Dolichyl Diphosphate Oligosaccharide Research Articles

Dolichol-dependent synthesis of chitobiosyl proteins and their further mannosylation. A second route for glycosylation of proteins in rat-spleen lymphocytes?

Incubation of whole lymphocytes with UDP-N-acetyl [3H]glucosamine used as the only precursor leads to the formation of dolichyl diphosphate [3H]chitobiose, DolPP-(GlcNAc)2, and dolichyl diphosphate N-acetyl-[3H]glucosamine, DolPP-GlcNAc. Although very few dolichyl diphosphate oligosaccharides are formed, a high level of radioactivity is recovered with proteins and has been characterized, using hydrazinolysis procedure, as [3H]chitobiosyl and N-acetyl[3H]glucosaminyl units. Addition of tunicamycin inhibits, to the same extent, both the synthesis of DolPP-(GlcNAc)1-2 and the incorporation of the N-acetyl[3H] glucosaminyl residues onto proteins, indicating that these carbohydrate units are transferred onto proteins acceptors from their dolichol derivatives. Chase experiments have indicated that, in fact, the DolPP-(GlcNAc)1-2 were utilized in two ways: either their transfer onto proteins or their degradation into water-soluble saccharidic material. Moreover, the transfer reaction appears to be a slow process compared to the degradation since the radioactivity chased from the DolPP-(GlcNAc)1-2 is not recovered on proteins. This fact allows to show that part of the [3H]chitobiose previously bound to proteins is further converted into oligomannosidic glycans in the presence of GDP-mannose. This direct mannosylation of chitobiosyl-proteins may represent a second route for the N-glycosylation of proteins.

Phosphorylated dolichols in aging

The age-associated changes in the levels and synthesis of dolichyl phosphate and dolichyl diphosphate derivatives were investigated in brain and liver of 057B1/NNia mice. The total chloroform/methanol (2:1, v/v)-extractable phosphorylated dolichols of brain increased from 1.01 micrograms/g at 3 months to 5.22 micrograms/g at 28 months of age. The long-chain dolichyl diphosphate oligosaccharide (Dol-PP-oligo) levels of brain increased from 0.82 microgram/g in 3 months to 2.8 micrograms/g in 28-month-old animals. However, in liver and in kidney, the levels of these components were unaffected by age. Incorporation of labelled glucose from UDP-glucose into dolichyl phosphate glucose and Dol-PP-oligo in brain microsomes was unaffected by age, whereas, in liver microsomes, the rates of synthesis of both components increased by 50-150%. The increased rate of synthesis and lack of accumulation of Dol-PP-oligo in liver suggest an active utilization and/or catabolism of these glycoprotein precursors. The accumulation of Dol-PP-oligo in aging brain may reflect its decreased utilization for N-glycosylation and/or reduced catabolism.

Recovery of dolichyl diphosphate oligosaccharide in methanolic aqueous phase prepared from rat liver microsomal fractions.

The synthesis of dolichyl diphosphate oligosaccharide was studied by incubating rat liver microsomes (microsomal fractions) with GDP-[14C]mannose, UDP-glucose, UDP-N-acetylglucosamine and [3H]dolichol phosphate. The labelled products obtained by the first step of extraction of the microsomes in methanolic aqueous phase (MAP fraction in chloroform/methanol/water; 3:2:1, by vol.) and in CMW fraction (chloroform/methanol/water; 10:10:3, by vol.) obtained by extraction of the interphase after the first step of extraction were analysed on a DEAE-cellulose column. With the progress of incubation, the radioactivity in unchanged GDP-mannose decreased, whereas the labelled dol-P-P-oligo in the MAP fraction increased about 5-6-fold. The lipid oligosaccharide in this fraction accounted for about 50-60% of the GDP-mannose used, whereas the recovery of the labelled lipid oligosaccharide in the CMW fraction was about 10%. The lipid oligosaccharide from both reactions after mild acid hydrolysis were analysed by gel filtration on Bio-Gel P-4. The oligosaccharide from the MAP fraction gave a peak of higher Mr distinctly separate from the lower-Mr peak obtained from the CMW fraction. Microsomes incubated with labelled lipid oligosaccharide from the MAP fraction showed incorporation of the label into endogenous protein.

Biosynthesis of lipid-linked oligosaccharides in embryonic liver. Glucosylation of mannose containing dolichyl diphosphate oligosaccharide

A maximum rate of dolichyl phosphate [14C]glucose synthesis from 55-day embryos was achieved at 16 nM concentration of exogenous dolichyl phosphate and exceeded about 3 times that without addition of dolichyl phosphate. The highest values of [14C]glucose incorporation from UDP-[14C]glucose into dolichyl phosphate [14C]glucose, dolichyl diphosphate [14C]Glc-oligosaccharides and protein were reached at 5 min time point of incubation of liver microsomes both from embryos and sows. The radioactive incorporation into proteins was about 7-fold higher in liver microsomes from sows compared to that from embryos, probably due to the greater content of acceptor proteins in microsomes from sows. The enzymatic transfer of Glc3-oligosaccharide from a lipid carrier to endogenous protein acceptor in microsomes from pig embryonic and adult livers was considerably faster than the removal of glucose residues during the initial stages of processing of protein-bound oligosaccharides. One labelled compound was discovered in the CHCl3-CH3OH-H2O (1:1:0.3, by vol) extract after incubation of liver microsomes from embryos and sows with UDP-[14C]glucose. On the basis of its mobility on the chromatogram it appears to be GlcNAc2Man9Glc3.

Effect of bis-(p-nitrophenyl) phosphate on the biosynthesis and the utilization of lipid-intermediates.

Incubations of rat spleen lymphocytes with the required labelled nucleotide sugars lead to the formation of the various lipid-intermediates involved in the N-glycosylation of proteins. The effect of bis-(p-nitrophenyl) phosphate on the different reactions involved in the dolichol pathway has been studied. Although dolichyl phosphate mannose, dolichyl phosphate glucose and dolichyl diphosphate N-acetylglucosamine synthesis is not affected at all by bis-(p-nitrophenyl) phosphate (20 mM), this product inhibits completely the addition of the second N-acetylglucosamine residue on the dolichyl diphosphate N-acetylglucosamine acceptor. The addition of the five innermost mannose residues from GDP-mannose as donor is also strongly abolished. However, the addition of the more distal sugars, i.e. the four mannose residues using dolichyl phosphate mannose as donors and the additional glucose residues are only slightly affected. The reactions involved in the utilization of dolichyl diphosphate oligosaccharide, i.e. transfer to the proteins or degradation into soluble phospho-oligosaccharides, are also strongly inhibited. Thus bis-(p-nitrophenyl) phosphate appears to affect only the reactions involving the presence of dolichyl diphosphate sugar as substrate.

The effect of flavomycin on the synthesis and transfer of lipid-linked saccharides in pig brain.

Particulate membrane fractions from pig brain catalyse the synthesis of lipid-linked sugar derivatives of the dolichyl phosphate pathway. Flavomycin, a phosphoglycolipid antibiotic produced by various species of streptomycetes, interferes with the formation of these glycolipids to a different extent. The formation of dolichyl phosphate glucose was shown to be most susceptible to the antibiotic, being blocked by about 50% in the presence of 0.2mm-flavomycin, whereas the synthesis of dolichyl diphosphate N-acetylglucosamine, dolichyl diphosphate chitobiose and dolichyl diphosphate chitobiosyl mannose required higher concentrations to achieve a comparable inhibition. Although the formation of dolichyl phosphate mannose was hardly affected, the accumulation of oligosaccharides with five to seven sugar units was observed, when dolichyl diphosphate oligosaccharides were synthesized with GDP-[(14)C]mannose in the presence of 1mm-flavomycin. This indicates that the inhibition of the synthesis of larger-sized oligosaccharides, known to be mediated by lipid-bound mannose, was not caused by an actual deficiency in dolichyl phosphate mannose. At flavomycin concentrations that inhibited the formation of dolichyl phosphate glucose by 50%, the transfer of lipid-linked saccharides to either the hexapeptide Tyr-Asn-Gly-Thr-Ser-Val or endogenous protein acceptors was hardly influenced. The mode of action of flavomycin is still obscure, but seems not to be of a competitive nature, since the inhibition was unaffected by increasing concentrations of dolichyl phosphate. Some evidence indicates that, besides a direct interaction of the antibiotic with some transferases, a non-specific incorporation into the membrane and alteration of its properties might be responsible for those inhibitory effects on all enzymes which were observed at high concentrations of flavomycin.

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.

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 biosynthesis of glucose containing insect lipid linked oligosaccharide and its possible role in glycoprotein assembly.

Microsome enriched Ceratitis capitata extracts synthesized a glucosylated lipid linked oligosaccharide. Its properties were closely related to those of the previously described insect mannosylated dolichyl diphosphate oligosaccharides and almost the same as those of the rat liver dolichyl-diphosphate-(GlcNAc)2-(Man)9-(Glc)1-3. The saccharide moiety of the latter was transferred to an unknown endogenous protein-like acceptor by the fly extracts. These represent the first evidence of a protein glycosylation in a pluricellular invertebrate through dolichyl derivatives.

Presence in a plant of a compound similar to the dolichyl diphosphate oligosaccharide of animal tissue.

A compound with properties identical with the glucose-containing dolichyl diphosphate oligosaccharide present in animal tissues was detected in alfalfa roots incubated with [14C]glucose. The products of mild acid hydrolysis behaved the same on paper chromatography, on treatment with specific glucosidases and on N-deacetylation.

Fate of oligosaccharide-lipid intermediates synthesized by resting rat-spleen lymphocytes.

Using conditions to avoid the utilization of labelled precursors by intracellular glycosyltransferases, experiments are described demonstrating that intact rat-spleen lymphocytes are capable of utilizing exogenous GDP-mannose and UDP-N-acetylglucosamine to synthesize dolichyl monophosphate mannose and dolichyl diphosphate oligosaccharides. Kinetic and chase experiments show that dolichyl diphosphate oligosaccharides are either utilized for the transfer of their carbohydrate moieties to protein acceptors or further degraded. Since glycosylation of proteins is limited in resting lymphocytes, the degradation pathway appears as a major event in the fate of the dolichyl diphosphate oligosaccharides synthesized in vitro. These dolichyl diphosphate oligosaccharides are degraded into phospho-oligosaccharides and oligosaccharides which are released in the medium. This enzymatic cleavage of the phosphodiester bond is inhibited by bacitracin. The phospho-oligosaccharides are susceptible to alkaline phosphatase giving neutral oligosaccharides and they are cleaved by endo-N-acetyl-beta-D-glucosaminidase H leaving N-acetylglucosamine 1-phosphate and neutral oligosaccharides. These data suggest that splitting of the phosphodiester bond of colichyl diphosphate oligosaccharides, dephosphorylation and/or endo-N-acetyl-beta-D-glucosaminidase hydrolysis of the phosphorylated oligosaccharides could represent the beginning of the catabolic pathway of dolichyl diphosphate oligosaccharides.

Addition of glucose to dolichyl diphosphate oligosaccharide and transfer to protein.

The glycosylation of asparagine residues in proteins is known to occur by transfer from a dolichyl diphosphate oligosaccharide containing glucose. Paper chromatography allowed the separation of oligosaccharides (obtained by acid hydrolysis of the dolichyl diphosphate derivative) containing 1, 2 and 3 glucose residues. Using this procedure it was found that the addition of all three glucoses to the dolichyl diphosphate oligosaccharide occur with dolichyl phosphate glucose as donor. Furthermore only the compound with three glucoses was used as donor in the transfer to protein. The addition of glucose to exogenous dolichyldiphosphate oligosaccharide labelled by transfer from radioactive guanosine diphosphate mannose was detected.

Microsomal glucosidases acting on the saccharide moiety of the glucose-containing dolichyl diphosphate oligosaccharide

The microsomal glucosidases which act on the oligosaccharide that contains 2 N-acetylglucosamine, 9 mannose and 1–3 glucose residues have been studied. Two fractions were separated by differential solubility in detergent and phosphate solutions or by gel filtration. One of the fractions removed glucose from the oligosaccharide containing three glucose residues, and another fraction acted on the compounds containing one and two glucose residues. Both fractions were free of mannosidase. Some properties of the enzymes are described.

The Dolichol Pathway of Protein Glycosylation in Rat Liver

Incorporation of N-acetylglucosamine into endogenous lipid and protein acceptors was investigated on heavy microsomes from rat liver, incubated with UDP-N-acetyl[14C]glucosamine and GDP-mannose in the absence of detergent. This subcellular preparation derived for 95% or more from the rough endoplasmic reticulum and was devoid of Golgi components which contain the enzyme that adds the peripheral N-acetylglucosamine units to glycoproteins. The label was found almost exclusively in dolichyl diphosphate N-acetylglucosamine, except when the subcellular preparation was treated with pyrophosphate and subsequently incubated with the nucleotide sugars in the presence of GTP. Then, the incorporation of N-acetylglucosamine was considerably enhanced, and the additional label was associated with dolichyl diphosphate N,N'-diacetylchitobiose, with dolichyl diphosphate oligosaccharides and with proteins. The time-course of N-acetylglucosamine incorporation in these products was compatible with the pathway of dolichyl diphosphate glycoconjugates for the biosynthesis of the core portion of saccharide chains linked to asparagine residues of glycoproteins. The addition of GDP-mannose to the incubation medium was required to produce labeled dolichyl diphosphate oligosaccharides, but not to incorporate N-acetylglucosamine in protein. It is concluded that rough microsomes are capable of assembling dolichol-linked oligosaccharides from exogenous nucleotide precursors and of transferring N,N'-diacetylchitobiose, or its mannosylated derivatives, from the lipid intermediate to endogenous proteins. However, these metabolic activities are hindered in the original subcellular preparation, and in the absence of GTP. Although the earliest perceptible effect produced jointly by the treatment with pyrophosphate and by GTP was the synthesis of dolichyl diphosphate N,N'-diacetylchitobiose, the primary action of these factors remains uncertain. They may stimulate directly the reaction forming dolichyl diphosphate N,N'-diacetylchitobiose from dolichyl diphosphate N-acetylglucosamine, or activate the synthesis of this latter intermediate from a particular pool of dolichyl monophosphate which is readily converted afterwards into disaccharide and oligosaccharide derivatives and glycosylates protein. The requirement for GTP might have a functional meaning, for GTP acted maximally at a concentration distinctly lower than its actual concentration in liver. The detachment of ribosomes from rough vesicles was the major alteration induced by treatment with pyrophosphate. It is suggested that the removal of ribosomes unmasks the membrane sites where GTP acts.

The Dolichol Pathway of Protein Glycosylation in Rat Liver

Incorporation of mannose into endogenous lipid and protein acceptors of the rat liver endoplasmic reticulum was investigated by incubating rough microsomes with GDP‐[14C]mannose in the absence of detergent. Labeled dolichyl monophosphate mannose was readily formed from the endogenous dolichyl monophosphate. In contrast, the label was not detectable in dolichyl diphosphate oligosaccharides and it was incorporated into protein only to a small extent, except when preparations previously treated with pyrophosphate were incubated in the presence of UDP‐N‐ acetylglucosamine and GTP. Then, incorporation of label into such lipid and protein derivatives was considerably enhanced. GTP could not be substituted by other nucleotides. The requirement for UDP‐N‐acetylglucosamine, and other experimental evidence, demonstrate that mannosylated oligosaccharides which contain internal N‐acetylglucosamine units are entirely assembled on dolichyl diphosphate from the exogenous nucleotide sugars. The kinetics of mannose incorporation into dolichol‐linked oligosaccharides and into protein suggest that the protein acceptors are mainly mannosylated through transfer of oligosaccharides preassembled on dolichyl diphosphate. In heavy microsomes which have not been treated with PPi, or upon incubation in the absence of GTP, the amount of dolichyl phosphate which can accept N‐acetylglucosamine 1‐phosphate is less than that which can accept mannose. However, when PPi‐treated preparations are incubated with UDP‐N‐acetylglucosamine and GDP‐mannose in the presence of GTP, a larger amount of dolichyl phosphate is mobilized in the form of various saccharide derivatives of dolichyl diphosphate, and the amount of dolichyl monophosphate mannose is correspondingly decreased. It seems, therefore, that the membranes contain a particular pool of dolichyl monophosphate, which readily forms dolichyl monophosphate mannose, but reacts with UDP‐N‐acetylglucosamine only under appropriate conditions. This pool could act as precursor of the glycoside derivatives of dolichyl diphosphate which transfer their saccharide moiety to proteins.

Polyprenyl phosphates as coenzymes in protein and oligosaccharide glycosylation

Partly saturated polyprenols of the dolichol type occur in all branches of the plant kingdom although in green plants they are quantitatively minor compared with the shorter polyprenols of the ficaprenol and betulaprenol type. Nevertheless, in all members of the plant kingdom so far studied it is the dolichyl phosphates that function in glycosyl transfer. In yeasts, dolichyl phosphate mannose is primarily an intermediate in the O-mannosylation of some membrane-bound proteins. Further mannosylation of the mannoprotein does not involve dolichyl phosphate. Yeast membrane preparations also catalyse the sequential transfer from the appropriate nucleoside diphosphate sugar of N -acetylglucosaminyl phosphate, N -acetylglucosamine, several mannose residues and several glucose residues to dolichyl monophosphate to form a dolichyl diphosphate oligosaccharide. The oligosaccharide is then transferred en bloc to protein to form an N-glycosidically linked glycoprotein. The transfer of mannose and glucose to the dolichyl diphosphate oligosaccharide is probably via dolichyl monophosphate sugar derivatives. Tunicamycin inhibits specifically the transfer of JV-acetylglucosamine phosphate which in turn blocks protein N-glycosylation. Evidence for corresponding processes in O- and N-glycosylation of proteins of hyphal fungi is also available but much less well established. The N-glycosylation of proteins by membrane preparations of green plants appears to occur by a process very similar to that found in yeast. In addition, the biosynthesis of β1-3 and β1-4 linked oligoglucans and also of an algal cellulose primer (a glucoprotein) via dolichyl phosphate glucose has been reported. Possible consequences of these phenomena are discussed.

Action of glycosidases on the saccharide moiety of the glucose—containing dolichyl diphosphate oligosaccharide

Action of glycosidases on the saccharide moiety of the glucose—containing dolichyl diphosphate oligosaccharide

Oligosaccharides containing glucose and mannose in glycoproteins of the thyroid gland.

The glucose-containing oligosaccharides formed by calf thyroid slices incubated with radioactive glucose were studied. A compound soluble in chloroform/methanol/water, 1:1:0.3 (vol/vol), was found that was indistinguishable from the previously described glucose-containing dolichyl diphosphate oligosaccharide formed by liver microsomes. Glycopeptides were prepared by treating the glycoproteins with pronase, the amino acids were removed with alkaline borohydride, and the products were examined by paper electrophoresis and chromatography. A saccharide equal to that which occurs in the glucose-containing dolichyl diphosphate oligosaccharide could not be detected but glucose was found in oligosaccharides that seemed to be smaller by about three to five monosaccharide residues. The same results were obtained by direct treatment of the glycoproteins with alkaline borohydride.