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.     

Genetic alterations of ribonuclease-sensitive glycoprotein subunits of amino acid transport systems in Neurospora crassa conidia.

Neurospora crassa conidia have multiple and constitutive amino acid transport systems. Extraction by KCl releases amino acid-binding glycoproteins which have been purified by arginine affinity chromatography. Disappearance of certain fractions is coordinate with genetic lesions which reduce amino acid transport. Two such affinity fractions contain radioactivity when cells are grown on l-[¹⁴C]phenylalanine or on [¹⁴C]uridine, but not when cells are grown on [¹⁴C ]glucosamine. One purified arginine-binding fraction (B) contains 113 amino acid residues per minimum molecular weight. This glycoprotein also contains eight types of neutral sugar residues. No amino sugars were detected. Electrophoresis of crude extracts reveals five major Coomassie blue-staining species. The number of species is reduced, and the electrophoretic pattern is altered in extracts from transport-deficient strains. Tryptic “fingerprints” of these extracts indicate that mutations that reduce transport result in amino acid substitutions in the extractable glycoproteins. Nondialyzable material which absorbs light in the 260-nm region becomes dialyzable after digestion with RNase. Digestion of conidia with RNase reduces the amount of l-phenylalanine accumulated by the cells after 10 min of incubation with the amino acid.     

Stimulation of the biosynthesis of membrane glycoproteins from zajdela ascites hepatoma cells by Robinia lectin

Membrane glycoprotein biosynthesis of ascites hepatoma cells is followed by [¹⁴C]glucosamine and [³H]leucine incorporation into cells in culture. The rate of incorporation is strongly increased by the addition of Robinia lectin in culture medium. Labeled glycoproteins are released from lectin stimulated and non-stimulated ceils by trypsin digestion. Studies of labeled trypsinates on sodium dodecyl sulfate gel electrophoresis and Sephadex G-200 filtration exhibit two fractions both labeled with [¹⁴C]glucosamine and [³H]leucine and having different molecular weights, one over 200 000 and the other about 2000. Identical results are obtained when external membrane glycoproteins are solubilized by sodium deoxycholate. Comparison of surface glycoproteins isolated by trypsinization from control cells labeled with [³H]glucosamine and from lectin stimulated cells labeled with [¹⁴C]glucosamine displays no significant qualitative differences between glycoprotein fractions released from both cell groups.     

Isolation and Characterization of Glucosamine-containing Storage Glycoproteins from the Cotyledons of Phaseolus aureus

Cotyledons of Phaseolus aureus contain protein-bound glucosamine which is metabolized during germination. The glucosamine is present in storage glycoproteins, and these are concurrently metabolized along with the glucosamine. These glycoproteins are associated with protein bodies. Characterization of the glucosamine-containing storage proteins showed them to be identical with vicilin and legumin, the major storage proteins of the Leguminosae. Phaseolus aureus vicilin has a sedimentation constant of 8.0S and is made up of four nonidentical subunits. It contains 0.2% glucosamine and 1% mannose. Legumin has a sedimentation constant of 11.3S and is made up of three nonidentical subunits. It contains about 0.1% glucosamine.     

Self-assembly of a plant cell wall in vitro

A method has been developed by which the cell wall of Chlamydomonas reinhardi may be dissociated into its components, and then reassembled in vitro into a product that is chemically and structurally identical to the original cell wall. Chaotropic agents, such as lithium chloride and sodium perchlorate, separate the wall into two fractions, an insoluble amorphous inner wall layer, which retains its integrity (7.5% by weight of the complete wall) and a salt-soluble fraction containing the homogeneous glycoproteins responsible for the outer crystalline layers of the cell wall. Removal of the salt from dissociated walls by dialysis leads to the rapid recovery of complete reassembled cell walls. The conditions necessary for successful reconstitution of the cell wall in vitro include the presence of a suitable surface, across which a decreasing salt gradient exists, and the presence of both the salt-insoluble and the salt-soluble components. The salt-soluble glycoproteins alone can self-assemble under various conditions to form fragments that have the crystalline structure characteristic of the outer layers of the complete cell wall. Both the inner wall layer and the salt-soluble glyco-proteins have similar bulk amino acid and sugar (arabinose, galactose, mannose) compositions and both contain hydroxyproline. On the basis of the in vitro reconstitution of the cell wall we discuss certain aspects of in vivo cell wall morphogenesis. This communication describes the first case in which a plant cell wall has been reconstructed in vitro, and indicates that components of very large cellular structures are capable of being built by a simple self-assembly process.     

Isolation and charaterization of surface glycoproteins from L-1210, P-388 and HeLa cells

A method is described that permits the rapid extration of the cell surface glycoproteins of two murine leukemic cells, the P-388 and the L-1210 cells as well as those of the human adenocarcinoma cells, the HeLa cells.Proof of the surface location of these glycoproteins is provided by labeling the intact cells; (a) with ¹²⁵I by the lactoperoxidase iodination technique; (b) with ³H by the galactose oxidase-reductive tritiation method. Most of these glycoproteins were also shown to incorporate radioactive glucosamine and fucose. By these criteria as well as by the distribution of molecular weights, the surface glycoproteins of the two murine cells are indistinguishable; however, they differ markedly from the surface glycoproteins of HeLa cells. The extracts of the murine cells wee shown to contain lectin receptor activity as determined by their ability to inhibit the lectin-induced agglutination of the intact cells.     

A microapparatus for liquid hydrogen fluoride solvolysis: Sugar and amino sugar composition of Erysiphe graminis and Triticum aestivum cell walls

The assembly and use of a simple and safe apparatus for HF solvolysis of microgram amounts of cell walls, polysaccharides, or glycoproteins are described. Using this apparatus the cell wall composition of Erysiphe graminis was compared with that of its wheat host. The HF solvolysis combined with TFA posthydrolysis considerably increased sugar yields compared with TFA hydrolysis alone, due mainly to increased yields of glucose from wheat, and glucosamine from Erysiphe, corresponding to cellulose and chitin, respectively. A potentially useful method for determining amounts of fungal hyphae in plant tissue is also provided.     

Halococcus morrhuae: A sulfated heteropolysaccharide as the structural component of the bacterial cell wall

The qualitative and quantitative composition of purified cell walls of Halococcus morrhuae CCM 859 was determined. Glucose, mannose, galactose; glucuronic and galacturonic acids; glucosamine, galactosamine, gulosaminuronic acid; acetate, glycine and sulfate are found as major constituents. The amino sugars are N-acetylated. It was not possible to fractionate the cell wall in chemically different polymers. Evidence is presented that the major cell wall polymer of this strain is a complex heteroglycan which seems, like the peptidoglycan of most bacteria, to be responsible for the rigidity and stability of the cell wall. In addition it could be proved that this heteroglycan is sulfated and therefore differs considerably from previously described bacterial cell wall polymers.     

Rates of degradation of glycoproteins from normal and regenerating rat livers: A study using double isotopes

The average decay rates (half-lives) of mixed glycoproteins were measured using double isotopes of fucose and glucosamine and compared to those of mixed overall proteins measured with leucine and NaH¹⁴CO₃ in whole homogenates and plasma membranes from normal and regenerating rat livers. A large reutilization of leucine was observed under both normal and regenerating conditions. Fucose seems to be recycling most predominantly in regenerating liver, whereas glucosamine was found to be very little if not at all reutilized under both conditions. Comparison of the results obtained with NaH¹⁴CO₃ and glucosamine demonstrated that glycoproteins from normal liver homogenate are degraded at a faster rate than mixed proteins. Contrary to that of mixed proteins, the half-life of glycoproteins remains unchanged during liver regeneration, and the use of glucosamine revealed that the degradation of plasma membrane glycoproteins is identical to that found in whole homogenate under both normal and regenerating conditions. Finally, the relative degradation rates of fractionated plasma membrane proteins and glycoproteins were evaluated under the same conditions. During liver regeneration some readjustments are observed in the relative degradation rates of individual species which suggest that the synthesis and degradation of the various surface membrane glycoproteins proceed at rates that are controlled independently.     

Chemical Studies on the Cell Walls of Leptospira biflexa Strain Urawa and Treponema pallidum Strain Reiter

The preparation and chemical poperties of the cell walls of Leptospira biflexa Urawa and Treponema pallidum Reiter are described. Both cell walls are composed mainly of polysaccharides and peptidoglycans. The data of chemical analysis indicate that the cell wall of L. biflexa Urawa contains rhamnose, arabinose, xylose, mannose, galactose, glucose and unidentified sugars as neutral sugars, and alanine, glutamic acid, α,ε-diaminopimelic acid, glucosamine and muramic acid as major amino acids and amino sugars. As major chemical constituents of the cell wall of T. pallidum Reiter, rhamnose, arabinose, xylose, mannose, galactose, glucose, alanine, glutamic acid, ornithine, glycine, glucosamine and muramic acid have been detected. The chemical properties of protein and polysaccharide fractions prepared from the cells of T. pallidum Reiter were also partially examined.     

Effect of Inhibition of Glycosylation on the Appearance of a 60 kD Membrane Glycopolypeptide in Endomembrane Fractions of Soybean Root

Endomembrane (endoplasmic reticulum, Golgi apparatus, plasma membrane) proteins of soybean (Glycine max) root cells are highly glycosylated. We investigated whether N-linked oligosaccharide moieties are essential for the correct intracellular transport of plant endomembrane glycoproteins. Excised roots were incubated with tunicamycin, to block cotranslational glycosylation of proteins, and dual labeled with [³H]glucosamine and [³⁵S] (methionine, cysteine). In the presence of tunicamycin, the incorporation of glucosamine into membrane proteins was inhibited by 60 to 90% while amino acid incorporation was only slightly affected. Autoradiograms of two-dimensionally separated polypeptides from each endomembrane fraction revealed the presence of at least one new polypeptide in tunicamycin-treated tissue. The new polypeptide was of the same isoelectric point but lower molecular weight than a preexisting polypeptide. The new polypeptide was unreactive to concanavalin A, as opposed to the preexisting polypeptide, suggesting the absence of the glycan portion. Trifluoromethanesulfonic acid and N-glycanase were used to cleave the carbohydrate from the preexisting concanavalin A binding polypeptide. In each case a deglycosylated polypeptide of the same isoelectric point and molecular weight as the new polypeptide from tunicamycin-treated tissue resulted. Since the absence of carbohydrate from the new endomembrane polypeptide did not prevent its appearance on autoradiograms of Golgi and plasma membrane, intracellular transport and intercalation of newly synthesized glycoproteins into plant cell membranes may not require the presence of polysaccharide moieties.     

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.     

Cell Wall Antigens of Pneumocystis carinii Trophozoites and Cysts: Purification and Carbohydrate Analysis of these Glycoproteins

We have purified and biochemically analyzed individual cell wall glycoproteins of Pneumocystis carinii. Our results show that corresponding core glycoproteins constitute the cell wall antigens in both trophozoites and cysts, and glycosylation of these glycoproteins does not appear to be significantly altered during development. Cysts and trophozoites in rat-derived organism preparations were separated from each other by counterflow centrifugal elutriation, then treated with Zymolyase to obtain the cell wall fractions. Gel electrophoresis patterns of these fractions from both life-cycle stages were qualitatively similar. Ten major antigenic glycoproteins in these fractions were purified by preparative continuous elution gel electrophoresis. All ten glycoproteins from cysts and trophozoites contained mannose, glucose, galactose. and N-acetylglucosamine, and some contained traces of fucose. The glycoproteins of cysts had more mannose than their trophozoite counterparts. The trophozoite glycoproteins differed from those of the cyst by the presence of xylose. To examine the species-specificity of glycoprotein glycosylation, preparations of human-derived P. carinii (comprised of mixed life-cycle stages) were also examined and found to contain the same sugars as those found in rat-derived organisms. Most of the purified rat-derived glycoproteins bound Concanavalin A, which was abolished by treatment with N-glycanase. This suggested that the majority of the oligosaccharides were N-linked to the proteins, but attempts to identify carbohydrate linkage sites by amino acid sequencing were hampered by apparent modifications of residues. The peptides derived by cyanogen bromide cleavage revealed distinct size patterns for each glycoprotein, suggesting that they were distinct proteins. Most of the glycoproteins reacted with monoclonal antibodies which recognize a highly conserved epitope on rat P. carinii. Four of the individually purified glycoprotein preparations elicited in vitro cellular immune responses, implicating their involvement in the recognition of P. carinii by host T cells. The identification and characterization of P. carinii cell wall proteins will be helpful in analyzing the relationship of the organism to its mammalian host. Supplementary key words. Biochemical analysis, developmental stages, opportunistic pathogen, structure.     

Lignin Depletion Enhances the Digestibility of Cellulose in Cultured Xylem Cells

Plant lignocellulose constitutes an abundant and sustainable source of polysaccharides that can be converted into biofuels. However, the enzymatic digestion of native plant cell walls is inefficient, presenting a considerable barrier to cost-effective biofuel production. In addition to the insolubility of cellulose and hemicellulose, the tight association of lignin with these polysaccharides intensifies the problem of cell wall recalcitrance. To determine the extent to which lignin influences the enzymatic digestion of cellulose, specifically in secondary walls that contain the majority of cellulose and lignin in plants, we used a model system consisting of cultured xylem cells from Zinnia elegans . Rather than using purified cell wall substrates or plant tissue, we have applied this system to study cell wall degradation because it predominantly consists of homogeneous populations of single cells exhibiting large deposits of lignocellulose. We depleted lignin in these cells by treating with an oxidative chemical or by inhibiting lignin biosynthesis, and then examined the resulting cellulose digestibility and accessibility using a fluorescent cellulose-binding probe. Following cellulase digestion, we measured a significant decrease in relative cellulose content in lignin-depleted cells, whereas cells with intact lignin remained essentially unaltered. We also observed a significant increase in probe binding after lignin depletion, indicating that decreased lignin levels improve cellulose accessibility. These results indicate that lignin depletion considerably enhances the digestibility of cellulose in the cell wall by increasing the susceptibility of cellulose to enzymatic attack. Although other wall components are likely to contribute, our quantitative study exploits cultured Zinnia xylem cells to demonstrate the dominant influence of lignin on the enzymatic digestion of the cell wall. This system is simple enough for quantitative image analysis, but realistic enough to capture the natural complexity of lignocellulose in the plant cell wall. Consequently, these cells represent a suitable model for analyzing native lignocellulose degradation.     

The N-Linked Outer Chain Mannans and the Dfg5p and Dcw1p Endo-α-1,6-Mannanases Are Needed for Incorporation of Candida albicans Glycoproteins into the Cell Wall

A biochemical pathway for the incorporation of cell wall protein into the cell wall of Neurospora crassa was recently proposed. In this pathway, the DFG-5 and DCW-1 endo-α-1,6-mannanases function to covalently cross-link cell wall protein-associated N-linked galactomannans, which are structurally related to the yeast outer chain mannans, into the cell wall glucan-chitin matrix. In this report, we demonstrate that the mannosyltransferase enzyme Och1p, which is needed for the synthesis of the N-linked outer chain mannan, is essential for the incorporation of cell wall glycoproteins into the Candida albicans cell wall. Using endoglycosidases, we show that C. albicans cell wall proteins are cross-linked into the cell wall via their N-linked outer chain mannans. We further demonstrate that the Dfg5p and Dcw1p α-1,6-mannanases are needed for the incorporation of cell wall glycoproteins into the C. albicans cell wall. Our results support the hypothesis that the Dfg5p and Dcw1p α-1,6-mannanases incorporate cell wall glycoproteins into the C. albicans cell wall by cross-linking outer chain mannans into the cell wall glucan-chitin matrix.     

The structure of a mannitol teichoic acid from Bifidobacterium bifidum ssp. Pennsylvanicum

The isolation and analysis of the cell wall and cell wall fractions of Bifidobacterium bifidum ssp. pennsylvanicum are presented. With lysozyme a solubilized cell wall fraction is obtained which contains muramic acid, glucosamine, rhamnose, glucose, mannitol, phosphate and all peptidoglycan amino acids. Its composition did not change with culture age. A glycogen-like glucose polymer which is of cytoplasmic origin is identified in the insoluble cell wall fraction. The solubilized cell wall fraction contains a glucosylated rhamnose polymer which is linked by glycosidic bonds to the peptidoglycan fragments. This polymer is a 1,2-linked or an alternating, 1,2/1,3-linked α-rhamnose chain substituted on average at every second rhamnose residue with an α-linked glucose molecule. Various experiments gave evidence that mannitol and phosphate are present in 4,6-linked mannitol phosphate oligomers which are linked by phosphodiester bonds to the glucosylated rhamnose polymer. These oligomers may fulfill the functions of the more common wall teichoic acids.     

Analyses of cell surface and secreted proteins of primary cultures of mouse extraembryonic membranes

Procedures have been developed for primary culture of 13th day mouse parietal and visceral endoderm, yolk sac mesoderm, and amnion cells. We have analyzed cell surface and secreted proteins of these cultures by labeling the cells with radioactive iodine, glucosamine, or amino acids, and/or by immunofluorescence. Cell surface and secreted proteins of visceral endoderm, yolk sac mesoderm, and amnion cells resemble each other closely, whereas parietal endoderm cells are strikingly different. Unlike the other cell types, parietal endoderm cells synthesize and secrete substantial quantities of a protein tentatively identified as procollagen. These cells also secrete a number of other glycoproteins not observed in the media from the other cultures. It is proposed that the procollagen and one or more of the other unique, secreted glycoproteins are normally constituents of Reichert's membrane. Compared to the other cultures, parietal endoderm cells appear to be deficient in production of LETS protein. However, parietal endoderm—Reichert's membrane complexes analyzed by immunofluorescence directly after dissection from the uterus show an abundant association with LETS protein. It is not clear whether this LETS protein is actually synthesized by the parietal endoderm cells themselves. If so, it is possible that this protein is rapidly degraded after its secretion in parietal endoderm primary cultures. The studies reported here represent a first step in the characterization of cell surface properties of embryonic and extraembryonic cell types. The information already accumulated should be useful in investigations aimed at identification of cells derived from blastocysts and teratocarcinomas in vitro.     

Incorporation of [C]Glucosamine and [C]Mannose into Glycolipids and Glycoproteins in Cotyledons of Pisum sativum L

Developing pea cotyledons incorporate radioactivity in vivo from [¹⁴C]glucosamine and [¹⁴C]mannose into glycolipids and glycoproteins. Several different lipid components are labeled including neutral, ionicnonacidic, and acidic lipids. The acidic lipids labeled in vivo appear similar to the polyisoprenoid lipid intermediates formed in vitro in pea cotyledons. Radioactivity from [¹⁴C]glucosamine and [¹⁴C]mannose is also incorporated into glycopeptides. Considerable redistribution of [¹⁴C]mannose into other glycosyl components found in endogenous glycoproteins is observed. An N-acetylglucosamine to asparagine glycopeptide linkage has been isolated from [¹⁴C]glucosamine-labeled glycoproteins.     

The formation of glycoproteins in tissues of higher plants. Specific labelling with d-[l-14C]glucosamine

1. Radioactivity from d-[l-(14)C]glucosamine is incorporated into ethanol-insoluble compounds of high molecular weight in a number of plant tissues, including roots of corn (Zea mays), callus cells of sycamore (Acer pseudoplatanus), axenic cultures of duckweed (Lemna minor) and germinating seedlings of corn, broad bean (Vicia faba) and barley (Hordeum vulgare). 2. Except in the case of Lemna, where some of the radioactivity was recovered in glucose, hydrolysis of these ethanol-insoluble materials with acid released [(14)C]glucosamine as the major radioactive product. 3. The labelled compounds isolated from Zea roots and the Acer cells are believed to be glycoproteins rather than polysaccharides on the basis of their solubility properties, their charge characteristics and their susceptibility to hydrolysis by 0.5m-potassium hydroxide and by the proteases trypsin and Pronase. Further, radioactive peptides were isolated and purified after Pronase treatment and shown to contain glucosamine as well as a number of amino acids. 4. The experiments therefore indicate that d-[(14)C]glucosamine can be used as a specific precursor of the amino sugar units of plant as well as animal glycoproteins.     

Oxidative coupling of tyrosine and ferulic acid residues: Intra- and extra-protoplasmic occurrence,predominance of trimers and larger products,and possible role in inter-polymeric cross-linking

I discuss the range of oxidative phenolic coupling products formed from the tyrosine residues of cell wall glycoproteins and from the feruloyl residues of wall polysaccharides possibly by the action of peroxidases and/or laccases. In the cases of both tyrosine- and ferulate-coupling, the coupling products are not confined to dimers but include trimers and probably higher oligomers, which are sometimes predominant. Thus, some previous assays, in which specifically dimers were monitored, will have underestimated the extent of phenolic coupling. The possibility is discussed that some of the phenolic coupling products, in both glycoproteins and polysaccharides, are inter-polymeric and that they may therefore act as cross-links in the cell wall. The limitations in the evidence for this hypothesis are stressed. The sub-cellular site of oxidative phenolic coupling is discussed. In-vivo radiolabelling of cultured maize cells with [¹⁴C]cinnamate has shown that, especially in young, rapidly growing cultures, much oxidative coupling of feruloyl-arabinoxylans occurs within the endomembrane system, before secretion of the polysaccharides into the cell wall. Appreciable feruloyl coupling within the cell wall depended on the supply of H₂O₂ and on culture age. The situation with tyrosine coupling in glycoproteins is also debated. Although peroxidase activity has long been known to occur in the endomembrane system, the recent finding of intraprotoplasmic feruloyl coupling provided the first evidence that peroxidases (and/or laccases) may act in this sub-cellular location in vivo. I draw attention to the distinction between peroxidase action (in vivo) and activity (assayed in vitro), and to the unknown origin of H₂O₂ within the endomembrane system.