Glycoprotein Metabolism in the Cotyledons of Pisum sativum during Development and Germination

The glycoprotein nature of legumin and vicilin, the reserve globulins in the cotyledons of Pisum sativum was studied. Legumin from mature seed was found to contain 1% neutral sugars (mannose and glucose) and 0.1% amino sugar (glucosamine), whereas vicilin contained 0.3% neutral sugar (mannose) and 0.2% amino sugar (glucosamine). On the basis of the incorporation of ¹⁴C-labeled glucosamine, it appeared that not all of the component subunits of the reserve proteins are glycosylated to the same extent. In addition, it has been established that glycosylation occurs after peptide synthesis. During seed development there was a change in neutral sugars and amino sugar ratio in vicilin. During germination, the neutral sugars and the amino sugar content of the glycoproteins declined. These findings are discussed in relation to the synthesis and degradation of the glycosyl component of the glycoproteins.     

Biosynthesis of the Sindbis Virus Carbohydrates

The sequence in which sugars are added to the Sindbis virus glycoproteins was studied. Infected cells contain three glycosylated virus-specific proteins: the two virion glycoproteins and the immediate precursor to the smaller virion glycoprotein. Larger Sindbis-specific proteins are not glycosylated. The cell-associated forms of both of the virion glycoproteins contain glucosamine, mannose, galactose, and fucose. The glycosylated precursor contains only glucosamine, mannose, and some galactose. The conversion of precursor to virion protein involves both the addition of galactose and fucose and the loss of mannose. The apparent extent of glycosylation of each virus-specific protein is not influenced by the host cell.     

Site of initial glycosylation of mannoproteins from Saccharomyces cerevisiae.

The cellular site of initial glycosylation of proteins from Saccharomyces cerevisiae has been studied. Short pulses of [U-14C]mannose label the ribosomal fraction of the yeast. Most of the label was associated with polysomes; monosomes contained only a small amount of radioactivity. All of the radioactivity present in the polysomal fraction was accounted by mannose and smaller amounts of glucose and glucosamine. Puromycin treatment detached more than 50% of the radioactivity from the polysomes; treatment of polysomes at pH 10.0 also caused the release of radioactivity. These results indicate that initial sugar binding occurs while the nascent polypeptide chains are still growing on the ribosomes. When the cells were preincubated with 2-deoxy-D-glucose, incorporation of [U-14C]mannose into the polysomes and the cell wall was inhibited, whereas its incorporation into membrane fractions was unimpaired. It was concluded that 2-deoxy-D-glucose inhibited the synthesis of glycoproteins by interference with the initial glycosylation steps at the ribosomal level.     

Carbohydrate composition of lymphocyte plasma membrane from pig mesenteric lymph node.

Pig lymphocyte plasma membrane isolated from mesenteric lymph node contained 69 mug of carbohydrate/mg dry wt., which was made up of neutral sugar, amino sugar and sialic acid in the molar proportions 5:1.7:1. The neutral sugar comprised fucose, ribose, mannose, glucose, galactose and inositol (molar proportions 2:9:11:15:26:1), and the amino sugar glucosamine and galactosamine (molar ratio 2:1). The ribose was most probably derived from RNA. All of the fucose and mannose and almost all of the glucosamine were associated with the membrane protein whereas the membrane lipid contained all of the inositol. The remaining sugars were distributed in various ratios between the protein and lipid fractions.     

Purification and molecular characterization of rat intestinal brush border membrane dipeptidyl aminopeptidase IV

Dipeptidyl aminopeptidase IV (EC 3.4.14.-) was solubilized from a particulate membrane fraction of rat intestinal mucosa with Triton X-100. The solubilized enzyme was purified to homogeneity following ammonium sulfate fractionation, chromatography on DEAE-Sepharose and hydroxyapatite, gel filtration and preparative polyacrylamide gel electrophoresis. The final enzyme preparation had a specific activity of 55 units/mg protein representing a 1373 fold purification over the starting material. Purity was judged by polyacrylamide gel electrophoresis and double immunodiffusion. The molecular weight of the native undenatured enzyme was estimated to be 230000 by gel filtration and polyacrylamide gel electrophoresis. Electrophoresis under denaturing conditions (sodium dodecyl sulfate) indicated that the protein consists of two identical 98 kDa subunits. Dipeptidyl aminopeptidase IV is a glycoprotein containing approx. 8% carbohydrate by weight. A detailed analysis of the individual sugar components demonstrated that fucose, galactose, glucose, mannose, sialic acid and hexosamine sugars were present. The nature of the constituent asparagine linked oligosaccharide side chains was further examined following cleavage from the peptide backbone by hydrazinolysis. Following high voltage paper electrophoresis approx. 80% of the isolated oligosaccharide was found with the neutral fraction while the remaining 20% consisted of a single acidic component. Gel filtration of the neutral oligosaccharide fraction indicated that it contains approx. 19 sugar residues.     

Some Properties and Characterization of Rice Seed Hemagglutinin

The phytohemagglutinin of rice seed has been purified by a sequence of steps involving fractionation with ammonium sulfate and successive chromatography on DEAE-and eMcellulose and finally gel filtration on Bio-Gel P-100. The purified rice seed hemagglutinin was shown to be homogeneous by electrophoresis on polyacrylamide gel and its molecular weight was 10,000, calculated from both the Ve/Vo value of gel filtration on Bio-Gel P-100 and the sum of the individual constituents (amino acids, sugars and metals). In addition to amino acid, the rice seed hemagglutinin contained 26.8% covalently bound carbohydrate which was identified and quantitated by gas chromatography of the acetylated alditols. Glucose was the predominant sugar with lesser amounts of glucosamine, xylose, and mannose also being present. And the rice seed hemagglutinin contained 1 g atom of calcium per molecule. The molecular weight of the rice seed hemagglutinin is smallest compared with some of phytohemagglutinins isolated from leguminous seeds and other plant sources. The rice seed hemagglutinin has the blastogenetic activity for human peripheral lymphocytes as well as Phaseolus vulgaris phytohemagglutinins or concanavalin A, jack bean hemagglutinin.     

Determination of fermentable carbohydrate from the upper gastrointestinal tract by using colectomized rats.

The primary aim of this study was to characterize the carbohydrate that would be supplied to the colon for fermentation under physiological conditions. Colectomized rats were fed fiber-free diets or diets containing 5% (wt/wt) gum arabic. Four (fucose, galactose, glucosamine, and galactosamine) of 11 analyzed sugars accounted for 77% of the total sugar in ileal excreta from colectomized rats fed fiber-free diets. The three sugars in gum arabic, rhamnose, arabinose, and galactose, accounted for 84% of the total sugars in gum arabic ileal excreta. Comparisons of the sugar compositions of the ileal excreta, the water-soluble fractions of the excreta, and three gel filtration fractions of the water-soluble material with those of the water-soluble fraction of rat mucosa, the acetone-soluble fraction of pancreas, and pancreatin suggested that the major source of endogenous carbohydrate is mucin. Gum arabic increased the daily excretion of the four mucin-derived sugars (fucose, galactose, glucosamine, and galactosamine) by the colectomized rats from 473 mumol per day to 634 mumol per day. We conclude that mucin is the major endogenous carbohydrate excreted from the upper gut and that gum arabic increases the amount of this endogenous carbohydrate.     

Determination of fermentable carbohydrate from the upper gastrointestinal tract by using colectomized rats.

The primary aim of this study was to characterize the carbohydrate that would be supplied to the colon for fermentation under physiological conditions. Colectomized rats were fed fiber-free diets or diets containing 5% (wt/wt) gum arabic. Four (fucose, galactose, glucosamine, and galactosamine) of 11 analyzed sugars accounted for 77% of the total sugar in ileal excreta from colectomized rats fed fiber-free diets. The three sugars in gum arabic, rhamnose, arabinose, and galactose, accounted for 84% of the total sugars in gum arabic ileal excreta. Comparisons of the sugar compositions of the ileal excreta, the water-soluble fractions of the excreta, and three gel filtration fractions of the water-soluble material with those of the water-soluble fraction of rat mucosa, the acetone-soluble fraction of pancreas, and pancreatin suggested that the major source of endogenous carbohydrate is mucin. Gum arabic increased the daily excretion of the four mucin-derived sugars (fucose, galactose, glucosamine, and galactosamine) by the colectomized rats from 473 mumol per day to 634 mumol per day. We conclude that mucin is the major endogenous carbohydrate excreted from the upper gut and that gum arabic increases the amount of this endogenous carbohydrate.     

Regulation of late functions in Salmonella bacteriophages P22 and L studied by assaying endolysin synthesis.

The glycopeptides obtained by pronase digestion of two ecotropic strains of murine leukemia virus (MuLV) were compared by gel filtration. Four different glycopeptide size classes, designated G(1), G(2), G(3), and G(4), with molecular weights of approximately 5,100, 2,900, 2,200, and 1,500, respectively, were shown to be associated with Rauscher MuLV virions grown in JLS-V9 cells. Various sugar precursors, including glucosamine, galactose, fucose, and mannose were incorporated into G(1) and G(2), suggesting that these are complex (type I) glycopeptides. The two smaller glycopeptide size classes, G(3) and G(4), were shown to be mannoserich (type II) glycopeptides. G(4) was more sensitive to digestion with endo-beta-N-acetylglucosaminidase H than G(3), suggesting that the core of G(3) may contain fewer mannose residues. Glycopeptides of the same size class as G(1) and G(2) were associated with both Rauscher MuLV and AKR-MuLV grown in III6A (mouse embryo) cells. Previous studies have shown that gp52, a proteolytic cleavage product of gp70, possessed primarily G(1) glycopeptides and that gp52 was more highly sulfated than gp70. We observed that G(1) is approximately twofold more highly sulfated than G(2), explaining the observed difference in sulfation of gp52. The unusually large size of G(1) suggested that infection with MuLV may alter the host cell glycosylation pattern. To test this possibility, glycopeptides from Sindbis virions grown in uninfected and Rauscher MuLV-infected JLS-V9 cells were compared, and no differences were observed. G(1) was not detected in Sindbis virions, indicating that acquisition of G(1) depends on properties of the virus-coded polypeptide backbone of the gp70 molecule.     

Studies on thyroid cell surface glycoproteins: Isolation of plasma membranes and characterization of carbohydrate units

Plasma membranes were isolated from calf thyroid microsomes and further resolved into two subfractions by sucrose density gradient centrifugations. The lighter and major membrane fraction was obtained in a yield of 10 mg/100 g of thyroid and was enriched 38-fold with respect to 5′-nucleotidase activity compared to the homogenate. It differed from the denser plasma membrane fraction in containing greater amounts of phospholipid and cholesterol but had a similar total carbohydrate content (16 mg/100 mg protein) and monosaccharide composition. The membranes were found to retain most (80%) of their carbohydrate after delipidation. The major protein-bound sugars present in the lighter membrane fraction expressed as micromoles per 100 mg of peptide were: galactose 24, mannose 17, fucose 3, glucosamine 23, galactosamine 4, and sialic acid 9. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of the lipid-free membranes revealed at least 18 protein bands and 3 periodic acid-Schiffreactive glycoprotein components. Incubation of the delipidated membranes with Pronase resulted in the solubilization of 95% of the saccharide portion which upon filtration through Bio-Gel P-6 and P-10 columns yielded several glycopeptide fractions. While some of the carbohydrate was found in glycopeptides which appeared to contain the well-known complex and polymannose asparagine-bound oligosaccharides, as well as small O-glycosidically linked units, approximately half was recovered in high molecular weight components which contained galactose and glucosamine as their principal sugar constituents, and which were similar in composition to glycopeptides recently isolated (T. Krusius, J. Finne, and H. Rauvala, 1978, Eur. J. Biochem.92, 289–300) from human erythrocyte membranes.     

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.     

The carbohydrate components of arterial basement-membrane-like material. Studies on rabbit aortic myomedial cells in culture.

The carbohydrate composition of arterial basement-membrane-like material was investigated. Basement-membrane-like material was isolated from cultures of aortic myomedial cells by a sonication/differential-centrifugation technique. Purified basement-membrane-like material contained a total of 5% sugars, comprising glucose, galactose, mannose, fucose, sialic acid, glucosamine and galactosamine in the approximate molar proportions 3.2:3.5:3.4:3.2:1:5.5:3.1. In addition, small amounts of xylose were found. Analyses for uronic acid showed that glycosaminoglycans comprised about 1% of isolated basement-membrane-like material. The carbohydrate composition indicated the presence of complex-type oligosaccharides in addition to hydroxylysine-linked disaccharides. [3H]Glucosamine-labelled glycopeptides obtained by proteinase digestion and gel filtration were resistant to endo-beta-N-acetylglucosaminidase D, but more than 10% were susceptible to alpha-mannosidase, demonstrating the presence of high-mannose-type oligosaccharides. The distribution of carbohydrates among peptides of basement-membrane-like material on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was investigated after labelling with [3H]mannose, [3H]fucose, [3H]galactose and [3H]glucosamine. Among peptides that appeared to carry carbohydrates were a proteoglycan(s) and seven glycoproteins in the molecular-weight range 120 000-700 000.     

Inhibition of glycosylphosphatidylinositol anchor formation by mannosamine.

Many eucaryotic cell surface proteins are anchored to the plasma membrane via a glycosylphosphatidylinositol (GPI), of which the core region is highly conserved from protozoa to mammalian cells. Previous studies (Lisanti, M. P., Field, M. C., Caras, I. W., Menon, A. K., and Rodiguez-Boulan, E. (1991) EMBO J. 10, 1969-1977) showed that mannosamine blocked the expression of a recombinant GPI-anchored protein in Madin-Darby canine kidney cells and converted this protein to an unpolarized secretory product. In the present study, we examined the effect of mannosamine on the formation of the glycan portion of the GPI anchor precursors. This amino sugar inhibited the incorporation of mannose into the glycan portion, and the inhibition was dose-dependent. Mannosamine was shown to be incorporated into the glycan as mannosamine, probably mostly in the second mannose position and thereby to block the further addition of mannose and other anchor components. The products formed in the presence of this drug were characterized by gel filtration and high resolution TLC both before and after deamination with nitrous acid and dephosphorylation by HF. Galactosamine and trehalosamine were inactive in this system, whereas glucosamine also inhibited mannose incorporation into GPI intermediates.     

Crystallization and partial characterization of Staphylococcus aureus hyaluronate lyase

Staphylococcus aureus, strain 1801, hyaluronate lyase was purified and crystallized to homogeneity as ascertained by chromatography and disc-gel electrophoresis. Purification procedures included sequential ammonium sulfate fractionation, acetone precipitation, Sephadex gel filtration, and ion exchange chromatography. During its passage through the cation exchange column, the hyaluronate lyase was resolved into two minor and one major fraction. The major peak, which was found to be cationic, was further characterized and designated as Fraction III. Carbohydrate analysis showed the presence of neutral sugars galactose, glucose, and mannose in the ratio of 1:3:6. Amino sugars galactosamine and glucosamine (or mannosamine) were present in a ratio of 1:1. Quantitative amino acid analysis of the Fraction III showed a relative abundance of the basic amino acids lysine and histidine.     

Interference with glycosylation of glycoproteins. Inhibition of formation of lipid-linked oligosaccharides in vivo.

Influenza-virus-infected cells were labelled with radioactive sugars and extracted to give fractions containing lipid-linked oligosaccharides and glycoproteins. The oligosaccharides linked to lipid were of the 'high-mannose' type and contained glucose. In the glycoprotein fraction, radioactivity was associated with virus proteins and found to occur predominantly in the 'high-mannose' type of glycopeptides. In the presence of the inhibitors 2-deoxy-D-glucose, 2-deoxy-2-amino-D-glucose (glucosamine), 2-deoxy-2-fluoro-D-glucose and 2-deoxy-2-fluoro-D-mannose incorporation of radiolabelled sugars into lipid- and protein-linked oligosaccharides was decreased. Kinetic analysis showed that the inhibitors affected first the assembly of lipid-linked oligosaccharides and then protein glycosylation after a lag period. During inhibition by deoxyglucose and the fluoro sugars lipid-linked oligosaccharides were formed that contained oligosaccharides of decreased molecular weight. No such aberrant forms were found during inhibition by glucosamine. In the case of inhibition by deoxyglucose it was shown that the aberrant oligosaccharides were not transferred to protein. Inhibition of formation of lipid-linked oligosaccharides by deoxyglucose and fluoro sugars was antagonized by mannose, in which case oligosaccharides of normal molecular weight were formed. The inhibition by glucosamine was reversed by its removal from the medium. The reversible effects of these inhibitors exemplify their usefulness as tools in the study of glycosylation processes.     

Glucosamine itself mediates reversible inhibition of protein glycosylation. A study of glucosamine metabolism at inhibitory concentrations in influenza-virus-infected cells

The metabolism of glucosamine in chick embryo fibroblasts was studied at different concentrations of the amino sugar added to the culture medium. In glucose-containing medium the well-known metabolites, UDP-N-acetylglucosamine, N-acetylglucosamine 6-phosphate and N-acetylglucosamine, are detectable after inhibition of glycosylation resulting from glucosamine treatment. Especially when the cells were infected with influenza virus, high intracellular concentrations of non-metabolized glucosamine are demonstrable in addition. Removal of the inhibitor from the medium results in release of the block of influenza virus glycoprotein glycosylation within 10 min. The onset of glycosylation is paralleled by a rapid reduction of intracellular levels of glucosamine without significant changes in the concentration of its metabolites. Furthermore, concentrations of GDP-mannose, UDP-glucose, and UDP-galactose remain constant for at least 30 min after reversal of the block. It is concluded that glucosamine as such exerts its effect on glycosylation, rather than one of its metabolites being responsible for this effect.     

Effect of tunicamycin on the glycosylation of rhodopsin

The incorporation of [³H]glucosamine, [³H]mannose, and [³⁵S]methionine into rhodopsin was investigated in retinas which had been incubated in the presence and absence of the antibiotic, tunicamycin. In its presence, the incorporation of glucosamine was inhibited 70% and mannose, 96% compared to controls. In the presence of tunicamycin the attachment of glucosamine to core-region sites was virtually eliminated. The formation of unglycosylated rhodopsin was also indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and concanavalin A-Sepharose chromatography. These findings are consistent with the participation of the lipid-linked pathway in the glycosylation of this well-characterized intrinsic glycoprotein of the membranes of the disk of the rod outer segment. As indicated by the incorporation of [³⁵S]methionine, the synthesis of rhodopsin apoprotein was inhibited by a much lesser amount. This suggests that the glycosylation of rhodopsin is not required for its insertion into the disk membrane.     

Chemical characterization of the proteins and glycoproteins of mouse liver plasma membranes solubilized by sequential extraction with aqueous and organic solvents

1. A procedure for the stepwise fractionation of the proteins of mouse liver plasma membranes is described. 2. Of the membrane protein 20-25% was soluble in 50mm-sodium carbonate-bicarbonate buffer (pH9.7). This fraction contained a large number of proteins but only 1 major glycoprotein. It was low in sialic acid, amino sugars and phospholipid. 3. Extraction of the alkali-insoluble residue with aq. 33% pyridine solubilized an additional 30-35% of the membrane protein. The pyridine-soluble membrane components were enriched in sialic acid and glucosamine and it was shown that this procedure resulted in the selective extraction of glycoproteins. 4. Gel filtration in sodium dodecyl sulphate resolved the pyridine-soluble proteins into five fractions of decreasing molecular weight and an inverse relationship between molecular weight and sialic acid content was indicated.     

Carbohydrate Composition of Vesicular Stomatitis Virus

Analysis by gas-liquid chromatography of the trimethylsilylated sugar residues of purified vesicular stomatitis virus grown in L cells or chick embryo cells revealed the presence in the whole virion of four hexoses (glucose, galactose, mannose, and fucose), two hexosamines (glucosamine and galactosamine), and 34 to 40% neuraminic acid. The isolated viral glycoprotein was devoid of galactosamine and fucose, both of which sugars were present in whole virions presumably as part of the membrane glycolipids.     

An Impaired Uptake of Glucosamine in Tunicamycin Resistant Chinese Hamster Ovary Cells

Tunicamycin resistant mutants (TM^R) were isolated and characterized from Chinese hamster ovary cells. One feature of this TM^R mutants was a marked decrease in incorporation of radioactive glucosamine, both into membrane glycoproteins and G protein of vesicular stomatitis virus.

The cellular uptake and incorporation into acid insoluble materials of various radioactive substances, including glucosamine, galactosamine, mannose, 2-deoxyglucose and leucine, was examined for the purpose of determination whether the reduced incorporation of radioactive glucosamine into glycoproteins was due to a defect in the glycosylation step or decreased uptake of glucosamine by cells.

While incorporation of glucosamine and 2-deoxyglucose into acid insoluble fractions was reduced strikingly in the mutants, the incorporation of mannose and leucine were the same as in the parent cells.

The uptake of glucosamine in TM^R cells was lower than that in the wild type cells, and the Km value for glucosamine uptake differed between the mutants and wild type cells. There was no obvious difference in the uptake of 2-deoxyglucose and mannose.