Mannose-6-phosphate: a regulator of LLO destruction.

Oligosaccharyltransferase (OT) catalyzes the signature reaction of the asparagine-linked glycosylation pathway, namely, the transfer of preformed glycans from the lipid-linked oligosaccharide Glc3Man9GlcNAc2-P-P-Dolichol (G3M9Gn2-LLO) to appropriate asparaginyl residues on acceptor polypeptides. We have identified a reaction, possibly catalyzed by OT, that results in the hydrolysis or "transfer to water" of host LLOs in response to viral infection with release of a free G3M9Gn2 glycan. The loss of LLO ostensibly hinders N-glycosylation of viral polypeptides. This response is achieved by a novel stress-activated signaling pathway in which free mannose-6-phosphate (M6P) acts as a second-messenger. Here, we describe methods with permeabilized mammalian cells for activation of the M6P-regulated LLO hydrolysis, or transfer of glycan to water, in vitro.

Teaching dolichol-linked oligosaccharides more tricks with alternatives to metabolic radiolabeling.

The dolichol cycle involves synthesis of the lipid-linked oligosaccharide (LLO) Glc(3)Man(9)GlcNAc(2)-P-P-dolichol (G(3)M(9)Gn(2)-P-P-Dol), transfer of G(3)M(9)Gn(2) to asparaginyl residues of nascent endoplasmic reticulum (ER) polypeptides by oligosaccharyltransferase (OT), and recycling of the resultant Dol-P-P to Dol-P for new rounds of LLO synthesis. The importance of the dolichol cycle in secretory and membrane protein biosynthesis, ER function, and human genetic disease is now widely accepted. Elucidation of the fundamental properties of the dolichol cycle in intact cells was achieved through the use of radioactive sugar precursors, typically [(3)H]-labeled or [(14)C]-labeled d-mannose, d-galactose, or d-glucosamine. However, difficulties were encountered with cells or tissues not amenable to metabolic labeling, or in experiments influenced by isotope dilution, variable rates of LLO turnover, or special culture conditions required for the use of radioactive sugars. This article will review recently developed alternatives for LLO analysis that do not rely upon metabolic labeling with radioactive precursors, and thereby circumvent these problems. New information revealed by these methods with regard to regulation, genetic disorders, and evolution of the dolichol cycle, as well as caveats of radiolabeling techniques, will be discussed.

Potentiation of angiogenic switch in capillary endothelial cells by cAMP: A cross-talk between up-regulated LLO biosynthesis and the HSP-70 expression.

During tumor growth and invasion, the endothelial cells from a relatively quiescent endothelium start proliferating. The exact mechanism of switching to a new angiogenic phenotype is currently unknown. We have examined the role of intracellular cAMP in this process. When a non-transformed capillary endothelial cell line was treated with 2 mM 8Br-cAMP, cell proliferation was enhanced by approximately 70%. Cellular morphology indicated enhanced mitosis after 32-40 h with almost one-half of the cell population in the S phase. Bcl-2 expression and caspase-3, -8, and -9 activity remained unaffected. A significant increase in the Glc(3)Man(9)GlcNAc(2)-PP-Dol biosynthesis and turnover, Factor VIIIC N-glycosylation, and cell surface expression of N-glycans was observed in cells treated with 8Br-cAMP. Dol-P-Man synthase activity in the endoplasmic reticulum membranes also increased. A 1.4-1.6-fold increase in HSP-70 and HSP-90 expression was also observed in 8Br-cAMP treated cells. On the other hand, the expression of GRP-78/Bip was 2.3-fold higher compared to that of GRP-94 in control cells, but after 8Br-cAMP treatment for 32 h, it was reduced by 3-fold. GRP-78/Bip expression in untreated cells was 1.2-1.5-fold higher when compared with HSP-70 and HSP-90, whereas that of the GRP-94 was 1.5-1.8-fold lower. After 8Br-cAMP treatment, GRP-78/Bip expression was reduced 4.5-4.8-fold, but the GRP-94 was reduced by 1.5-1.6-fold only. Upon comparison, a 2.9-fold down-regulation of GRP-78/Bip was observed compared to GRP-94. We, therefore, conclude that a high level of Glc(3)Man(9)GlcNAc(2)-PP-Dol, resulting from 8Br-cAMP stimulation up-regulated HSP-70 expression and down-regulated that of the GRP-78/Bip, maintained adequate protein folding, and reduced endoplasmic reticulum stress. As a result capillary endothelial cell proliferation was induced.

Insulin up-regulates a Glc3Man9GlcNAc2-PP-Dol pool in capillary endothelial cells not essential for angiogenesis.

Endothelial cells line blood vessels, and their proliferation during neovascularization ( i.e., angiogenesis) is essential for a normal growth and development as well as for tumor progression and metastasis. Mechanistic details indicated that down-regulation of Glc(3)Man(9)GlcNAc(2)-PP-Dol level reduced angiogenesis and induced apoptosis in capillary endothelial cells (Martínez JA, Torres-Negrón I, Amigó LA, Banerjee DK, Cellular and Molec Biochem 45, 137-152 (1999)). Unlike in any other insulin-responsive cells, insulin reduced capillary endothelial cell proliferation by increasing the cell doubling time. But, when analyzed, the rate of lipid-linked oligosaccharide-PP-Dol (LLO) synthesis as well as its turnover ( i.e., t(1/2)) were increased in insulin treated cells. No major differences in their molecular size were observed. This corroborated with an enhanced glycosylation of Factor VIIIC, an N-linked glycoprotein (essential cofactor of the blood coagulation cascade) and a marker for the capillary endothelial cell. Increased LLO synthesis was independent of elevating either Dol-P level or Man-P-Dol synthase gene (dpm) transcription. Insulin however, enhanced 2-deoxy-glucose transport across the endothelial cell plasma membrane and caused increased secretion of Factor VIIIC, thus, supporting the existence of additional LLO pool(s), and arguing favorably that growth retardation of capillary endothelial cells by insulin turned a highly proliferative cell into a highly secretory cell.

The yeast ALG11 gene specifies addition of the terminal alpha 1,2-Man to the Man5GlcNAc2-PP-dolichol N-glycosylation intermediate formed on the cytosolic side of the endoplasmic reticulum.

The initial steps in N-linked glycosylation involve the synthesis of a lipid-linked core oligosaccharide followed by the transfer of the core glycan to nascent polypeptides in the endoplasmic reticulum (ER). Here, we describe alg11, a new yeast glycosylation mutant that is defective in the last step of the synthesis of the Man(5)GlcNAc(2)-PP-dolichol core oligosaccharide on the cytosolic face of the ER. A deletion of the ALG11 gene leads to poor growth and temperature-sensitive lethality. In an alg11 lesion, both Man(3)GlcNAc(2)-PP-dolichol and Man(4)GlcNAc(2)-PP-dolichol are translocated into the ER lumen as substrates for the Man-P-dolichol-dependent sugar transferases in this compartment. This leads to a unique family of oligosaccharide structures lacking one or both of the lower arm alpha1,2-linked Man residues. The former are elongated to mannan, whereas the latter are poor substrates for outerchain initiation by Ochlp (Nakayama, K.-I., Nakanishi-Shindo, Y., Tanaka, A., Haga-Toda, Y., and Jigami, Y. (1997) FEBS Lett. 412, 547-550) and accumulate largely as truncated biosynthetic end products. The ALG11 gene is predicted to encode a 63.1-kDa membrane protein that by indirect immunofluorescence resides in the ER. The Alg11 protein is highly conserved, with homologs in fission yeast, worms, flies, and plants. In addition to these Alg11-related proteins, Alg11p is also similar to Alg2p, a protein that regulates the addition of the third mannose to the core oligosaccharide. All of these Alg11-related proteins share a 23-amino acid sequence that is found in over 60 proteins from bacteria to man whose function is in sugar metabolism, implicating this sequence as a potential sugar nucleotide binding motif.

Expression and study of recombinant ExoM, a beta1-4 glucosyltransferase involved in succinoglycan biosynthesis in Sinorhizobium meliloti.

Here we report on the overexpression and in vitro characterization of a recombinant form of ExoM, a putative beta1-4 glucosyltransferase involved in the assembly of the octasaccharide repeating subunit of succinoglycan from Sinorhizobium meliloti. The open reading frame exoM was isolated by PCR and subcloned into the expression vector pET29b, allowing inducible expression under the control of the T7 promoter. Escherichia coli BL21(DE3)/pLysS containing exoM expressed a novel 38-kDa protein corresponding to ExoM in N-terminal fusion with the S-tag peptide. Cell fractionation studies showed that the protein is expressed in E. coli as a membrane-bound protein in agreement with the presence of a predicted C-terminal transmembrane region. E. coli membrane preparations containing ExoM were shown to be capable of transferring glucose from UDP-glucose to glycolipid extracts from an S. meliloti mutant strain which accumulates the ExoM substrate (Glcbeta1-4Glcbeta1-3Gal-pyrophosphate-polyprenol). Thin-layer chromatography of the glycosidic portion of the ExoM product showed that the oligosaccharide formed comigrates with an authentic standard. The oligosaccharide produced by the recombinant ExoM, but not the starting substrate, was sensitive to cleavage with a specific cellobiohydrolase, consistent with the formation of a beta1-4 glucosidic linkage. No evidence for the transfer of multiple glucose residues to the glycolipid substrate was observed. It was also found that ExoM does not transfer glucose to an acceptor substrate that has been hydrolyzed from the polyprenol anchor. Furthermore, neither glucose, cellobiose, nor the trisaccharide Glcbeta1-4Glcbeta1-3Glc inhibited the transferase activity, suggesting that some feature of the lipid anchor is necessary for activity.

Reduced utilization of Man5GlcNAc2-P-P-lipid in a Lec9 mutant of Chinese hamster ovary cells: analysis of the steps in oligosaccharide-lipid assembly.

Recently we reported that CHB11-1-3, a Chinese hamster ovary cell mutant defective in glycosylation of asparagine-linked proteins, is defective in the synthesis of dolichol [Quellhorst et al., 343:19-26, 1997: Arch Biochem Biophys]. CHB11-1-3 was found to be in the Lec9 complementation group, which synthesizes polyprenol rather than dolichol. In this paper, levels of various polyprenyl derivatives in CHB11-1-3 are compared to levels of the corresponding dolichyl derivatives in parental cells. CHB11-1-3 was found to maintain near normal levels of Man5GlcNAc2-P-P-polyprenol and mannosylphosphorylpolyprenol, despite reduced rates of synthesis, by utilizing those intermediates at a reduced rate. The Man5GlcNAc2 oligosaccharide attached to prenol in CHB11-1-3 cells and to dolichol in parental cells is the same structure, as determined by acetolysis. Man5GlcNAc2-P-P-polyprenol and Man5GlcNAc5-P-P-dolichol both appeared to be translocated efficiently in an in vitro reaction. Glycosylation of G protein was compared in vesicular stomatitus virus (VSV)-infected parent and mutant; although a portion of G protein was compared in vesicular stomatitus virus (VSV)-infected parent and mutant; although a portion of G protein was normally glycosylated in CHB11-1-3 cells, a large portion of G was underglycosylated, resulting in the addition of either one or no oligosaccharide to G. Addition of a single oligosaccharide occurred randomly rather than preferentially at one of the two sites.

Expression cloning of a novel suppressor of the Lec15 and Lec35 glycosylation mutations of Chinese hamster ovary cells.

Lec15 and Lec35 are recessive Chinese hamster ovary (CHO) cell glycosylation mutations characterized by inefficient synthesis and utilization, respectively, of mannose-P-dolichol (MPD). Consequently, Lec15 and Lec35 cells accumulate Man5GlcNAc2-P-P-dolichol and glucosaminyl-acylphosphatidylinositol. This report describes the cloning of a suppressor (termed SL15) of the Lec15 and Lec35 mutations from a CHO cDNA library by functional expression in Lec15 cells, employing phytohemagglutinin/swainsonine selection. The SL15 protein has a predicted molecular weight of 26,693 with two potential membrane spanning regions and a likely C-terminal endoplasmic reticulum retention signal (Lys-Lys-Glu-Gln). Lec15 cells transfected with SL15 have normal levels of MPD synthase activity in vitro and convert Man5GlcNAc2-P-P-dolichol to Glc0-3Man9GlcNAc2-P-P-dolichol in vivo. Surprisingly, SL15 also corrects the defective mannosylation in Lec35 cells. The SL15 protein bears no apparent similarity to Saccharomyces cerevisiae MPD synthase (the DPM1 protein), but is highly similar to the hypothetical F38E1.9 protein encoded on Caenorhabditis elegans chromosome 5. These results indicate a novel function for the SL15 protein and suggest that MPD synthesis is more complex than previously suspected.

Mannolipid donor specificity of glycosylphosphatidylinositol mannosyltransferase-I (GPIMT-I) determined with an assay system utilizing mutant CHO-K1 cells.

The microsomal enzyme glycosylphosphatidylinositol mannosyltransferase I (GPIMT-I) catalyses the transfer of a mannosyl residue from beta-mannosylphosphoryldolichol (beta-Man-P-Dol) to glucosamine-alpha(1,6)(acyl)phosphatidylinositol (GlcN-aPI) to form Man alpha(1,4)GlcN-aPI (ManGlcN-aPI), an intermediate in glycosylphosphatidylinositol (GPI) synthesis. While the transfer of [3H]mannosyl units to endogenous GlcN-aPI was not seen when membrane fractions from normal Chinese hamster ovary (CHO) K1 cells were incubated with exogenous [3H]Man-P-Dol, GPIMT-I activity could be characterized with an in vitro enzyme assay system employing membrane fractions from Lec15 or Lec35 cells. These CHO cell mutants apparently contain elevated levels of endogenous GlcN-aPI due to the inability to synthesize (Lec15) or utilize (Lec35) beta-Man-P-Dol in vivo. The presence of a saturated alpha-isoprene unit in the dolichyl moiety is required for optimal GPIMT-I activity since beta-mannosylphosphorylpolyprenol (beta-Man-P-Poly), which contains a fully unsaturated polyisoprenyl chain, was only 50% as effective as beta-[3H]Man-P-Dol as a mannosyl donor. When beta-[3H]-Man-P-Dol and alpha-[3H]Man-P-Dol were compared as substrates, GPIMT-I exhibited a strict stereospecificity for the mannolipid containing the beta-mannosyl-phosphoryl linkage. beta-[3H]Man-P-dolichols containing 11 or 19 isoprenyl units were equally effective substrates for GPIMT-I. Membrane fractions from Lec 9, a CHO mutant that apparently lacks polyprenol reductase activity and synthesizes very little beta-Man-P-Dol, but accumulates beta-Man-P-Poly, synthesized no detectable Man-GlcN-aPI when incubated with beta-[3H]Man-P-Dol in vitro. This indirect assay suggests that GlcN-aPI does not accumulate in Lec 9 cells, possibly because it is mannosylated via beta-Man-P-Poly, or perhaps the small amount of Man-P-Dol formed by the mutant in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Manipulation of the biosynthesis of protein-modifying glycoconjugates by the use of specific inhibitors.

Since it is possible to interfere with different steps in the dolichol pathway of protein glycosylation and in the processing of N-linked oligosaccharides information can be deduced as to the role of protein-bound carbohydrate by comparing the biochemical fates and functions of glycosylated proteins with their non-glycosylated counterparts, or with proteins exhibiting differences in the type of oligosaccharide side-chains. Cells infected with enveloped viruses are excellent model systems for studying the inhibition of protein glycosylation, since they contain a restricted number of glycoproteins, in some cases with well-defined functions. Tunicamycin, an antibiotic, as well as several sugar analogues have been found to inhibit the glycosylation of proteins by virtue of their antiviral properties. They interfere with different steps in the dolichol pathway of N-glycosylation resulting in a lack of functional lipid-linked oligosaccharide precursors. Trimming inhibitors, a second generation of inhibitors of glycosylation, interfere with the processing of oligosaccharides by specific glucosidases and mannosidases, resulting in a block of the conversion of high-mannose to complex-type oligosaccharides. Depending upon the compound used, glycoproteins contain glucosylated-high-mannose, high-mannose, or hybrid oligosaccharide structures instead of complex ones. Various effects of glycosylation inhibitors have been described: susceptibility to proteases, improper protein processing and misfolding of polypeptide chains, loss of biological activity, and alteration of the site of virus-budding. A third generation of inhibitors of glycosylation is emerging which are designed to interfere with late steps of N-glycosylation occurring after trimming, to bring about changes in the makeup of complex carbohydrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Control of N-linked oligosaccharide synthesis: cellular levels of dolichyl phosphate are not the only regulatory factor.

When MDCK cells were incubated in the presence of the protein synthesis inhibitor puromycin or cycloheximide, there was a rapid and concentration-dependent inhibition in the incorporation of [2-3H]mannose into lipid-linked oligosaccharide and into protein. However, mannose incorporation into dolichyl-P-mannose was not affected. Interestingly, these inhibitors did block [6-3H]glucosamine incorporation into dolichyl-PP-GlcNAc as well as into lipid-linked oligosaccharides. Similar results were obtained when other cell lines were used and also when inhibitors of protein glycosylation such as beta-hydroxynorvaline and beta-fluoroasparagine were used. Cells incubated in puromycin did not show any changes in the levels of sugar nucleotides, GDP-mannose or UDP-GlcNAc, or in the in vitro activities of the glycosyltransferases that add mannose to the lipid-linked oligosaccharides. The inhibition of mannose incorporation into lipid-linked oligosaccharides could not be overcome by addition of dolichyl-P to the inhibited cells, even though the addition of dolichyl-P to control cells stimulated mannose incorporation into dolichyl-P-mannose, lipid-linked oligosaccharides, and protein from 3- to 5-fold. Thus, limitations in the levels of dolichyl-P do not appear to be a major factor in this inhibition. On the other hand, addition of the tripeptide acceptor N-acyl-Asn-Try-Thr did overcome the puromycin inhibition to some extent, suggesting that accumulation of some intermediate such as lipid-linked oligosaccharides might be involved in the inhibition.

Evidence that the synthesis of glucosylphosphodolichol in yeast involves a 35-kDa membrane protein.

In an effort to identify the polypeptide chain of glucosylphosphodolichol synthase (EC 2.4.1.117), yeast microsomal membranes were allowed to react with 5-azido[beta-32P]UDPGlc, a photoactive analogue of UDPGlc, which is a substrate for this enzyme. Upon photolysis the 32P-labeled probe was shown to link covalently to a 35-kDa protein present in microsomal membranes prepared from several wild-type yeast strains. Binding was either reduced or absent in the microsomal membranes from two yeast mutants (alg5 and dpg1) that are known to be defective in the synthesis of glucosylphosphodolichol. The microsomes isolated from a heterozygous diploid strain alg5::dpg1 generated from these two mutants exhibited partial restoration of both the ability to photolabel the 35-kDa protein and the ability to catalyze the synthesis of glucosylphosphodolichol. Microsomal membranes from a mutant strain that synthesized glucosylphosphodolichol but lacked the ability to transfer the glucosyl residue to the growing lipid-linked oligosaccharide (alg6) exhibited labeling with 5-azido[beta-32P]UDPGlc comparable to that found in microsomes from the wild-type strain. In all cases photoinsertion of the probe into the 35-kDa protein correlated with the level of synthase assayed in the microsomal membranes. These results strongly support the conclusion that the 35-kDa protein labeled in these experiments is a component of glucosylphosphodolichol synthase.

Polyprenyl phosphates: synthesis and structure-activity relationship for a biosynthetic system of Salmonella anatum O-specific polysaccharide.

A series of polyprenyl phosphates with modified structure of polyprenyl residue was prepared through phosphorylation of polyprenyl trichloroacetimidates with phosphoric acid. Interaction of polyprenols with tetra-n-butylammonium dihydrogen phosphate and trichloroacetonitrile was found to represent a very efficient, simple and general method for the synthesis of polyprenyl phosphates. A procedure was developed for smooth conversion of polyprenyl pyrophosphates into the monophosphates through hydrolysis in the presence of 4-dimethylaminopyridine. The polyprenyl phosphates prepared were studied as substrates for the enzymes of Salmonella anatum O-specific polysaccharide biosynthesis. Correct stereochemistry of alpha- and beta-isoprenic units was found to be essential for substrate efficiency. At the more remote positions of the hydrocarbon chain just the presence of isoprenic units of any configuration seems necessary. Some changes in position of the phosphate group may be permissible without significant loss of substrate properties.

Biosynthesis of linkage units for teichoic acids in gram-positive bacteria: distribution of related enzymes and their specificities for UDP-sugars and lipid-linked intermediates.

The distribution and substrate specificities of enzymes involved in the formation of linkage units which contain N-acetylglucosamine (GlcNAc) and N-acetylmannosamine (ManNAc) or glucose and join teichoic acid chains to peptidoglycan were studied among membrane systems obtained from the following two groups of gram-positive bacteria: group A, including Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Staphylococcus aureus, and Lactobacillus plantarum; group B, Bacillus coagulans. All the membrane preparations tested catalyzed the synthesis of N-acetylglucosaminyl pyrophosphorylpolyprenol (GlcNAc-PP-polyprenol). The enzymes transferring glycosyl residues to GlcNAc-PP-polyprenol were specific to either UDP-ManNAc (group A strains) or UDP-glucose (group B strains). In the synthesis of the disaccharide-bound lipids, GlcNAc-PP-dolichol could substitute for GlcNAc-PP-undecaprenol. ManNAc-GlcNAc-PP-undecaprenol, ManNAc-GlcNAc-PP-dolichol, Glc-GlcNAc-PP-undecaprenol, Glc-GlcNAc-PP-dolichol, and GlcNAc-GlcNAc-PP-undecaprenol were more or less efficiently converted to glycerol phosphate-containing lipid intermediates and polymers in the membrane systems of B. subtilis W23 and B. coagulans AHU 1366. However, GlcNAc-GlcNAc-PP-dolichol could not serve as an intermediate in either of these membrane systems. Further studies on the exchangeability of ManNAc-GlcNAc-PP-undecaprenol and Glc-GlcNAc-PP-undecaprenol revealed that in the membrane systems of S. aureus strains and other B. coagulans strains both disaccharide-inked lipids served almost equally as intermediates in the synthesis of polymers. In the membrane systems of other B. subtilis strains as well as B. licheniformis and B. pumilus strains, however, the replacement of ManNAc-GlcNAc-PP-undecaprenol by Glc-GlcNAc-PP-undecaprenol led to a great accumulation of (glycerol phosphate)-Glc-GlcNAc-PP-undecaprenol accompanied by a decrease in the formation of polymers.

The function of galactosyl phosphorylpolyprenol in biosynthesis of lipoteichoic acid in Bacillus coagulans.

Incubation of UDP-[14C]galactose with membranes of Bacillus coagulans led to the formation of a radioactive glycolipid, which was tentatively characterized as beta-galactosyl phosphorylpolyprenol (Gal-P-prenol) on the basis of its chromatographic behavior and data from structural analysis of its sugar 1-phosphate moiety. The sugar moiety of [14C]Gal-P-prenol was shown to be incorporated into a membrane-bound polymer, which coincided with the diacyl form of lipoteichoic acid in its chromatographic behavior on columns of Sephacryl S-300, DEAE-Sephacel and octyl-Sepharose. Hydrogen fluoride hydrolysis of the polymer afforded an alpha-galactoside identical with Gal(alpha 1----2)Gro obtained from lipoteichoic acids. The incorporation of galactose residues from [14C]Gal-P-prenol into the polymer was greatly enhanced by exogenous lipoteichoic acids, especially of the diacyl and monoacyl forms. The optimal pH and metal concentration for the Gal-P-prenol formation, respectively, were found to be 8.4 and 10 mM (MgCl2), whereas those for the transfer of galactose from this lipid intermediate to polymer were 4.5 and 16 mM (CaCl2). The above results lead to the conclusion that Gal-P-prenol serves as the direct galactosyl donor in the synthesis of lipoteichoic acids in B. coagulans.

Glycosyltransfer by pea membranes from sugar nucleotides to added prenyl phosphates.

Pea membranes supplied with GDP-[14C]mannose, UDP-N-[14C]acetylglucosamine or UDP-[14C]glucose catalyze the transfer of 14C-labeled sugars or sugar phosphates to endogenous lipid acceptors as well as to exogenously added dolichyl phosphates. Fully unsaturated polyprenyl phosphates were not used as effective acceptors by this system. Mannosyl-P-dolichol was formed most rapidly in the presence of long-chained dolichyl-P while mannosyl-PP-, glucosyl-PP- and GlcNAc-PP-dolichol were preferentially formed from relatively short-chained dolichyl phosphate acceptors. Glucosyl-PP- and mannosyl-PP-dolichol accumulated in the preparation without further metabolism, but GlcNAc-PP-dolichol was lengthened by addition of a second GlcNAc plus several [14C]mannose units to form an oligosaccharide fraction susceptible to the action of endoglycosidase H. This lipid-linked oligosaccharide could then be glycosylated in the presence of UDP-[14C]glucose to form a longer oligosaccharide. It is concluded that levels of endogenous dolichyl phosphates in pea membranes are rate-limiting for several of the key glycosyltransferases required for oligosaccharide assembly.

Stimulation of protein glycosylation in cultured hepatoma cells by polyprenyl phosphates.

The aim of this work was to selectively stimulate N-glycosylation of proteins in cultured AH 70Btc hepatoma cells. Dolichyl phosphate, alpha-dihydrodecaprenyl phosphate and solanesyl phosphate were entrapped in egg lecithin liposomes and supplied to the cells. After treatment with 0.6-15 nmol of the polyprenyl phosphates/3 ml/dish for 48 hr, the incorporations of [14C]glucosamine into N-linked saccharide chains of glycoproteins and into lipid intermediates were increased to various extents. The stimulation by alpha-dihydrodecaprenyl phosphate was most significant and 15 nmol of the lipid/dish increased the incorporation into N-linked saccharide chains of glycoproteins by about 80%. The incorporations of [14C]glucosamine into O-linked saccharide chains and of [14C]leucine into proteins were not changed significantly by alpha-dihydrodecaprenyl phosphate. When the N-linked glycopeptides prepared from the cells and those from the cells treated with the polyprenyl phosphate were compared by Sephadex G-50 column chromatography, no significant difference was observed. Analysis of reduced cellular glycoproteins by sodium dodecyl sulfate/polyacrylamide-gel electrophoresis showed that alpha-dihydrodecaprenyl phosphate increased the incorporation of [14C]glucosamine into N-linked saccharide chains of certain high-molecular-weight glycoproteins. In addition, the polyprenyl phosphate enhanced the adhesiveness of the cells to the substratum, probably as a result of the stimulation of N-glycosylation of proteins.

Transient methylation of dolichyl oligosaccharides is an obligatory step in halobacterial sulfated glycoprotein biosynthesis.

Biosynthesis of sulfated saccharides that are linked to asparagine residues in the cell surface glycoprotein of Halobacterium halobium via a glucose residue involves sulfated dolichyl-monophosphoryl oligosaccharide intermediates (Lechner, J., Wieland, F., and Sumper, M. (1985) J. Biol. Chem. 260, 860-866). During isolation and characterization of these lipid oligosaccharides we detected a group of related compounds containing additional unidentified sugar residues. Here we report that: 1) the unknown sugar residues were 3-O-methylglucose, linked peripherally to the lipid-saccharide intermediates; 2) the 3-O-methylglucose residues in the oligosaccharides occur only at the lipid-linked level but are absent at the protein-linked level; 3) cell surface glycoprotein biosynthesis in Halobacteria in vivo is drastically depressed when S-adenosylmethionine-dependent methylation is inhibited, indicating that methylation is an obligatory step during glycoprotein synthesis. We propose a mechanism for the transport of lipid oligosaccharides through the cell membrane, involving an intermediate stage in which the saccharide moieties are transiently modified with 3-O-methylglucose.

Transfer of oligosaccharide from oligosaccharide pyrophosphoryl dolichol to endogenous acceptor proteins in human breast malignant and normal tissues.

We have prepared dolichylpyrophosphoryl-[14C]-oligosaccharide (Dol-PP-oligosaccharide) from calf thyroid. Microsomal fractions from human breast tissues catalyzed the transfer of labeled oligosaccharide to endogenous acceptor proteins. Malignant tumors showed higher activity of the oligosaccharide transferring enzyme than normal tissue. With kojibiose (Kj), an inhibitor of (Glc3)-glucosidase, an increase in the radioactivity associated with glycoprotein was observed.

Dolichols and enzymatic formation of dolichyl phosphate sugars in the thymus of mice on neoplastic transformation.

The content of dolichol in thymus gland of mice increased up to four times upon malignant transformation evoked by X-ray irradiation of animals or/and in the case of spontaneous tumours as compared with normal thymus. Dolichyl phosphate mannose and dolichyl pyrophosphate N-acetylglucosamine were formed in homogenates of transformed thymus at a higher rate than in normal gland.