Biosynthesis of yeast mannoproteins. Synthesis of mannan outer chain and of dolichol derivatives.

The Saccharomyces cerevisiae mutants affected in the structure of mannan outer chain were found to synthesize dolichol diphosphate-linked oligosaccharides identical in size to those of the wild type strain. The mannosyl transferases involved in the synthesis of the outer chain had an absolute requirement for manganese ions and were activated when enzymatic preparations were stored at 2 degrees C, whereas the transferases responsible for the formation of dolichol monophosphate mannose and dolichol diphosphate oligosaccharides were drastically inactivated from the onset of storage and required magnesium or manganese ions, the former being more effective than the latter. Both sets of enzymes could be separated by ion exchange chromatography. In vitro conditions that enhanced the synthesis of dolichol monophosphate mannose did not stimulate the incorporation of mannose residues into the outer chain. It is concluded that dolichol monophosphate mannose is not an intermediate in the synthesis of the outer chain and that this part of mannan and the dolichol diphosphate oligosaccharides are synthesized by different mannosyltransferases.     

In vivo biosynthesis of the saturated isoprene unit of dolichyl phosphate

Reduction of the e-isoprene unit of polyprenols to form dolichols was studied in vivo using³H-polyprenol derivatives as substrates and liposomes as carriers. Liposomes containing labeled polyprenol, polyprenyl phosphate, or polyprenyl pyrophosphate were injected through the portal vein into the livers of rats under anesthesia. Uptake and conversion of the labeled compounds to dolichol derivatives was studied at different intervals. The greatest conversion to dolichol derivatives was found with polyprenyl pyrophosphate and polyprenyl monophosphate, with 31% and 8% of the absorbed dose converted respectively. Less than 0.2% of the absorbed polyprenol was converted to dolichol derivatives. These results suggest that the substrate for the -isoprene reductase involved in dolichol biosynthesis is either polyprenyl monophosphate or polyprenyl pyrophosphate, or both.     

The effects of triton X-100 on the transfer of mannose, glucose and n-acetylglusomine phosphate to dolichol monophosphate by preparations of rough and smooth endoplasmic reticulum and of mitochondria of rat liver.

Triton X-100 and exogenous dolichol monophosphate have been used to investigate the nature of enzymes responsible for the transfer of mannose, glucose and N-acetylglucosamine phosphate from nucleotide donors to dolichol monophosphate in vesicles derived from rough and smooth endoplasmic reticulum and mitochondria. Mitochondria were shown to contain the highest specific activities of these enzymes. The responses of the glycosyltransferases to increasing concentrations of Triton X-100 and the effect on these responses of exogenous dolichol monophosphate suggest that the enzymes for mannose and glucose transfer are less hydrophobic, and therefore less intrinsic, in the membrane than the enzyme for N-acetylglucosamine phosphate transfer. In smooth vesicles the results are consistent with mannosyl- and glucosyl-transferases being located at both inner and outer faces of the membrane. In rough vesicles and in mitochondria mannosyl- and glucosyl-transferases were confirmed at the outer face. There is, however, only one site of N-acetylglucosamine phosphate transfer, this being more hydrophobically located in the membrane than the other sites of glycosyl transfer. Mitochondrial enzyme activity closely resembled that of rough endoplasmic reticulum in response to Triton X-100 and exogenous dolichol monophosphate, and is probably associated with the outer membrane.     

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.     

Formation of lipid-bound oligosaccharides in yeast

Incubation of a membrane fraction from Saccharomyces cerevisiae with UDP-N-acetyl [¹⁴C] glucosamine catalyzes the tranfer of N-acetylglucosamine to an endeenous lipid fraction as well as a methanol-insoluble polymer. The glycolipid was shown to separate into three compounds by thin-layer chromatography. The biosynthesis of two of them could clearly be stimulated by the addition of dolichol monophosphate to the incubation mixture. Evidence is presented that the substances are dolichol pyrophosphate derivatives: dolichol pyrophosphate N-acetylglucosamine and dolichol pyrophosphate di-N-acetylchitobiose. The formation of the chitobiose-containing lipid was increased by reincubation of the glycolipid with non-radioactive UDP-N-acetylglucosamine.The same particulate preparation transferred mannose from GDPmannose to dolichol pyrophosphate di-N-acetylchitobiose, giving rise to a lipid-bound oligosaccharide. Molecular weight determination of the oligosaccharide moiety gave a value of 780, which is consistent with a tetrasaccharide containing two mannose subunits attached to di-N-acetylchitobiose.The methanol-insoluble radioactive product obtained in the presence of UDP-N-acetyl[¹⁴C]glucosamine was transformed by pronase treatment to a large extent into dialyzable material. It is suggested that the glycolipids described serve as intermediates in the glycosylation of yeast mannoproteins.     

Polyisoprenoid glycolipids involved in glycoprotein biosynthesis

Summary Until five years ago, it was believed that the oligosaccharide chains of most, if not all, glycoproteins were assembled by the stepwise transfer of single sugar residues from their nucleotide derivatives to growing oligosaccharide chains attached to a polypeptide core. It is now becoming widely accepted that polyisoprenol-linked mono- and oligosaccarides function as activated glycosyl carriers in the biosynthesis of some glycoproteins in animal tissues. The lipophilic glycosyl carrier of monosaccharides is the phosphomonoester of dolichol, the C_80-100-polyisoprenol, containing a saturated terminal isoprene unit. In this biosynthetic process, sugars are initially transferred to dolichol monophosphate from their nucleotide derivatives by membrane-associated glycosyltransferases. These dolichol-linked monosaccharides serve as glycosyl donors in the glycosylation of oligosaccharide phospholipids. It appears likely that dolichol is also the lipid moiety of the oligosaccharide intermediates. Detailed enzymatic studies with oligosaccharide phospholipids formed by rat liver, a mouse myeloma tumor and hen oviduct have revealed that these intermediates function as oligosaccharide donors in the assembly of at least one class of glycoproteins.The exact nature of the glycoproteins glycosylated by lipid intermediates and the sub-cellular site(s) of this assembly process remain to be established. The possibility, that the mannose and GlcNAc-containing core found in many glycoproteins, is assembled at the lipid-level is now being investigated.At the current rate of progress in this area of research, the identity of the glycoproteins glycosylatedvia lipid intermediates and the subcellular site of this assembly process will soon be known.An invited article.     

Glycosyl transfer to an acceptor lipid from insects.

Insect extracts were found to contain a lipid which becomes glycosylated when incubated with uridine diphosphate glucose or uridine diphosphate N-acetylglucosamine and microsomal enzymes of rat liver. The behaviour of the lipid on column or thin-layer chromatography and its stability to acid were equal to those of dolichol monophosphate. The glycosylated compounds were acid labile. Treatment with alkali of the acetylglucosaminyl compound produced a substance that migrated like a hexose phosphate on electrophoresis and that liberated acetylglucosamine on treatment with alkaline phosphatase. The behaviour of the insect glucosylated lipid on thin-layer chromatography and its stability to phenol were similar to dolichol monophosphate glucose and different from ficaprenyl monophosphate glucose. It is concluded that the insect glycosyl acceptor lipid is an α saturated polyprenyl phosphate.     

Enzymatic synthesis of polyprenol monophosphate mannose in insects

Summary The microsomal fraction of insects was found to contain an enzyme which transfers mannose from guanosine diphosphate mannose to an endogenous or exogenous insect lipid and to other acceptors such as dolichol monophosphate or ficaprenol monophosphate. This activity depended on the presence of Triton X-100 and magnesium ions, the optimal concentration of the latter being 10mM. The optimal temperature of the reaction was 25 °C and the maximal activity was obtained at pH 7.9. The mannolipid formed behaved as a monophosphodiester when chromatographed on DEAE-cellulose. Weak acid treatment of the product liberated mannose. Its behaviour both on thin layer and Sephadex G-150 chromatography would indicate the presence of a number of isoprenyl units similar to the dolichol and different from the ficaprenol derivative. Stability to phenol treatment indicated that the lipid fraction of the mannolipid is an±-saturated polyprenol phosphate similar to dolichol monophosphate.Abbreviations DoIMP dolichol monophosphate - FMP ficaprenol monophosphate - IGAL insect glycosyl acceptor lipid Dedicated to ProfessorLuis F. Leloir on the occasion of his 70th birthday.     

Evaluation of the anti-adhesive effect of milk fat globule membrane glycoproteins on Helicobacter pylori in the human NCI-N87 cell line and C57BL/6 mouse model

Background: The interest in non‐antibiotic therapies for Helicobacter pylori infections in man has considerably grown because increasing numbers of antibiotic‐resistant strains are being reported. Intervention at the stage of bacterial attachment to the gastric mucosa could be an approach to improve the control/eradication rate of this infection. Materials and Methods: Fractions of purified milk fat globule membrane glycoproteins were tested in vitro for their cytotoxic and direct antibacterial effect. The anti‐adhesive effect on H. pylori was determined first in a cell model using the mucus‐producing gastric epithelial cell line NCI‐N87 and next in the C57BL/6 mouse model after dosing at 400 mg/kg protein once or twice daily from day ?2 to day 4 post‐infection. Bacterial loads were determined by using quantitative real‐time PCR and the standard plate count method. Results: The milk fat globule membrane fractions did not show in vitro cytotoxicity, and a marginal antibacterial effect was demonstrated for defatted milk fat globule membrane at 256 μg/mL. In the anti‐adhesion assay, the results varied from 56.0 ± 5.3% inhibition for 0.3% crude milk fat globule membrane to 79.3 ± 3.5% for defatted milk fat globule membrane. Quite surprisingly, in vivo administration of the same milk fat globule membrane fractions did not confirm the anti‐adhesive effects and even caused an increase in bacterial load in the stomach. Conclusions: The promising anti‐adhesion in vitro results could not be confirmed in the mouse model, even after the highest attainable exposure. It is concluded that raw or defatted milk fat globule membrane fractions do not have any prophylactic or therapeutic potential against Helicobacter infection.     

Characterisation of the surface of bovine milk fat globule membrane using microelectrophoresis

The nature of the ionogenic groups on the surface of the milk fat globule membrane was studied by microelectrophoresis of intact fat globules after chemical and enzymic modification. The changes in pH-mobility curves effected by formaldehyde and 2,4-dinitrofluorobenzene showed that the membrane surface contained amine groups. These were identified as arising from lysine and arginine by chromatography of their dinitrophenyl derivatives. The contribution of N-acetylneuraminic acid and phosphate to the surface charge was demonstrated by their specific removal by neuraminidase and phospholipase C, respectively. After removal of N-acetylneuraminic acid and phosphate, anionogenic effects remained which were attributed to protein carboxyl groups. These groups could be partially esterified using diazomethane. The effect of sodium dodecyl sulphate and of ionic strength on electrophoretic mobility indicated that the surface contains little neutral lipid and is predominantly ionogenic. The results obtained concerning the nature of the surface of the milk fat globule membrane support the hypothesis that the milk fat globule membrane originates from the plasmalemma of the mammary alveolar cell.     

The role of dolichol monophosphate in sugar transfer

The specificity of the transfer of monosaccharides from sugar nucleotides to dolichol monophosphate catalyzed by liver microsomes was studied. Besides uridine diphosphate glucose, uridine diphosphate-N-acetylglucosamine and guanosine diphosphate mannose were found to act as donors for the formation of the respective dolichol monophosphate sugars. Uridine diphosphate galactose and uridine diphosphate-N-acetylgalactosamine gave negative results.     

Biosynthesis of Glycoproteins in the Human Pathogenic Fungus Sporothrix Schenckii: Synthesis of Dolichol Phosphate Mannose and Mannoproteins by Membrane-Bound and Solubilized Mannosyl Transferases

A membrane fraction obtained from the filamentous form of Sporothrix schenckii was able to transfer mannose from GDP-Mannose into dolichol phosphate mannose and from this inTermediate into mannoproteins in coupled reactions catalyzed by dolichol phosphate mannose synthase and protein mannosyl transferase(s), respectively. Although the transfer reaction depended on exogenous dolichol monophosphate, membranes failed to use exogenous dolichol phosphate mannose for protein mannosylation to a substantial extent. Over 95% of the sugar was transferred to proteins via dolichol phosphate mannose and the reaction was stimulated several fold by Mg²⁺ and Mn²⁺. Incubation of membranes with detergents such as Brij 35 and Lubrol PX released soluble fractions that transferred the sugar from GDP-Mannose mostly into mannoproteins, which were separated by affinity chromatography on Concanavilin A–Sepharose 4B into lectin-reacting and non-reacting fractions. All proteins mannosylated in vitro eluted with the lectin-reacting proteins and analytical electrophoresis of this fraction revealed the presence of at least nine putative mannoproteins with molecular masses in the range of 26–112 kDa. The experimental approach described here can be used to identify and isolate specific glycoproteins mannosylated in vitro in studies of O-glycosylation.     

Subcellular sites of enzymes catalyzing the phosphorylation-dephosphorylation of dolichol in the central nervous system

The subcellular locations of several enzymes involved in dolichyl monophosphate (Dol-P) metabolism in brain have been investigated. Dolichol kinase is highly enriched in a heavy microsomal fraction from calf brain, while 71% of the Dol-P phosphatase activity was recovered with the light microsomes. Lower amounts of the phosphatase activity were also found in the heavy microsomal, mitochondrial-lysosomal, and synaptic plasma membrane fractions. Since the light microsomal fraction also contained substantial acetylcholinesterase activity, an axon plasma membrane marker, an axolemma-enriched fraction, was prepared from rat brain by a second procedure. A comparison with microsomal and mitochondrial-lysosomal fractions revealed that the axolemma-enriched fraction contained the highest specific activity of Dol-P phosphatase, indicating that the enzyme was present in the axon plasma membrane. The tunicamycin-sensitive UDP-N-acetylglucosamine:Dol-P N- acetylglucosaminylphosphotransferase , glucosyl- phosphoryldolichol (Glc-P-Dol) synthase, Glc-P-Dol:oligosaccharide glucosyltransferase, and the oligosaccharyltransferase were all found predominantly in the heavy microsomes. These results indicate that the enzymes responsible for the initiation and termination of biosynthesis, as well as the transfer of dolichol-linked oligosaccharides, reside in the rough endoplasmic reticulum (ER) of central nervous tissue. Evidence that at least some Dol-P molecules formed by dolichol kinase are accessible to multiple glycosyltransferases in the rough ER of brain is also presented.     

Intramembranous arrangement of the glycosylating systems in rough and smooth microsomes from rat liver

The distribution of mannosyl-, glucosaminyl- and glucosyltransferases in rough and smooth microsomes isolated from rat liver homogenate has been investigated. Amphomycin and tunicamycin were used as inhibitors of dolichol-mediated glycosylation, and diazobenzene sulfonate and proteolytic enzymes were used as nonpenetrating surface probes. Under in vitro conditions only 20-30% of the proteins glycosylated are of the secretory type. Nonpenetrating surface probes, which interact with components on the outer surface of rough microsomal vesicles, decrease glycosylation of both secretory and membrane proteins to a great extent. Inhibitor sensitive glycosylation is present in both the outer and inner compartments of the microsomal membranes. In contrast, the surface probes and the inhibitors of dolichol-mediated glycosylation do not significantly affect protein glycosylation in smooth microsomes. When dolichol phosphate sugars were used as substrates, instead of nucleotide sugars, the probes used inhibited protein glycosylation in both subfractions. Glycosylation of externally added Lipidex-bound dolichol monophosphate and of ovalbumin were in agreement with the above results. It appears that both rough and smooth microsomes may possess several types of glycosylating pathways. The most prominent of these in rough microsomes under the conditions used is the dolichol mono- and pyrophosphate-mediated glycosylation of endogenous proteins, where the enzymes involved in the initial steps are distributed at the outer surfaces of the microsomal vesicles. The dominating pathway in smooth microsomes appears to function in completion of the oligosaccharide chain of the protein and this process does not involve lipid intermediates and cannot be influenced by nonpenetrating surface probes.     

Glycosylation of myelin glycoproteins in peripheral nerve via lipid intermediates

Membrane preparations from chick peripheral nervous system (PNS) catalyzed the transfer of [³H]glucose from UDP-[³H]glucose into glucosylphosphoryl dolichol. The initial rate of glucosylphosphoryl dolichol formation in a non-myelin membrane fraction from actively myelinating chick PNS was 11 fold higher than that from adult. Exogenous dolichyl monophosphate stimulated glucosylphosphoryl dolichol synthesis in both fractions. The higher level of glucosylphosphoryl dolichol synthesis corresponded to the onset of myelination in chick PNS. Exogenous dolichyl monophosphate also stimulated the labeling of glucosylated oligosaccharide lipids and glycoproteins in the fraction. On SDS polyacrylamide gel electrophoresis, the relative mobility of the major and minor radioactive glycoprotein corresponded with that of the P0 and PASII glycoprotein in PNS myelin, respectively. The results suggest that myelin glycoproteins in PNS are glycosylated via lipid intermediates.     

Enzymic characteristics of fat globule membranes from bovine colostrum and bovine milk

Fat globule membranes have been isolated from bovine colostrum and bovine milk by the dispersion of the fat in sucrose solutions at 4 degrees C and fractionation by centrifugation through discontinuous sucrose gradients. The morphology and enzymic characteristics of the separated fractions were examined. Fractions comprising a large proportion of the total extracted membrane were thus obtained having high levels of the Golgi marker enzymes UDP-galactose N-acetylglucosamine beta-4-galactosyltransferase and thiamine pyrophosphatase. A membrane-derived form of the galactosyltransferase has been solubilized from fat and purified to homogeneity. This enzyme is larger in molecular weight than previously studied soluble galactosyltransferases, but resembles in size the galactosyltransferase of lactating mammary Golgi membranes. In contrast, when fat globule membranes were prepared by traditional procedures, which involved washing the fat at higher temperatures, before extraction, galactosyltransferase was not present in the membranes, having been released into supernatant fractions, When the enzyme released by this procedure was partially purified and examined by gel filtration, it was found to be of a degraded form resembling in size the soluble galactosyltransferase of milk. The release is therefore attributed to the action of proteolytic enzymes. Our observations contrast with previous biochemical studies which suggested that Golgi membranes do not contribute to the milk fat globule membrane. They are, however, consistent with electron microscope studies of the fat secretion process, which indicate that secretory vesicle membranes, derived from the Golgi apparatus, may provide a large proportion of the fat globule membrane.     

Topological investigations. Study of the trypsin sensitivity of the N-acetylglucosaminyl and mannosyl-transferase activities located in the outer mitochondrial membrane

The trypsin sensitivity of the mitochondrial N-acetylglucosaminyl and mannosyltransferase activities involved in the N-glycoprotein biosynthesis through dolichol intermediates as well as the N-acetylglucosaminyl-transferase activity involved in direct N-glycosylation were examined in mitochondria and isolated outer mitochondrial membrane preparations. The trypsin action on mitochondrial membrane was checked by measuring the activities of marker enzymes (rotenone-insensitive NADH cytochrome c reductase, adenylate kinase, and monoamine oxidase). Glycosyl-transferase activities of both N-glycosylation pathways were insensitive to trypsin action and consequently were located in the outer mitochondrial membrane. Based on the activator effect of the trypsin on these enzyme activities, the results suggested two distinct orientations of their active sites. As regards the N-glycoprotein biosynthesis pathway through dolichol intermediates, the dolicholphosphoryl-mannose and dolichol-pyrophosphoryl-di-N-acetylchitobiose synthases would be oriented outside while the oligomannosyl-synthase and the oligomannosyl-transferase would be rather oriented inside in the outer membrane. The N-acetylglucosaminyl-transferase involved in the direct transfer of N-acetylglucosamine from its nucleotide donor to a proteinic acceptor would be oriented outside in the outer membrane.     

Transfert du galactose dans les membranes des globules lipidiques du lait humain

Galactose transfer in the membranes of human milk fat globulesGalactosyltransferase which catalyzes the transfer from UDP-galactose to either endogeneous glycoproteins, free N-actylglucosamine or N-acetylglucosaminyl residues in the carbohydrate portion of glycoproteins, or to glucose when α-lactalbumin is added, occurs in human milk fat globule membranes. Various treatments (washing of membranes, freezing and thawing) did not affect this activity. In the presence of Triton X-100, the enzyme shows appreciable latency. This detergent was then used to solubilize the enzyme and to study its main characteristics. A competition and a heat stability experiment show that only one enzyme acts on two substrates (free N-acetylglucosamine or desialyzed and degalactosylated fetuin). UDP-galactose hydrolase activities were very low compared to those of the bovine milk fat globule membranes. Other characteristic enzymes of Golgi vesicles were found in human milk fat globules membranes. It is of interest to find out whether this is the result of contamination with cytoplasmic particles or whether it reflects the participation of Golgi vesicles in human milk fat globule secretion.     

Dolichol alters dynamic and static properties of mouse synaptosomal plasma membranes

Dolichols are isoprenologues that are found in almost all tissues and whose biochemical function, aside from dolichol phosphate precursors, is not known. In addition, an understanding of the organizational and dynamic properties of dolichols in biological membranes has not been forthcoming. The purpose of the experiments reported here were to examine the effects of dolichol on the physical properties of mouse synaptic plasma membranes (SPM). Differential polarized phase fluorometry indicated that dolichol both fluidized and rigidified SPM. Membrane areas detected by diphenylhexatriene and trans-parinaric acid were selectively fluidized and rigidified, respectively. It also was found that the spin label, 5-doxyl stearic acid indicated that dolichol reduced membrane fluidity. These results report for the first time a structural effect of dolichol on a biological membrane.     

Mannosylation of glycoproteins and dolichol derivatives in fibroblasts from patients with cystic fibrosis

Increased incorporation of mannose into endogenous glycoprotein fractions has been found in whole cell lysates and crude membrane preparations of cultured skin fibroblasts from patients with cystic fibrosis (1.3–2.3-times normal) when GDP[¹⁴C]mannose served as the mannosyl donor. In contrast, the incorporation of mannose from GDPmannose into lipid fractions containing dolichol phosphate and dolichol pyrophosphate oligosaccharides as well as the incorporation of mannose from dolichol phospho[³H]mannose into both glycoproteins and dolichol derivatives were not significantly different among cell preparations from patients with cystic fibrosis and normal controls. Mannosyltransferase activity toward exogenous glycoproteins as well as the activities of soluble and membranous α-mannosidase and β-mannosidase appeared to be normal and could not account for the observed differences. The altered incorporation of mannose into endogenous glycoprotein may reflect changes in glycosylation processes other than mannosylation.