The effect of castanospermine on the synthesis of synaptic glycoproteins by rat brain slices

Slices were prepared from rat forebrains and the incorporation of [³H]mannose and [³⁵S]methionine into proteins and glycoproteins determined. The incorporation of methionine continued to increase for up to 8 hours whereas mannose incorporation was maximal between 2 and 4 hours and declined thereafter. Glycopeptides prepared by pronase digestion of [³H]mannose-labeled glycoproteins were digested with endoglucosaminidase H (endo H) and analysed by gel filtration. The major endo H-sensitive oligosaccharide eluted in a position similar to standard Man₈GlcNAc. In the presence of castanospermine, which inhibits glucosidase I, the first enzymatic step in the processing of N-linked oligosaccharides, a new endo H-sensitive glycan similar in size to standard Glc₃Man₉GlcNAc₂ accumulated. Synaptic membranes (SMs) were isolated from slices which had been incubated with either [³H]mannose or [³⁵S]methionine in the presence and absence of castanospermine. In the presence of inhibitor the relative incorporation of [³H]mannose into high-mannose glycans of synaptic glycoproteins was increased. The incorporation of newly synthesized, [³⁵S] methioninelabeled, Con A-binding glycoproteins into SMs was not affected by the addition of inhibitor. Many of the glycoproteins synthesized in the presence of castanospermine exhibited a decreased electrophoretic mobility indicative of the presence of altered oligosaccharide chains. The results indicate that changes in oligosaccharide composition produced by castanospermine had little effect on the subsequent transport and incorporation of glycoproteins into synaptic membranes.To whom to address reprint requests.     

The effect of glycoprotein-processing inhibitors on the secretion of glycoproteins by Madin-Darby canine kidney cells

The effects of various glycoprotein-processing inhibitors on the biosynthesis and secretion of N-linked glycoproteins was examined in cultured Madin-Darby canine kidney (MDCK) cells. Since incorporation of [2-3H]mannose into lipid-linked saccharides and into glycoproteins was much greater in phosphate-buffered saline (PBS) than in serum-supplemented basal medium (BME), most experiments were done in PBS. Castanospermine, an inhibitor of glucosidase I, caused the formation of glycoproteins having mostly Glc3Man7-9(GlcNAc)2 structures; deoxymannojirimycin, an inhibitor of mannosidase I, gave mostly glycoproteins with Man9(GlcNAc)2 structures; swainsonine, an inhibitor of mannosidase II, caused the accumulation of hybrid types of oligosaccharides. Castanospermine and swainsonine, either in PBS or in BME medium, had no effect on the incorporation of [2-3H]mannose or [5,6-3H]leucine into the secreted glycoproteins and, in fact, there was some increase in mannose incorporation in their presence. These inhibitors also did not affect mannose incorporation into cellular glycoproteins nor did they affect the biosynthesis as measured by mannose incorporation into lipid-linked saccharides. On the other hand in PBS medium, deoxymannojirimycin, at 25 micrograms/mL, caused a 75% inhibition in mannose incorporation into secreted glycoproteins, but had no effect on the incorporation of [3H]leucine into the secreted glycoproteins. Since deoxymannojirimycin also strongly inhibited mannose incorporation into lipid-linked oligosaccharides in PBS, the decreased amount of radioactivity in the secreted and cellular glycoproteins may reflect the formation of glycoproteins with fewer than normal numbers of oligosaccharide chains, owing to the low levels of oligosaccharide donor. However, in BME medium, there was only slight inhibition of mannose incorporation into lipid-linked saccharides and into cellular and secreted glycoproteins.     

Sugar-mediated ligand-receptor interactions in the immune system

Most molecules involved in the recognition and elimination of pathogens by the immune system are glycoproteins. Oligosaccharides attached to glycoproteins initiate biological functions through mechanisms that involve multiple interactions of the monosaccharide residues with receptors. For example, calreticulin, a quality-control lectin-like chaperone, interacts with glucosylated mannose glycans presented by empty major histocompatibility complex (MHC) class I molecules, retaining them in the endoplasmic reticulum (ER) until antigenic peptide is loaded. Clusters of specific IgG glycoforms, present in increased amounts in rheumatoid arthritis, bind mannose-binding lectin (MBL), providing a potential route to inflammation through activation of the complement pathway. Secretory IgA glycans bind gut bacteria, and an unusual cluster of mannose residues on gp120, the surface coat protein of the HIV virus, is recognized by the novel 'domain-swapped' IgG 2G12 serum antibody.     

Effect of tunicamycin on herpes simplex virus glycoproteins and infectious virus production.

The antibiotic tunicamycin, which blocks the synthesis of glycoproteins, inhibited the production of infectious herpes simplex virus. In the presence of this drug, [14C]glucosamine and [3H]mannose incorporation was reduced in infected cells, whereas total protein synthesis was not affected. Gel electrophoresis of [2-3H]mannose-labeled polypeptides failed to detect glycoprotein D or any of the other herpes simplex virus glycoproteins. By use of specific antisera we demonstrated that in the presence of tunicamycin the normal precursors to viral glycoproteins failed to appear. Instead, lower-molecular-weight polypeptides were found which were antigenically and structurally related to the glycosylated proteins. Evidence is presented to show that blocking the addition of carbohydrate to glycoprotein precursors with tunicamycin results in the disappearance of molecules, possibly due to degradation of the unglycosylated polypeptides. We infer that the added carbohydrate either stabilizes the envelope proteins or provides the proper structure for correct processing of the molecules needed for infectivity.     

Biosynthesis of the D2 cell adhesion molecule: pulse-chase studies in cultured fetal rat neuronal cells

D2 is a membrane glycoprotein that is believed to function as a cell adhesion molecule (CAM) in neural cells. We have examined its biosynthesis in cultured fetal rat brain neurones. We found D2-CAM to be synthesized initially as two polypeptides: Mr 186,000 (A) and Mr 136,000 (B). With increasing chase times the Mr of both molecules increased to 187,000-201,000 (A) and 137,000-158,000 (B). These were similar to the sizes of D2-CAM labeled with [14C]glucosamine, [3H]fucose and [14C]mannosamine, indicating that the higher Mr species are glycoproteins. In the presence of tunicamycin, which specifically blocks the synthesis of high mannose cores, Mr were reduced to 175,000 (A) and 124,000 (B). Newly synthesized A and B are susceptible to degradation by endo-beta-N-acetyl-glucosaminidase H, which specifically degrades high mannose cores, but they are resistant to such degradation after 150 min of posttranslational processing. Hence, we deduce that A and B are initially synthesized with four to five high mannose cores which are later converted into N-linked complex oligosaccharides attached to asparagine residues. However, no shift of [35S]methionine radioactivity between A and B was detected with different pulse or chase times, showing that these molecules are not interconverted. Thus, our data indicate that the neuronal D2-CAM glycoproteins are derived from two mRNAs.     

The effect of deoxymannojirimycin on the processing of the influenza viral glycoproteins

Deoxymannojirimycin (dMM) was tested as an inhibitor of the processing of the oligosaccharide portion of viral and cellular N-linked glycoproteins. The NWS strain of influenza virus was grown in MDCK cells in the presence of various amounts of dMM, and the glycoproteins were labeled by the addition of 2-[3H]mannose to the medium. At levels of 10 micrograms/ml dMM or higher, most of the viral glycopeptides became susceptible to digestion by endoglucosaminidase H, and the liberated oligosaccharide migrated mostly like a Hexose9GlcNAc on a calibrated column of Bio-Gel P-4. This oligosaccharide was characterized as a typical Man9GlcNAc by a variety of chemical and enzymatic procedures. Deoxymannojirimycin gave rise to similar oligosaccharide structures in the cellular glycoproteins. In both the viral and the cellular glycoproteins, this inhibitor caused a significant increase in the amount of [3H]mannose present in the glycoproteins. Deoxymannojirimycin did not inhibit the incorporation of [3H]leucine into protein in MDCK cells, nor did it affect the yield or infectivity of NWS virus particles. However, its effect on mannose incorporation into lipid-linked saccharides depended on the incubation time, the virus strain, and the cell line. Thus, high concentrations of dMM showed some inhibition of mannose incorporation into lipid-linked oligosaccharides with the NWS strain in a 3-h incubation, but no inhibition was observed after 48 h of incubation. On the other hand, the PR8 strain was much more sensitive to dMM inhibition, and mannose incorporation into lipid-linked oligosaccharides was strongly inhibited when the virus was raised in chick embryo cells, but less inhibition was observed when this virus was grown in MDCK cells. Nevertheless, in these cases also, the major oligosaccharide structure in the glycoproteins was the Man9GlcNAc2 species.     

Processing of the rough endoplasmic reticulum membrane glycoproteins of rotavirus SA11

The synthesis and oligosaccharide processing of the glycoproteins of SA11 rotavirus in infected Ma104 cells was examined. Rotavirus assembles in the rough endoplasmic reticulum (RER) and encodes two glycoproteins: VP7, a component of the outer viral capsid, and NCVP5, a nonstructural protein. A variety of evidence suggests the molecules are limited to the ER, a location consistent with the high mannose N-linked oligosaccharides modifying these proteins. VP7 and NCVP5 were shown to be integral membrane proteins. In an in vitro translation system supplemented with dog pancreas microsomes, they remained membrane associated after high salt treatment and sodium carbonate-mediated release of microsomal contents. In infected cells, the oligosaccharide processing of these molecules proceeded in a time-dependent manner. For VP7, Man8GlcNAc2 and Man6GlcNAc2 were the predominant intracellular species after a 5-min pulse with [3H]mannose and a 90 min chase, while in contrast, trimming of NCVP5 halted at Man8GlcNAc2. VP7 on mature virus was processed to Man5GlcNAc2. It is suggested that the alpha-mannosidase activities responsible for the formation of these structures reside in the ER. In the presence of the energy inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP), processing of VP7 and the vesicular stomatitis virus G protein was blocked at Man8GlcNAc2. After a 20-min chase of [3H]mannose-labeled molecules followed by addition of CCCP, trimming of VP7 could continue while processing of G protein remained blocked. Thus, an energy-sensitive translocation step within the ER may mark the divergence of the processing pathways of these glycoproteins.     

The effect of glycoprotein-processing inhibitors on fucosylation of glycoproteins

The influenza viral hemagglutinin contains L-fucose linked alpha 1,6 to some of the innermost GlcNAc residues of the complex oligosaccharides. In order to determine what structural features of the oligosaccharide were required for fucosylation or where in the processing pathway fucosylation occurred, influenza virus-infected MDCK cells were incubated in the presence of various inhibitors of glycoprotein processing to stop trimming at different points. After several hours of incubation with the inhibitors, [5,6-3H]fucose and [1-14C]mannose were added to label the glycoproteins, and cells were incubated in inhibitor and isotope for about 40 h to produce mature virus. Glycopeptides were prepared from the viral and the cellular glycoproteins, and these glycopeptides were isolated by gel filtration on Bio-Gel P-4. The glycopeptides were then digested with endo-beta-N-acetylglucosaminidase H and rechromatographed on the Bio-Gel column. In the presence of castanospermine or 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine, both inhibitors of glucosidase I, most of the radioactive mannose was found in Glc3Man7-9GlcNAc structures, and these did not contain radioactive fucose. In the presence of deoxymannojirimycin, an inhibitor of mannosidase I, most of the [14C]mannose was in a Man9GlcNAc structure which was also not fucosylated. However, in the presence of swainsonine, an inhibitor of mannosidase II, the [14C]mannose was mostly in hybrid types of oligosaccharides, and these structures also contained radioactive fucose. Treatment of the hybrid structures with endoglucosaminidase H released the [3H]fucose as a small peptide (Fuc-GlcNAc-peptide), whereas the [14C]mannose remained with the oligosaccharide. The data support the conclusion that the addition of fucose linked alpha 1,6 to the asparagine-linked GlcNAc is dependent upon the presence of a beta 1,2-GlcNAc residue on the alpha 1,3-mannose branch of the core structure.     

Glycosylation in vitro of Semliki-Forest-virus and influenza-virus glycoproteins and its suppression by nucleotide-2-deoxy-hexose

Cell-free enzyme preparations from cultured fibroblasts infected with Semliki forest virus or fowl plague virus (an influenza A virus) incorporate [14C]-mannose into dolichol-phosphate-mannose, lipid-linked oligosaccharides and into endogenous virus-specific glycoproteins. When GDP-2-deoxy-D-[14C]glucose serves as substrate 2-deoxy-D-[14C]glucose is transferred to dolichol phosphate yielding dolichol-monophosphate-2-deoxy-D-[14C]glucose. UDP-2-deoxy-D-[14C]glucose gives rise also to a lipid which, however, is not a polyprenol derivative. The transfer of [14C]mannose to lipid-extractable fractions and glycoproteins in vitro is blocked by GDP-2-deoxy-D-glucose. It can be restored by exogenous dolichol monophosphate only with regard to the formation of dolichol-monophosphate-[14C]mannose-labelled oligosaccharides into glycoproteins. UDP-2-deoxy-D-glucose has no inhibitory effect on transfer reactions of [14C]mannose from GDP-[14C]mannose into various lipid fractions or into glycoprotein. It is concluded therefore, that the inhibition of glycosylation brought about by 2-deoxyglucose in vivo is caused by an interference of its GDP derivative with the formation of a correct lipid-oligosaccharide.     

Glucose requirement for induction by sodium butyrate of the glycoprotein hormone alpha subunit in HeLa cells

Butyric acid produces multiple effects on mammalian cells in culture, including alterations in morphology, depression of growth rate, increased histone acetylation, and modified production of various proteins and enzymes. The latter effect is exemplified by the induction in HeLa cells of the glycoprotein hormone alpha subunit by millimolar concentrations of the fatty acid. This report demonstrates that increased subunit accumulation in response to sodium butyrate is strikingly dependent on the presence of glucose (or mannose) in the growth medium. In contrast, basal levels of subunit synthesis are only marginally affected when the culture medium is supplemented with one of a variety of hexoses. An increase in the accumulation of HeLa alpha does not occur in medium containing pyruvate as the energy source, and sustained induction requires the simultaneous and continued presence of both glucose and butyrate. The effects of butyrate on HeLa cell morphology and subunit induction can be separated, since the latter is glucose-dependent while the former is not. Failure of butyrate to induce alpha in medium containing pyruvate does not result from restricted subunit secretion, since the levels of intracellular alpha are not increased disproportionately relative to those in the medium. The hexoses which support induction of HeLa alpha (glucose greater than or equal to mannose greater than galactose greater than fructose) are identical to those which have been shown previously to stimulate the glucosylation of lipid-linked oligosaccharides and enhance the synthesis of certain glycoproteins. Labeling of various glycosylation intermediates with [3H]mannose indicates that in glucose medium there is a decrease in the level of radioactivity associated with both dolicholpyrophosphoryl oligosaccharide and cellular glycoproteins and a concomitant increase in the fraction of label recovered in secreted glycoproteins. Butyrate also causes a decrease in [3H]mannose-labeled cellular glycoproteins and an increase in tritiated extracellular glycoproteins, particularly in glucose medium. Likewise, glucose stimulates the incorporation of [3H]glucosamine into immunoprecipitable alpha subunit relative to the bulk of HeLa-secreted glycoproteins, and this is further enhanced by butyrate. However, as demonstrated by lectin chromatography of conditioned media, a nonglycosylated subunit does not accumulate in pyruvate medium, either in the absence or presence of butyrate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biosynthesis of an unglycosylated envelope glycoprotein of Rous sarcoma virus in the presence of tunicamycin.

Cells stably infected with Rous sarcoma virus were treated with tunicamycin to prevent the glycosylation of the precursor (pr92gp) to the two viral envelope glycoproteins gp85 and gp35. Pretreatment of the cells for 4 h with the antibiotic resulted in a 90% reduction in [3H]mannose incorporation into total cellular glycoproteins, intracellular viral glycoproteins, and released virus particles. Protein synthesis and virus particle formation were not significantly affected by the treatment. A new polypeptide made in the presence of the drug was identified by immunoprecipitation of pulse-labeled cell lysates with monospecific anti-gp85 and anti-gp35 sera. This polypeptide, migrating on sodium dodecyl sulfate-polyacrylamide gels as a molecule of 62,000 daltons (pr62), contained no [3H]mannose, was labeled with [S35]methionine and [3H]arginine, could not be chased into the higher-molecular-weight glycosylated form, and contained the same [3H]arginine tryptic peptides as pr92gp. The unglycosylated pr62 was still detectable 2 h after the pulse labeling of the cells. The lack of glycosylation of pr62 did not seem to reduce its stability. No clear evidence for the incorporation of this molecule or its cleavage products into viral particles could be obtained. To code for an envelope polypeptide of 62,000 daltons, only about 1,500 nucleotides or 15% of the total coding capacity of the virus are needed.     

Relationships among dolichyl phosphate, glycoprotein synthesis, and cell culture growth

Following treatment of Chinese hamster ovary cells with inhibitors of mevalonate biosynthesis in the presence of exogenous cholesterol, the cellular concentration of phosphorylated dolichol and the incorporation of [3H]mannose into dolichol-linked saccharides and N-linked glycoproteins declined coincident with a decline in DNA synthesis. Addition of mevalonate to the culture medium increased rates of mannose incorporation into lipid-linked saccharides and restored mannose incorporation into N-linked glycoproteins to control levels within 4 h. After an additional 4 h, synchronized DNA synthesis began. Inhibition of the synthesis of lipid-linked oligosaccharides and N-linked glycoproteins by tunicamycin prevented the induction of DNA synthesis by mevalonate, indicating that glycoprotein synthesis was required for cell division. The results suggest that the rate of cell culture growth may be influenced by the level of dolichyl phosphate acting to limit the synthesis of N-linked glycoproteins.     

Monosaccharide Sequence of Protein-Bound Glycans of Uukuniemi Virus

Uukuniemi virus, a member of the Bunyaviridae family, was grown in BHK-21 cells in the presence of [³H]mannose. The purified virions were disrupted with sodium dodecyl sulfate and digested with pronase. The [³H]mannose-labeled glycopeptides of the mixture of the two envelope glycoproteins G1 and G2 were characterized by degrading the glycans with specific exo-and endoglycosidases, by chemical methods, and by analyzing the products with lectin affinity and gel chromatography. The glycopeptides of Uukuniemi virus fell into three categories: complex, high-mannose type, and intermediate. The complex glycopeptides probably contained mainly two NeuNAc-Gal-GlcNAc branches attached to a core (Man)₃(GlcNAc)₂ peptide. The high-mannose-type glycans were estimated to contain at least five mannose units attached to two N-acetylglucosamine residues. Both glycan species appeared to be similar to the asparagine-linked oligosaccharides found in many soluble and membrane glycoproteins. The results suggested that the intermediate glycopeptides contained a mannosyl core. In about half of the molecules, one branch appeared to be terminated in mannose, and one appeared to be terminated in N-acetylglucosamine. Such glycans are a novel finding in viral membrane proteins. They may represent intermediate species in the biosynthetic pathway from high-mannose-type to complex glycans. Their accumulation could be connected with the site of maturation of the members of the Bunyaviridae family. Electron microscopic data suggest that the virions bud into smooth-surfaced cisternae in the Golgi region. The relative amounts of [³H]mannose in the complex, high-mannose-type, and intermediate glycans were 25, 62, and 13%, respectively, which corresponded to the approximate relative number of oligosaccharide chains of 2:2.8:1, respectively, in the roughly equimolar mixture of G1 and G2. Endoglycosidase H digestion of isolated [³⁵S]methionine-labeled G1 and G2 proteins suggested that most of the complex and intermediate chains were attached to G1 and that most of the high-mannose-type chains were attached to G2.     

Biosynthesis of the mannose 6-phosphate recognition marker in transport-impaired mouse lymphoma cells. Demonstration of a two-step phosphorylation

The biosynthesis of the mannose 6-phosphate recognition marker has been studied in transport-impaired mouse lymphoma cells to determine the subcellular location of the processing enzymes and to characterize the biosynthetic intermediates. Cells were labeled with [2-3H]mannose and chased at a low temperature (15 or 20 degrees C) or at 37 degrees C in the presence of m-chlorocarbonylcyanide phenylhydrazone to disrupt transport of the pulse-labeled molecules within the secretory apparatus. Both treatments inhibited the migration of the pulse-labeled glycoproteins to the Golgi apparatus as measured by the production of complex-type asparagine-linked oligosaccharides. Despite this inhibition in protein transport, acid hydrolases were phosphorylated. Structural analysis of the phosphorylated oligosaccharides indicated that the transport-impaired cells produced a single species of phosphorylated high mannose oligosaccharide; essentially all of the molecules contain a single phosphodiester group that is restricted to the alpha 1,6 branch of the oligosaccharide. The results suggest that synthesis of mannose 6-phosphate-bearing high mannose oligosaccharides occurs in an ordered, compartmentalized posttranslational process. The initial phosphorylation of newly synthesized acid hydrolases occurs at a pre-Golgi site and results in the production of high mannose-type units that contain a single phosphodiester group. In a subsequent compartment, probably within the Golgi apparatus, the monophosphorylated units may be converted to diphosphorylated forms. Finally, at a site distal to the phosphorylation reactions the diesters are hydrolyzed to reveal the mannose 6-phosphate recognition marker.     

Metabolism of 2-deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose in yeast and chick-embryo cells.

2-Deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose have been prepared by tritiation of the corresponding unlabeled 2-fluoro sugars. The tritiated 2-fluoro sugars are phosphorylated and activated by UTP and by GTP to yield UDP-2-deoxy-2-fluoro-D-[3H]glucose, UDP-2-deoxy-2-fluoro-D-[3H]mannose, GDP-2-deoxy-2-fluoro-D-[3H]glucose and GDP-2-deoxy-2-fluoro-D-[3H]mannose in both cell types. The nucleotide derivatives could also be labeled in the nucleotide moiety by feeding the cells with [14C]uridine or [14C]guanosine in the presence of unlabeled 2-fluoro sugar. No evidence was obtained for metabolic steps in which the six-carbon chain of 2-fluoro sugars was not preserved. No epimerisation of the label to 2-deoxy-2-fluoro-D-[3H]galactose could be observed by radioactive gas-liquid chromatography of the enzymatic cleavage products of the different 2-fluoro sugar metabolites isolated from either cell type. Yeast and chick embryo cells both incorporate 2-deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose specifically into glycoproteins, although this incorporation is very low when compared to the incorporation of 2-deoxy-D-[3H]glucose.     

The use of 1-deoxymannojirimycin to evaluate the role of various alpha-mannosidases in oligosaccharide processing in intact cells

The mannose analogue, 1-deoxymannojirimycin, which inhibits Golgi alpha-mannosidase I but not endoplasmic reticulum (ER) alpha-mannosidase has been used to determine the role of the ER alpha-mannosidase in the processing of the asparagine-linked oligosaccharides on glycoproteins in intact cells. In the absence of the inhibitor, the predominant oligosaccharide structures found on the ER glycoprotein 3-hydroxy-3-methylglutaryl-CoA reductase in UT-1 cells are single isomers of Man6GlcNAc and Man8GlcNAc. In the presence of 150 microM 1-deoxymannojirimycin, the Man8GlcNAc2 isomer accumulates indicating that the 1-deoxymannojirimycin-resistant ER alpha-mannosidase is responsible for the conversion of Man9GlcNAc2 to Man8GlcNAc2 on reductase. The processing of Man8GlcNAc2 to Man6GlcNAc2, however, must be attributed to a 1-deoxymannojirimycin-sensitive alpha-mannosidase. When cells were radiolabeled with [2-(3)H]mannose for 15 h in the presence of 1-deoxymannojirimycin and then further incubated for 3 h in nonradioactive medium without inhibitor, the Man8GlcNAc2 oligosaccharides which accumulated during the labeling period were partially trimmed to Man6GlcNAc. This finding suggests that a second alpha-mannosidase, sensitive to 1-deoxymannojirimycin, resides in the crystalloid ER and is responsible for trimming the reductase oligosaccharide chain from Man8GlcNAc2 to Man6GlcNAc2. To determine if ER alpha-mannosidase is responsible for trimming the oligosaccharides of all glycoproteins from Man9GlcNAc to Man8GlcNAc, the total asparagine-linked oligosaccharides of rat hepatocytes labeled with [2-(3)H]mannose in the presence or absence of 1.0 mM 1-deoxymannojirimycin were examined. the inhibitor prevented the formation of complex oligosaccharides and caused a 30-fold increase in the amount of Man9GlcNAc2 and a 13-fold increase in the amount of Man8GlcNAc2 present on secreted glycoproteins. This result suggests that only one-third of the secreted glycoproteins is initially processed by ER alpha-mannosidase, and two-thirds are processed by Golgi alpha-mannosidase I or another 1-deoxymannojirimycin-sensitive alpha-mannosidase. The inhibitor caused only a 2.6-fold increase in the amount of Man9GlcNAc2 on cellular glycoproteins suggesting that a higher proportion of these glycoproteins are initially processed by the ER alpha-mannosidase. We conclude that some, but not all, hepatocyte glycoproteins are substrates for ER alpha-mannosidase which catalyzes the removal of a specific mannose residue from Man9GlcNAc2 to form a single isomer of Man8GlcNAc2.     

Intracellular movement of two mannose 6-phosphate receptors: return to the Golgi apparatus

We have used Chinese hamster ovary (CHO) cells and a murine lymphoma cell line to study the recycling of the 215-kD and the 46-kD mannose 6-phosphate receptors to various regions of the Golgi to determine the site where the receptors first encounter newly synthesized lysosomal enzymes. For assessing return to the trans-most Golgi compartments containing sialyltransferase (trans-cisternae and trans-Golgi network), the oligosaccharides of receptor molecules on the cell surface were labeled with [3H]galactose at 4 degrees C. Upon warming to 37 degrees C, the [3H]galactose residues on both receptors were substituted with sialic acid with a t1/2 approximately 3 hrs. Other glycoproteins acquired sialic acid at least 8-10 times slower. Return of the receptors to the trans-Golgi cisternae containing galactosyltransferase could not be detected. Return to the cis/middle Golgi cisternae containing alpha-mannosidase I was measured by adding deoxymannojirimycin, a mannosidase I inhibitor, during the initial posttranslational passage of [3H]mannose-labeled glycoproteins through the Golgi, thereby preserving oligosaccharides which would be substrates for alpha-mannosidase I. After removal of the inhibitor, return to the early Golgi with subsequent passage through the Golgi complex was measured by determining the conversion of the oligosaccharides from high mannose to complex-type units. This conversion was very slow for the receptors and other glycoproteins (t1/2 approximately 20 h). Exposure of the receptors and other glycoproteins to the dMM-sensitive alpha-mannosidase without movement through the Golgi apparatus was determined by measuring the loss of mannose residues from these proteins. This loss was also slow. These results indicate that both Man-6-P receptors routinely return to the Golgi compartment which contains sialyltransferase and recycle through other regions of the Golgi region less frequently. We infer that the trans-Golgi network is the major site for lysosomal enzyme sorting in CHO and murine lymphoma cells.     

Involvement of Lipid-linked Oligosaccharides in Synthesis of Storage Glycoproteins in Soybean Seeds

Membrane preparations from developing soybean (var. Prize) cotyledon tissue, at the time of synthesis of storage glycoproteins, catalyze the sequential assembly of lipid-linked oligosaccharides from uridine-5'-diphospho-N-acetyl-d-[6-(3)H] glucosamine and guanosine-5'diphospho-d-[U-(14)C]mannose. The maximum size of lipid-linked oligosaccharide that accumulates contains the equivalent of 10 saccharide units on the basis of Bio-Gel P-2 gel filtration studies. These lipid-linked oligosaccharides show similar characteristics to polyisoprenyl diphosphate derivatives on diethylaminoethyl-cellulose chromatography and are potential intermediates in glycoprotein biosynthesis in this tissue. These glycolipids do not appear to turn over in pulse-chase experiments and no completed storage glycoproteins were detected among the products of these incubations.Tissue slices from cotyledons at the same stage of development synthesize lipid-linked oligosaccharides from [(3)H]mannose and [(3)H]glucosamine with sizes equivalent to 1, 7, 10, and approximately 15 saccharide units. In pulse-chase experiments, the lipid-linked saccharides with the equivalent of 1 and 10 units rapidly turnover, whereas those with 7 and 15 units do not. Examination of the higher oligosaccharide peaks (10 and 15) by Bio-Gel P-4 gel filtration shows them to comprise 2 distinct subsets of oligosaccharides containing different proportions of glucosamine and mannose units. Tissue slices synthesize products which resemble the completed 7S storage glycoproteins as judged by similarity of molecular weight and precipitation with specific antisera. Analysis of the oligosaccharides obtained by hydrazinolysis of glycoproteins shows the presence of a similar size "high-mannose" type N-linked oligosaccharides as in other glycoproteins from animal and plant cells.     

Characterization of the N- and O-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni schistosomula

This report describes the structural analyses of the O- and N-linked oligosaccharides contained in glycoproteins synthesized by 48-hr-old Schistosoma mansoni schistosomula. Schistosomula were prepared by mechanical transformation of cercariae and were then incubated in media containing either [2-3H] mannose, [6-3H]glucosamine, or [6-3H]galactose to metabolically radiolabel the oligosaccharide moieties of newly synthesized glycoproteins. Analysis by SDS-polyacrylamide gel electrophoresis and fluorography demonstrated that many glycoproteins were metabolically radiolabeled with the radioactive mannose and glucosamine precursors, whereas few glycoproteins were labeled by the radioactive galactose precursor. Glycopeptide were prepared from the radiolabeled glycoproteins by digestion with pronase and fractionated by chromatography on columns of concanavalin A-Sepharose and pea lectin-agarose. The structures of the oligosaccharide chains in the glycopeptides were analyzed by a variety of techniques. The major O-linked sugars were not bound by concanavalin A-Sepharose and consisted of simple O-linked monosaccharides that were terminal O-linked N-acetylgalactosamine, the minor type, and terminal O-linked N-acetylglucosamine, the major type. The N-linked oligosaccharides were found to consist of high mannose- and complex-type chains. The high mannose-type N-linked chains, which were bound with high affinity by concanavalin A-Sepharose, ranged in size from Man6GlcNAc2 to Man9GlcNAc2. The complex-type chains contained mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. No sialic acid was present in any metabolically radiolabeled glycoproteins from schistosomula.     

Biochemical characterization and biosynthesis of the uterine milk proteins of the pregnant sheep uterus

The uterine milk proteins (UTM-proteins), a pair of basic glycoproteins with similar isoelectric points and molecular weights (57,000 and 55,000), are secreted by the endometrium of the pregnant ewe. Peptide mapping of the two species of UTM-proteins demonstrated them to be structurally related. Furthermore, pulse-chase and continuous-labeling experiments indicated that both are produced from a common precursor of lower molecular weight. Purified UTM-proteins were found to be rich in basic amino acids, low in tyrosine, and apparently lacking in tryptophan. The proteins were about 5.6-5.7% carbohydrate by weight and bound the lectin, concanavalin A. UTM-proteins synthesized in vitro incorporated D-[3H]glucosamine. Analysis of [3H]glucosamine-labeled glycopeptides of Pronase-digested UTM-proteins by gel filtration indicated that most radioactivity is associated with one size class of oligosaccharide. UTM-proteins secreted by the endometrium in the presence of tunicamycin, an N-glycosylation inhibitor, were of lower molecular weight than those from control endometria, indicating that sugar chains are attached to the protein core via N-linkages to asparagine. UTM-proteins synthesized in culture incorporated [32P]orthophosphate, and tunicamycin inhibited this incorporation. Analysis of hydrolyzed UTM-proteins by paper chromatography indicated that much of the 32P was associated with mannose 6-phosphate. Because this moiety is the so-called lysosomal recognition marker and is present on uteroferrin, the acid phosphatase of porcine uterine secretions, we tested UTM-proteins for several enzymatic activities characteristic of lysosomes, but none was found. In conclusion, the UTM-proteins are related glycoproteins that, like porcine uteroferrin, contain mannose 6-phosphate, a result which suggests that secretion of glycoproteins with phosphorylated oligosaccharide chains may be a common feature of the progestational uterus.