Abnormal synthesis of mannose 1-phosphate derived carbohydrates in carbohydrate-deficient glycoprotein syndrome type I fibroblasts with phosphomannomutase deficiency

In fibroblasts from five patients with carbohydrate-deficient glycoprotein syndrome type 1, the incorporation of [2-3H] mannose into mannose phosphates, GDP-mannose, GDP-fucose, dolichol-P-mannose, lipid- linked oligosaccharides, and glycoprotein fraction was determined. We observed a 3- to 5-fold reduction of incorporation of radioactivity into mannose 1-phosphate, GDP-mannose, GDP-fucose, dolichol-P-mannose, and nascent glycoproteins. The incorporation of radioactivity into mannose 6-phosphate was normal. The formation of lipid linked oligosaccharides was only slightly affected (</=20%), but their size was severely reduced, mostly containing five or fewer residues. As a consequence, truncated oligosaccharides were transferred to newly synthesized glycoproteins. The metabolic changes can be explained by a deficiency of phosphomannomutase activity, which was reduced to </=10% of control.      

Carbohydrate-deficient glycoprotein syndrome type 1: correction of the glycosylation defect by deprivation of glucose or supplementation of mannose

In the carbohydrate deficient glycoprotein syndrome (CDGS) type 1 glycoproteins with less and shorter N-linked oligosaccharides are synthesized due to a deficiency of phosphomannomutase. Glucose deprivation or mannose addition are shown to partially or fully correct the size of oligosaccharides incorporated into lipid linked oligosaccharides and nascent glycoproteins in skin fibroblasts from CDGS type 1 patients with a phosphomannomutase defect. The corrective effect is ascribed to regulatory mechanisms and/or metabolic pathways that bypass phosphomannomutase.     

Transport of glutamine by rat kidney brush-border membrane vesicles.

I studied the glycosylation in vivo of a viral envelope protein, the glycoprotein of vesicular stomatitis virus (VSV), by pulse labelling of virus-infected HeLa cells with 3H-labelled monosaccharides (mannose, glucosamine). Radioactivity was incorporated into the fraction of membrane-bound polyribosomes, although metabolic conversion of [3H]-mannose into amino acids was negligible. Dissociation of bound polyribosomes revealed that the radioactively co-purified with the peptidyl-tRNA. The nascent peptides were released by alkaline hydrolysis, immunoprecipitated and analysed by polyacrylamide-gel electrophoresis. It is apparent from the size distribution of the [3H]mannose-labelled nascent chains that attachment of carbohydrate starts when approximately half of the amino acid sequence of the viral glycoprotein has been synthesized.     

Metformin-stimulated mannose transport in dermal fibroblasts

The biguanide drug metformin stimulates AMP-activated protein kinase, a master regulator of cellular energy metabolism, and has antihyperglycemic activity due to attenuation of gluconeogenesis in hepatocytes and 2-fold stimulation of glucose transport by skeletal muscle. Here we identify a metformin-stimulated d-mannose transport (MSMT) activity in dermal fibroblasts. MSMT increased mannose uptake 1.8-fold and had greater affinity for mannose than basal mannose transport activity. It was attributed to robust stimulation of a transporter expressed weakly in untreated cells. MSMT was not explained by greater glucose transporter activity because metformin unexpectedly decreased transport of 2-deoxy-d-glucose and 3-O-methyl-d-glucose by fibroblasts. Effective inhibitors of MSMT retained specificity for the 3-, 4-, and 6-OH groups of the mannose ring but not the 2-OH group. Thus, MSMT could be strongly inhibited by glucose and 2-deoxy-d-glucose even though the latter was not a good transport substrate. MSMT was significant because in the presence of 2.5 microm mannose, metformin corrected experimentally induced deficiencies in the synthesis of glucose(3)mannose(9)GlcNAc(2)-P-P-dolichol and N-linked glycosylation. MSMT was also identified in congenital disorder of glycosylation types Ia and Ib fibroblasts, and metformin acted synergistically with 100 microm mannose to correct lipid-linked oligosaccharide synthesis and N-glycosylation in the Ia cells. In conclusion, metformin activates a novel fibroblast mannose-selective transport system. This suggests that AMP-activated protein kinase may be a regulator of mannose metabolism and implies a therapy for congenital disorders of glycosylation-Ia.     

Oligosaccharide Chains of Avian RNA Tumor Virus Glycoproteins Contain Heterogeneous Oligomannosyl Cores

Chicken embryo fibroblasts (C/E phenotype) infected with subgroups B and C of the Prague strain of Rous sarcoma virus were radiolabeled with either [6-(3)H]-glucosamine or [2-(3)H]mannose, and virus was purified from the growth medium. The large envelope glycoprotein, gp85, was the only major radiolabeled component of purified virus. Pronase-digested glycopeptides from purified virus were analyzed by a combination of (i) gel filtration with columns of Sephadex G15/G50 and Bio-Gel P4 and (ii) enzymatic digestion of the oligosaccharide chains with specific exoglycosidases and endo-beta-N-acetylglucosaminidases. The rather broad molecular weight distribution (approximately 2,000 to 4,000) for glycopeptides in these studies and previous studies in other laboratories was shown to represent actual heterogeneity in the carbohydrate moieties: (i) the glycopeptides contained both mannose-rich, neutral chains and complex, acidic chains with terminal sialic acid; and (ii) both classes of asparagine-linked carbohydrate structures exhibited heterogeneity in the size of the oligomannosyl core (a mixture of approximately 5 to 9 mannose units for the neutral structures, and 3 or 5 mannose units for the acidic structures). With the [2-(3)H]mannose-labeled glycopeptides from Rous sarcoma virus, Prague strain subgroup C, most of the oligosaccharide chains were high-molecular-weight, acidic structures, with similar numbers of 3-mannose and 5-mannose core structures.     

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.     

Effect of swainsonine on the processing of the asparagine-linked carbohydrate chains of alpha 1-antitrypsin in rat hepatocytes. Evidence for the formation of hybrid oligosaccharides

The biosynthesis of the proteinase inhibitor alpha 1-antitrypsin has been studied in rat hepatocyte primary cultures. Newly synthesized alpha 1-antitrypsin was found in hepatocytes as a glycoprotein of an apparent molecular weight of 49,000 carrying oligosaccharide side chains of the high mannose type. In the hepatocyte medium a secreted alpha 1-antitrypsin of an apparent molecular weight of 54,000 could be identified as a glycoprotein with carbohydrate chains of the complex type. Pulse-chase experiments revealed a precursor-product relationship for the two forms of alpha 1-antitrypsin. When the hepatocytes were treated with swainsonine, an intracellular form of alpha 1-antitrypsin with an apparent molecular weight of 49,000 indistinguishable from that of control cells was found. However, the alpha 1-antitrypsin secreted from swainsonine-treated hepatocytes was different from that present in control media. It was characterized by a lower apparent molecular weight (51,000), a higher amount of [3H]mannose incorporation, half as much incorporation of [3H]galactose, and the same amount of [3H]fucose incorporation compared to alpha 1-antitrypsin of control media. In contrast to the 54,000 complex type alpha 1-antitrypsin from control media the 51,000 alpha 1-antitrypsin from the medium of swainsonine-treated cells was found to be susceptible to the action of endoglucosaminidase H, even when fucose was attached to the proximal GlcNAc residue. alpha 1-Antitrypsin secreted from swainsonine-treated cells combines features usually associated with either high mannose or complex type oligosaccharides and therefore represents a hybrid structure. In spite of its effect on the carbohydrate part of alpha 1-antitrypsin swainsonine did not impair the secretion of the incompletely processed glycoprotein.     

Carbohydrate-free carboxypeptidase Y is transferred into the lysosome-like yeast vacuole

Carboxypeptidase Y, localized in the lysosome-like yeast vacuole, has been metabolically labeled with [2-³H]mannose. After immunoprecipitation the carbohydrate moieties were released by treatment with endo-β-N-acetyl-glucosaminidase H and separated by paper electrophoresis. Evidence for the presence of phospho-monoester and -diester groups in the molecule has been obtained. In the latter phosphate links C-1 of mannose or of mannosyl 1,3-mannose to C-6 of a mannose residue within a larger oligomannose moiety. In the presence of tunicamycin yeast cells synthesize a carbohydrate-free carboxypeptidase Y, which could be traced after metabolic labeling with [¹⁴C]-phenylalanine. The carbohydrate-free enzyme was segregated into the vacuoles to the same extent as the intact glycoprotein.     

Studies on incorporation of mannose into rat alpha 1-acid glycoprotein: evidence for precursor forms in rough membranes

Rats were given pulse injections of D-[14C]mannose and were killed at various times up to 60 min after injection. Rough, smooth, and Golgi fractions were prepared from liver, and alpha 1-acid glycoprotein was isolated from Lubrol extracts of the fractions. The kinetics of incorporation of D-[14C]mannose into total protein, Lubrol protein, and alpha 1-acid glycoprotein showed that proteins associated with rough fractions had particularly high specific radioactivities at early times of incorporation. One explanation for the kinetic data is that glycoproteins contain a high mannose content at early times of assembly of oligosaccharide chains. This idea was confirmed in the case of alpha 1-acid glycoprotein by isolation of a high mannose containing precursor species of alpha 1-acid glycoprotein from rough fractions of liver. This species contained 56 residues of hexose (mainly mannose) compared with 35 residues of hexose (roughly equal amounts of mannose and galactose) which are found in the native protein. It is proposed that the high mannose precursor is a form of alpha 1-acid glycoprotein that exists at an early stage in assembly of the glycoprotein and which contains largely unprocessed carbohydrate chains. In addition, evidence is presented from amino acid analyses and gel electrophoresis of the high mannose precursor and another fraction from which it is formed by limited tryptic treatment, that pro-forms of alpha 1-acid glycoprotein with extensions of the polypeptide chain may also exist.     

Recent evidence for the participation of vitamin A in glycoprotein synthesis.

Recent evidence supports the concept that vitamin A plays some role in glycoprotein synthesis in a large-variety of tissues examined. Its involvement may be through participation of a retinol-linked sugar, mannosyl retinyl phosphate (MRP). Upon injection of [3H]retinol and [14C]mannose into rats, [14C, 3H]MRP could be isolated from liver and intestinal mucosa, and identified by chromatographic and hydrolytic experiments. The enzyme system that forms MRP from GDP-mannose and retinyl phosphate was located primarily in rough endoplasmic reticulum of fractionated liver cells, with some activity also in smooth membranes and Golgi apparatus. Vitamin A deficiency resulted in depressed synthesis of the rat serum glycoprotein alpha 1-macroglubin (alpha 1-MG), as shown by a decline in labeling. Analysis of the labeled alpha 1-MG from serum of normal and vitamin A-deficient rats showed this to be the result of a defect in glycosylation. The specific activity ratio (deficient:normal) of the alpha 1-MG of serum declined progressively with development of the deficiency, as a result of underglycosylation. Complete carbohydrate analysis of the alpha 1-MG of normal and deficient serum revealed a sugar loss in this glycoprotein as a result of vitamin A deficiency.     

D-mannose transport and metabolism in isolated enterocytes

D-mannose transport and metabolism has been studied in enterocytes isolated from chicken small intestine. In the presence of Na(+), the mannose taken up by the cells either remains free, is phosphorylated, is catabolized to H(2)O, or becomes part of membrane components. The mannose remaining free in the cytosol is released when the cells are transferred to an ice bath. The Na(+)-dependent D-mannose transport is electrogenic and inhibited by ouabain and dinitrophenol; its substrate specificity differs from SGLT-1 transporter. The Glut2 transporter inhibitors phloretin and cytochalasin B added following 30-min mannose uptake reduced the previously accumulated D-mannose, whereas these two agents increased the cell to external medium 3-O-methyl-glucose (3-OMG) concentration ratio. D-mannose efflux rate from preloaded D-[2-(3)H]-mannose enterocytes is Na(+)-independent. Phloretin did not affect D-mannose efflux rate, whereas it inhibited that of 3-OMG. Neither mannose uptake nor efflux rate were affected by fructose. It is concluded that part of the mannose taken up by the enterocytes is rapidly metabolized and that enterocytes have two D- mannose transport systems: one is concentrative and Na(+)-dependent and the other is Na(+)-independent and passive.     

Regulation of Myelination: Biosynthesis of the Major Myelin Glycoprotein by Schwann Cells in the Presence and Absence of Myelin Assembly

Schwann cell biosynthesis of the major myelin glycoprotein, P0, was investigated in the crush-injured adult rat sciatic nerve, where there is myelin assembly, and in the permanently transected nerve, where there is no myelin assembly. Endoneurial fractions from desheathed rat sciatic nerves distal to the crush were compared with similar fractions from the permanently transected nerves at 7, 14, 21, 28, and 35 days after injury. The Schwann cell expression of this asparagine-linked glycoprotein was evaluated after sodium dodecyl sulfate-pore gradient electrophoresis by Coomassie Blue and silver stain and by autoradiography after direct overlay of radioiodinated lectins [wheat germ agglutinin, gorse agglutinin, and concanavalin A (Con A)]. As evaluated by these parameters, the concentration of P0 after crush decreased and subsequently increased as a function of time after injury, corresponding to the events of demyelination and remyelination. After permanent transection, the P0 concentration decreased following the same time course found after crush. At subsequent time points, P0 could not be detected with Coomassie Blue stain, silver stain, or wheat germ agglutinin. Both gorse agglutinin and Con A, however, showed binding to P0. Radioactive precursor incorporation studies with [3H]fucose or [3H]-mannose into endoneurial slices at 35 days posttransection revealed active oligosaccharide processing of P0 glycoprotein by Schwann cells in this permanent transection model. Compared with other Schwann cell glycoproteins in the transected nerve, the highest level of incorporation of [3H]mannose was found in P0 which accounted for 42.7% of the incorporated label. In contrast, incorporation of [3H]mannose into endoneurial slices at 35 days after crush accounted for only 13.3% in P0. In addition, higher levels of Con A binding were observed in P0 in the transected nerve compared with the contralateral control or the crushed nerve. Both the [3H]fucose incorporation and gorse agglutinin binding to P0 in the transected nerve suggest posttranslational processing of this glycoprotein in the Golgi apparatus; however, the absence of wheat germ agglutinin binding, the high level of mannose incorporation, and the high level of binding by Con A imply that additional processing steps are required prior to its assembly into myelin.     

Influence of vitamin A status and DDT on vitamin A-dependent protein mannosylation in rat liver

Male Wistar rats of different vitamin A status (total depletion to moderate deficiency) were administered DDT (5 mg/kg/day) or vehicule (corn oil) i.p. daily for 14 days. Vitamin A-dependent protein mannosylation was measured either by in vivo incorporation of [3H]mannose into liver glycoprotein or by in vitro assay of incorporation of [14C]mannose into mannosylretinyl phosphate. Vitamin A deficiency resulted in a significantly impaired in vivo incorporation of mannose in liver glycoprotein but had no effect on the in vitro transport of mannose via retinyl phosphate. Although DDT induced an increase synthesis of liver proteins in smooth endoplasmic reticulum and caused a diminution of the hepatic vitamin A content, it did not affect vitamin A-dependent protein mannosylation.     

Mannose supplementation corrects GDP-mannose deficiency in cultured fibroblasts from some patients with Congenital Disorders of Glycosylation (CDG)

Congenital Disorders of Glycosylation (CDG) are human deficiencies in glycoprotein biosynthesis. Previous studies showed that 1 mM mannose corrects defective protein N-glycosylation in cultured fibroblasts from some CDG patients. We hypothesized that these CDG cells have limited GDP-mannose (GDP-Man) and that exogenous mannose increases the GDP-Man levels. Using a well established method to measure GDP-Man, we found that normal fibroblasts had an average of 23.5 pmol GDP-Man/10(6) cells, whereas phosphomannomutase (PMM)-deficient fibroblasts had only 2.3-2.7 pmol/10(6) cells. Adding 1 mM mannose to the culture medium increased the GDP-Man level in PMM-deficient cells to approximately 15.5 pmol/10(6) cells, but had no significant effect on GDP-Man levels in normal fibroblasts. Similarly, mannose supplementation increased GDP-Man from 4.6 pmol/10(6) cells to 24.6 pmol/10(6) cells in phosphomannose isomerase (PMI)-deficient fibroblasts. Based on the specific activity of the GDP-[(3)H]Man pool present in [2-(3)H]mannose labeled cells, mannose supplementation also partially corrected the impaired synthesis of mannosylphosphoryldolichol (Man-P-Dol) and Glc(0)(-)(3)Man(9)GlcNAc(2)-P-P-Dol. These results confirm directly that deficiencies in PMM and PMI result in lowered cellular GDP-Man levels that are corrected by the addition of mannose. In contrast to these results, GDP-Man levels in fibroblasts from a CDG-Ie patient, who is deficient in Man-P-Dol synthase, were normal and unaffected by mannose supplementation even though mannose addition was found to correct abnormal lipid intermediate synthesis in another study (Kim et al. [2000] J. Clin. Invest., 105, 191-198). The mechanism by which mannose supplementation corrects abnormal protein N-glycosylation in Man-P-Dol synthase deficient cells is unknown, but this observation suggests that the regulation of Man-P-Dol synthesis and utilization may be more complex than is currently understood.     

Synthesis of cellular and extracellular glycoproteins by cultured human keratinocytes and their response to retinoids

The glycoproteins synthesized by human keratinocytes cultured on 3T3 feeder layers were studied by metabolic labelling. Keratinocytes freed of feeder cells synthesized a complex pattern of cellular and extracellular glycoproteins that was distinct from that of 3T3 cells, dermal fibroblasts and epidermal melanocytes. The effect of low concentrations of all-trans-retinoic acid and arotinoid ethyl ester on glycoprotein synthesis was examined in keratinocyte cultures depleted of vitamin A. Treatment with either retinoid resulted in a 2-3-fold increase in the amount of D-[3H]glucosamine-labelled material in the culture medium. Gel electrophoresis revealed increased incorporation of D-[3H]glucosamine into extracellular glycoproteins of Mr 245,000, 170,000, 140,000, 130,000, 120,000 and 105,000 as well as into glycosaminoglycans in retinoid-treated cultures. The labelling of extracellular glycoproteins with L-[3H]leucine and L-[35S]methionine was also increased by retinoids suggesting increased synthesis of these components rather than an effect on their glycosylation. The Mr 245 000 glycoprotein was identified as keratinocyte-derived fibronectin by immunoblotting, immunoprecipitation and specific binding to gelatin. The results show that retinoids increase the synthesis of glycoprotein as well as glycosaminoglycan components of the extracellular matrix in human keratinocyte cultures. It is suggested that retinoids select for a population of cells that synthesize relatively large amounts of glycosaminoglycan, fibronectin and other as yet unidentified extracellular glycoproteins.     

Localization and partial composition of the oligosaccharide units on the propeptide extensions of type I procollagen

Type I procollagen secreted by matrix-free chick embryo tendon cells was labeled with L-[3,3'-3H] cystine and purified by DEAE-cellulose chromatography. After bacterial collagenase digestion, the NH2- and COOH-terminal propeptides were partially characterized by ion exchange chromatography and gel filtration. Similar experiments were then conducted after labeling with either D-[6-3H] glucosamine, D-[2-3H] mannose, or D-[U-14C] glucose. On the basis of these studies and subsequent carbohydrate analysis, it was concluded that the COOH-terminal peptide contained greater than 90% of the radioactive carbohydrate which consisted predominantly of glucosamine and mannose with traces of galactosamine and galactose. Only radioactive glucosamine could be detected in the NH2-terminal propeptide. Under conditions which inhibit hydroxylation of lysine and glycosylation of hydroxylysine, unhydroxylated procollagen (protocollagen) could still be labeled with [3H] glucosamine and [3H] mannose. This suggested that glycosylation of the propeptides is at least initiated at the level of the rough endoplasmic reticulum.     

Biosynthesis of a glycosylated keratin by human keratinocytes

Human keratinocytes, cultured in the presence of D-[1-14C]glucosamine, incorporated radioactivity into a cytoskeleton-associated glycoprotein with Mr 53,000. This glycoprotein co-purified with prekeratin when keratinocyte cytoskeletons were extracted with 0.1 M citric acid/0.1 M sodium citrate and subjected to isoelectric precipitation at pH 4.0. Analysis of the prekeratin polypeptides by two-dimensional gel electrophoresis revealed that the radioactivity was restricted to a single polypeptide with an isoelectric point in the pH range 4.5-5.5. Acid hydrolysis of prekeratin followed by paper chromatography of the hydrolysate showed that the radioactivity was incorporated as glucosamine and not by metabolic conversion to amino acids. Control experiments showed that the radioactivity associated with the glycoprotein of Mr 53,000 was not the result of adsorbed glycolipids or non-enzymatic labelling. In contrast to the incorporation of D-[1-14C]glucosamine and D-[6-3H]glucosamine, no appreciable amounts of L-[6-3H]fucose, D-[2-3H]mannose or 32PO4 were incorporated into this glycoprotein. The immunological relationship of the glycoprotein of Mr 53,000 to the keratins was demonstrated by its reactivity with both polyclonal and monoclonal antisera to keratin.     

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.     

Direct utilization of mannose for mammalian glycoprotein biosynthesis

Direct utilization of mannose for glycoprotein biosynthesis has not been studied because cellular mannose is assumed to be derived entirely from glucose. However, animal sera contain sufficient mannose to force uptake through glucose-tolerant, mannose-specific transporters. Under physiological conditions this transport system provides 75% of the mannose for protein glycosylation in human hepatoma cells despite a 50- to 100-fold higher concentration of glucose. This suggests that direct use of mannose is more important than conversion from glucose. Consistent with this finding the liver is low in phosphomannose isomerase activity (fructose-6-P<->mannose-6-P), the key enzyme for supplying glucose-derived mannose to the N-glycosylation pathway. [2- 3H] Mannose is rapidly absorbed from the intestine of anesthetized rats and cleared from the blood with a t1/2of 30 min. After a 30 min lag, label is incorporated into plasma glycoproteins, and into glycoproteins of all organs during the first hour. Most (87%) of the initial incorporation occurs in the liver, but this decreases as radiolabeled plasma glycoproteins increase. Radiolabel in glycoproteins also increases 2- to 6-fold in other organs between 1-8 h, especially in lung, skeletal muscle, and heart. These organs may take up hepatic- derived radiolabeled plasma glycoproteins. Significantly, the brain, which is not exposed to plasma glycoproteins, shows essentially no increase in radiolabel. These results suggest that mammals use mannose transporters to deliver mannose from blood to the liver and other organs for glycoprotein biosynthesis. Additionally, contrary to expectations, most of the mannose for glycoprotein biosynthesis in cultured hepatoma cells is derived from mannose, not glucose. Extracellular mannose may also make a significant contribution to glycoprotein biosynthesis in the intact organism.      

beta-Adrenergic stimulation alters oligosaccharide pyrophosphoryl dolichol metabolism in rat parotid acinar cells.

beta-Adrenergic stimulation of rat parotid acinar cells markedly increases [3H]mannose incorporation into N-linked glycoproteins [Kousvelari, Grant, Banerjee, Newby & Baum (1984) Biochem. J. 222, 17-24]. More than 90% of this protein-bound [3H]mannose was preferentially incorporated into four secretory glycoproteins. The ratio of [3H]mannose/[14C]leucine present in these individual proteins was 1.7-4-fold greater with isoproterenol-treated cells than with untreated controls. In isoproterenol-stimulated cells, [3H]mannose incorporation into mannosylphosphoryl dolichol and oligosaccharide-PP-dolichol was increased 2-3-fold over that observed in unstimulated cells. Similarly, formation of mannosylated oligosaccharide-PP-dolichol was increased approx. 4-fold in microsomes prepared from isoproterenol-treated cells. Also, turnover of oligosaccharide-PP-dolichol was significantly increased (5-fold) by beta-adrenergic stimulation; the half-life for oligosaccharide-PP-dolichol decreased from 6 min in control cells to 1.2 min in isoproterenol-stimulated cells. By 15 min after isoproterenol addition to acinar cells, the specific radioactivity of parotid oligosaccharide moieties increased about 3-fold over the value observed in the absence of the agonist. Taken together, these results strongly suggest that elevation of N-linked protein glycosylation in rat parotid acinar cells after beta-adrenoreceptor stimulation resulted from significant enhancement in the synthesis of mannosylphosphoryl dolichol and oligosaccharide-PP-dolichol and the turnover of oligosaccharide-PP-dolichol.