of a major population of dolichyl diphosphate oligosaccharides containing three glucose residues. Using these conditions, it is shown that the amount of phosphooligosaccharides generated from the splitting of dolichyl diphosphate oligosaccharides is greatly decreased and that the major part of these remaining phosphooligosaccharides do not contain glucose. These results show that the presence of glucosyl units prevent dolichyl diphosphate oligosaccharides from further degradation into phosphooligosaccharides.     

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.     

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.     

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.     

Increased hepatic dolichol and dolichyl phosphate-mediated glycosylation in rats fed cholesterol

The concentrations of dolichol and cholesterol in livers of rats maintained for 2 weeks on a diet enriched with cholesterol (1%) were significantly higher than those in animals on a normal diet. The incorporation of radioactive mevalonate into dolichol and into a dolichyl diphosphate oligosaccharide fraction by liver slices of the cholesterol-fed animals was increased over that of the control group. However, the incorporation of radioactive mevalonate into cholesterol was decreased, as was the incorporation of radioactive acetate into both dolichol and, more markedly, cholesterol. These results are consistent with cholesterol feeding causing partial inhibition of the cholesterol-biosynthetic pathway both at β-hydroxy-β-methylglutaryl coenzyme A reductase and at a step after farnesyl pyrophosphate formation, resulting in a greater flux of mevalonate to dolichol and an increase in pool sizes of precursors of β-hydroxy-β-methylglutaryl coenzyme A. Maximal activity of glycosyl transfer to dolichyl phosphate was greater in microsomal preparations from livers of cholesterol-fed animals compared with those of control animals. A corresponding higher degree of in vitro glycosylation of endogenous protein was also observed. It is concluded that the cholesterol-enriched diet caused an increase in the biosynthesis and concentration of dolichyl monophosphate which resulted in a higher level of N-glycosylation of protein. These effects were complicated by differences in the kinetics of glycosyl transfer and in its response to exogenous dolichyl monophosphate.     

The use of liposomes in aspergillus niger van tieghem mannosylation:in vitro studies

The use of dolichyl phosphate-containing liposomes in glycoprotein biosynthesis under conditions that control the transfer of exogenous dolichyl phosphate from liposomes to microsomes is described. The exogenously added dolichyl phosphate is available for mannosylation, and direct evidence for the role of dolichyl phosphate as intermediate in protein mannosylation was shown by use of liposomes containing dolichyl [¹⁴C]mannosyl phosphate.     

Fluoroglucose-inhibition of protein glycosylation in vivo. Inhibition of mannose and glucose incorporation into lipid-linked oligosaccharides

The effects of the glycosylation inhibitor 2-deoxy-2-fluoro-D-glucose on the formation of the lipid-linked oligosaccharides and monosaccharides that are involved in protein glycosylation were investigated. In chick embryo cells treated with fluoroglucose the formation of lipid-linked oligosaccharides cannot go to completion and oligosaccharides with decreased amounts of glucose and mannose can be detected. These oligosaccharides are probably biosynthetic intermediates and serve as acceptors of sugar residues while reversing fluoroglucose-inhibition by the addition of mannose and glucose to the culture medium. In contrast to deoxyglucose, fluoroglucose was not incorporated into lipid-linked oligosaccharides. Fluoroglucose inhibits the formation in vivo of dolichyl phosphate glucose and dolichyl phosphate mannose, but not the transfer of those sugar residues from the lipid monophosphate derivative to the lipid-linked oligosaccharides. The pool size of UDP-glucose, but not of GDP-mannose and UDP-N-acetylglucosamine, was decreased. Also, the formation of lipid-linked N-acetylglucosamine was not affected by fluoroglucose. Fluoroglucose was applied to deplete cellular membranes of endogenous lipid-linked mannose and glucose, and can possibly be used to discern different pathways of glycosylation.     

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.     

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.     

Effects of turpentine-induced inflammation on the synthesis of dolichol-linked intermediates of N-glycosylation and the phosphorylation of dolichol by CTP-dependent dolichol kinase

Inflammation was induced in rats by the subcutaneous injection of turpentine. Microsomes were prepared from the livers between 2 and 72 h after injection. Mannose and glucose incorporation into mannosyl and glucosyl dolichyl monophosphate was increased 2-fold over saline-injected controls 24 h after induction of inflammation. Synthesis of glycosylated dolichyl pyrophosphoryl oligosaccharides was also increased compared to controls. Extraction and assay of dolichol monophosphate from inflamed and control rat liver microsomes indicated that the endogenous levels of the lipid were elevated in the inflamed state. CTP-dependent phosphorylation of endogenous dolichol was also found to increase in microsomes from inflamed rats 24 h after injection of turpentine. When exogenous dolichol was added to the microsomal system an increase in phosphorylation was observed as early as 6 h after turpentine injection. Furthermore, the increase appeared to be biphasic, there being two peaks of elevated activity at 12 and 36-48 h after induction of inflammation. The earlier peak was the greater of the two. The results suggest that the increase in glycosylation of dolichol derivatives was due to greater amounts of endogenous dolichol monophosphate. The increase in dolichol monophosphate was itself due to greater availability of dolichol and an increase in the levels of CTP-dependent dolichol kinase.     

The formation of glycosidic bonds in yeast glycoproteins

Membranes of Saccharomyces cerevisiae were separated on urografin gradients. The specific activity of the light membranes (endoplasmic reticulum), the Golgi-like vesicles and the plasma membrane in transferring mannosyl residues from GDP-mannose to mannoproteins and to dolichyl monophosphate has been determined. The first mannose of the O-glycosidically linked manno-oligosaccharides is incorporated with the highest specific activity by the endoplasmic reticulum. The incorporation of the second to fourth mannosyl groups is catalysed with increasing activity also by the Golgi-like vesicles and the plasma membrane.The incorporation of mannosyl groups into weak alkali-stable positions (N-glycosidically linked chains) is carried out with almost the same specific activity by all three membrane fractions, however, dolicholdependent and-independent steps could not be distinguished as yet.The results are discussed in terms of a sequential addition of sugar residues along the route of export of the mannoproteins. The dolichol-dependent steps seem to occur on the endoplasmic reticulum and thus very carly in the event.Abbreviations GDP-mannose guanosine diphosphate mannose - Dol-P dolichyl monophosphate - Dol-P-mannose dolichyl monophosphate mannose     

Short-chain dolichols of defined chain length as cofactors in reactions of the microsomal dolichyl-phosphate cycle and transglycosylations.

1) The biological cofactor and carrier activities of dolichyl phosphates of low isoprene multiplicity (n) and defined geometry, (synthesized according to L. Jaenicke and H.-U. Siegmund, Chem. Phys. Lipids 51 (1989) 159-170), were assayed in different transfer reactions of the microsomal dolichyl-phosphate cycle against natural pig liver dolichyl phosphate (n = 18 to 20). 2) The apparent Michaelis constants and maximal velocities were determined from initial reaction rates for the transfer from GDP-mannose, UDP-N-acetylglucosamine, and UDP-glucose to the synthetic truncated dolichyl phosphates. They afford quantitative comparison and show increasing biological activities from dolichyl-6 phosphate to dolichyl-11 phosphate, which is about as active as the natural mixture. This is in accord with previous findings on the starting reactions of the cycle. 3) Truncated dolichyl diphosphochitobioses, biosynthesized in vitro from synthetic dolichyl phosphates, were used as acceptors for nucleoside diphosphohexoses in solubilized membranes. All of them show about the same activity. The kinetics and yield were determined for each of the transfers. Activity is increased by adding UDP-glucose. The inactive very short-chain dolichol compounds do not interfere with the transfer to active longer chain dolichols. 4) The oligosaccharides produced by transfer of mannose and glucose to truncated dolichyl diphosphate-bound chitobiose were isolated and analysed for sugar multiplicity. The heptasaccharide and the un-decasaccharide are accumulated most, pointing to the transport across the endoplasmic membranes (ER) as the rate limiting reaction. 5) The truncated dolichyl-diphosphate-bound oligosaccharides are transferred to protein(s) by the crude, solubilized microsomal preparation independent of chain length of the cofactor/carrier, yet with increasing yield as shown by enzyme immunoblot analysis.     

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.