Serum glycoconjugates in children with schizophrenia and conduct and adjustment disorders

The serum glycoproteins represented by the individual protein-bound carbohydrate components and glycosaminoglycans represented by the hexuronic acid contents were determined in the sera of black and Caucasian normal children and children with diagnoses of schizophrenia, conduct disorder, and adjustment disorder. There were no race-related or sex-related differences in glycoproteins and glycosaminoglycans in the sera of normal children. Although the serum glycosaminogltents were determined in the sera of black and Caucasian normal children and children with diagnoses of schizophrenia, conduct disorder, and adjustment disorder. There were no race-related or sex-related differences in glycoproteins and glycosaminoglycans in the sera of normal children. Although the serum glycosaminogltents were determined in the sera of black and Caucasian normal children and children with diagnoses of schizophrenia, conduct disorder, and adjustment disorder. There were no race-related or sex-related differences in glycoproteins and glycosaminoglycans in the sera of normal children. Although the serum glycosaminoglycans were significantly elevated in children with a diagnosis of schizophrenia, the levels were in normal range in children with conduct and adjustment disorders. All of the protein-bound carbohydrates were elevated in schizophrenic children. However, only arabinose and galactosamine were significantly elevated in children with a diagnosis of conduct disorder, while only galactosamine was elevated in children with adjustment disorder. The presence of arabinose in serum glycoprotein was confirmed by chemical ionization-mass spectrometry. The possible causes of the differential elevation of the glycoconjugates in psychiatric disorders in relation to the effect of stress and environment are discussed.     

Studies of the polysaccharides from sunflower heads

Extraction of sunflower heads with ammonium oxalate afforded water-soluble pectin material and water-insoluble glycoprotein material, the carbohydrate portion of which consisted of galacturonic acid and xylose residues; the pectin material defied fractionation with cetylpyridinium chloride. Extraction with hydrochloric acid (pH 1.5) afforded water-soluble and water-insoluble polysaccharide materials. The former, when fractionated with cetylpyridinium chloride, gave a glycoprotein, the carbohydrate moiety of which was composed of galacturonic acid, galactose (major), glucose, arabinose, and xylose, and also a rhamnan. The latter was a glycoprotein, the carbohydrate portion of which consisted of galactose (major), glucose, xylose, and rhamnose residues. Extraction of the sunflower heads with water also gave glycoprotein material, which was fractionated by paper electrophoresis into a glyco-protein, the carbohydrate moiety ofwhich was composed of galacturonic acid (minor), galactose, glucose, xylose, arabinose, and rhamnose (major) residues, and a heteropolysaccharide composed of galactose (major), glucose, xylose, and arabinose residues.     

Glycoproteins from ulva lactuca

Ulva lactuca yielded glycoprotein materials which differed significantly in their carbohydrate and protein moieties. The electrophoretic patterns of the isolated materials showed the presence in each of glycoprotein and polysaccharide components. All the glycoprotein components moved towards the cathode.The glycoprotein material isolated by extraction with NaOH was fractionated on a DEAE-cellulose column into four glycoprotein components and one protein component. All the glycoprotein components contained glucuronic acid, xylose, and rhamnose in different proportions, and in addition two of them contained glucose.     

Carbohydrate Composition of Variant-Specific Surface Antigen Glycoproteins from Trypanosoma brucei

SYNOPSIS. The carbohydrate of variant-specific surface antigen glycoproteins from bloodstream forms of 13 cloned variants of Trypanosoma brucei was analyzed by gas-liquid chromatography. The glycoproteins contained from 6 to 17% carbohydrate by weight, and all contained the same 4 sugars: mannose, galactose, glucose, and glucosamine (probably as N-acetyl-glucosamine). The glycoprotein from variant 048, strain 427 contained (±20%) 11 mannose, 4 galactose, 4 glucose, and 5 glucosamine residues/mole of glycoprotein (molecular weight 65,000). Glucose was an integral component of the glycoproteins, not dissociable by sodium dodecyl sulphate, 8 M urea, or 1 M acetic acid. Some of the glucose was dissociated by trichloroacetic acid. Most of the glycoproteins formed precipitin bands with concanavalin A in Ouchterlony double diffusion, but none formed such bands with wheat germ agglutinin or Ricinus communis lectin (molecular weight 120,000).     

Carbohydrate composition of variant-specific surface antigen glycoproteins from Trypanosoma brucei

The carbohydrate of variant-specific surface antigen glycoproteins from bloodstream forms of 13 cloned variants of Trypanosoma brucei was analyzed by gas-liquid chromatography. The glycoproteins contained from 6 to 17% carbohydrate by weight, and all contained the same 4 sugars: mannose, galactose, glucose, and glucosamine (probably as N-acetylglucosamine). The glycoprotein from variant 048, strain 427 contained (+20%) 11 mannose, 4 galactose, 4 glucose, and 5 glucosamine residues/mole of glycoprotein (molecular weight 65,000). Glucose was an intergral component of the glycoproteins, not dissociable by sodium dodecyl sulphate, 8 M urea, or 1 M acetic acid. Some of the glucose was dissociated by trichloroacetic acid. Most of the glycoproteins formed precipitin with concanavalin A in Ouchterlony double diffusion, but none formed such bands with wheat germ agglutinin or Ricinus communis lectin (molecular weight 120, 000).     

Lectin from rice

N-Acetyl-D-glucosamine-binding lectin was isolated and purified from rice by ammonium sulphate fractionation and affinity chromatography using N-acetyl-D-glucosamine linked Sepharose 6B column. It gave a single hand on Polyacrylamide disc gel. It was identified as a glycoprotein. The purified lectin dissociated into two components on Sephadex G-100 column chromatography,-a higher molecular weight fraction not containing any carbohydrate and a lower molecular weight glycoprotein fraction. The apparent molecular weights of these fractions were 85,000 and 14,500. The lectin agglutinated erythrocytes of human A,B,O groups and of several other mammals and its activity was inhibited only by N-acetyl-D-glucosamine. The glycopeptide isolated by pronase digestion of the lectin was homogeneous and did not possess agglutinating activity. It contained about 10% carbohydrate of which xylose, arabinose and glucose were the major components.     

Estimating glycoprotein carbohydrate chain structures by lectin reactivities in polyacrylamide gels

Complex mixtures of cellular glycoproteins contain a myriad of different carbohydrate chains that cannot be easily analyzed without rigorous purification of each individual glycoprotein. We have analyzed the carbohydrate chains in complex mixtures of cellular glycoproteins by separation using sodium dodecyl-sulfate polyacrylamide gel electrophoresis and interacting the gels with several 125I-labeled lectins. By use of in situ chemical modifications of the glycoproteins after their electrophoretic separation together with the known carbohydrate-binding specifities of several lectins, it has been possible to estimate glycoprotein carbohydrate chain structures. As an example we have examined the cellular glycoproteins of a ovary-colonizing metastatic variant of B16 melanoma and report the types of carbohydrate chains that are found on various melanoma glycoproteins.     

Developmental regulation of the carbohydrate composition of glycoproteins associated with central nervous system myelin

Abstract: Glycoproteins from central nervous system myelin were evaluated for developmental alterations in their carbohydrate composition by autoradiographic analysis of radioiodinated lectin binding after separation by high-resolution sodium dodecyl sulfate-pore gradient slab gel electrophoresis (SDS-PGE). Sixteen lectin-binding components were assessed in highly purified myelin preparations from 15-day, 18-day, and adult rat brains, using the lectins Triticum vulgaris (wheat germ agglutinin) and Ulex europeus (gorse agglutinin I). Developmental changes in lectin binding for individual glycoproteins were evaluated semiquantitatively by comparing densitometric scans of the auto radiographs. Both increases and decreases in lectin binding for individual components were observed as a consequence of development, as well as the appearance and disappearance of lectin binding to three low-molecular-weight components. No changes in electrophoretic mobility and hence glycoprotein molecular weight were observed in any components when using these lectins. These developmental changes in lectin binding suggest that increases in glycoprotein (receptor) density occur, as well as an elaboration of oligosaccharide branching for individual glycoproteins. In addition, the appearance of a new glycoprotein in the adult myelin membrane could imply a new functional role not present in the immature membrane. These observations suggest that dynamic alterations of myelin-associated glycoproteins occur during development. Such developmental regulation of membrane glycoproteins increases the significance of their potential role in myelination and myelin maintenance.     

Biogenesis of microsomal membrane glycoproteins in rat liver. I. Presence of glycoproteins in microsomes and cytosol

The glycoproteins of microsomes and cytosol were studied. Various washing procedures did not release the proteins from the microsomes, and immunological tests demonstrated that the sialoproteins are not serum components. Low concentrations of deoxycholate and incubation in 0.25 M sucrose solution liberated a small amount of microsomal sialoprotein and this fraction exhibited a high degree of labeling of protein-bound N-acetylneuraminic acid. A part of the glycoprotein fraction could not be solubilized, even with a high concentration of the detergent. Thoroughly perfused rat liver contained sialoproteins in the particle-free supernate. The level of sialoprotein present could not be due to contamination with serum or broken organelles. The high in vivo incorporation of [3H]glucosamine into protein-bound sialic acid of Golgi membranes and cytosol was paralleled by a delayed and lesser rate of incorporation into the rough and smooth microsomal membranes. This incorporation pattern suggests the possibility that the glycoproteins of cytosol and Golgi may later be incorporated into the membrane of the endoplasmic reticulum.     

Isolation and partial characterization of two antigenic glycoproteins from rye-grass (Lolium perenne) pollen.

Two glycoproteins have been purified from a buffer extract of rye-grass (Lolium perenne) pollen. Both migrated as single bands on sodium dodecyl sulphate/polyacrylamide gels. Glycoprotein 1 (0.8 mg/g of pollen) had a apparent mol.wt. of 33 000 and contained 95% protein and 5% carbohydrate. The monosaccharides glucose, galactose, mannose, arabinose and N-acetylglucosamine were present in the proportions 3:3:1:2:1. Glycoprotein 2 (0.4 mg/g of pollen) had an apparent mol. wt. of 68000 and contained 88% protein and 12% carbohydrate. The monosaccharides glucose, galactose, mannose, fucose, xylose, arabinose and N-acetylglucosamine were present in the proportions 4:7:13:5:8:6:6. This glycoprotein bound concanavalin A and Lotus tetragonolobus (asparagus pea) lectin. Radioallergosorbent (RAST) inhibition tests showed that Glycoprotein 1 is an effective allergen, whereas Glycoprotein 2 has less allergenic activity. A method for performing both lectin-binding assays and RAST inhibition tests using microtitre trays is described.     

Biosynthesis of abnormally glycosylated hepatoma secretory proteins in cell cultures

We studied, by electrophoretic techniques, the physiochemical properties of 4 glycoproteins, alpha 1-antitrypsin, alpha 1-antichymotrypsin, alpha 1-acid glycoprotein and transferrin synthesized by three different human hepatoma cell lines. A common feature was the export of glycoproteins with retarded electrophoretic mobility, indicating incomplete sialylation, and a predominance of atypical, highly branched carbohydrate chains. The abnormal glycosylation pattern may be specific for malignant transformation of hepatocytes and possibly related to the intracellular accumulation of some of these proteins in malignant cells.     

A galactose-rich, cell-wall glycoprotein from styles of Nicotiana alata

A basic, galactose-rich style glycoprotein (GaRSGP) encoded by a previously characterized style-specific cDNA (NaPRP4) has been isolated from the styles of Nicotiana alata and structurally characterized. The glycoprotein is associated with cell walls in the transmitting tract and is composed of approximately 25% (w/w) protein and 75% (w/w) carbohydrate. The purified glycoprotein appears as a smear of between 45–120 kDa on SDS—PAGE; the deglycosylated protein backbone has an apparent molecular weight of approximately 30 kDa. The glycoprotein is rich in the amino acids lysine, proline, and hydroxyproline and in the monosaccharides galactose and arabinose. It is one of only a few proline/hydroxyproline-rich glycoproteins (P/HRGPs) to be characterized both as a cDNA-clone and protein. Glycans are attached to the protein backbone through both O - and N -glycosidic linkages with the majority of the carbohydrate being O -linked and consisting of short, highly branched chains terminating primarily in galactose residues. A carbohydrate epitope(s) is found on both GaRSGP and another style-specific glycoprotein but not on glycoproteins from other tissues. This finding provides further evidence for the existence of a style-specific carbohydrate epitope(s) which may play a role in style function.     

Biochemical Characteristics of Pollen Wall Glycoproteins Separated on Ricin-Sepharose 4B in Cucurbita pepo

By affinity chromatography on ricin-Septarose 4B. pollen wall glycoproteins of Cucurbita pepo were isolated from pollen wall proteins which were prepared by extracting the pollen with isotonic Tris-HC1 buffer. Gas chromatographic analyses showed that the carbohydrate fraction of the pollen wall glycoproteins contained 0.7% rhamnose, 8.1% fucose, 5.1% arabinose, 5.1% xylose, 36.5% galactose, 36.2% glucose, and two unknown components. Isoelectric focusing PAGE showed that the pollen wall glycoproteins contained 6 components, with their respective pI at 3.8, 4.8, 5.4, 6.2, 6.7 and 7.6, and that the relative content of each of them was 40.6%, 15.6% 8.3%, 14.4%, 7,9%, and 13.2%, respectively. The amino acid composition of the 6 glycoproteins was determined with amino acid analyzer. Serine, glutamic acid and glycine were some of the predominent amino acids. These three, amino acids constitute more than 49% of the total amino acids.     

Concentrations of carbohydrate components of glycoproteins in rat serum and erythrocyte and leukocyte membranes in hyperthyroidism

The content of carbohydrate components of glycoproteins in blood serum and erythrocyte and leucocyte membranes was studied as affected of various doses of L-thyroxin. It is established that administration of L-thyroxin small doses increases the level of glycoprotein components in blood serum and cells. Administration of median and especially of high doses of the hormone already on the 12th of the experiment lowers sharply the components level in blood serum and increases it in the membranes of erythrocytes and to a less extent in the membranes of leucocytes. By the 24th day of the experiment the total content of carbohydrate components in the membranes decreases (16.3-25.7% below the normal level). A moderate positive correlation is established between the changes in the content of certain components of glycoproteins in the blood serum and cell membranes in rats with hyperthyreosis induced by administration of L-thyroxin small doses. In the rest cases such a regularity is observed only between the indices in the blood cell membranes.     

Origin of the minor glycoproteins of murine leukemia viruses.

Polyacrylamide gel electrophoretic analysis and immunoprecipitation were used to study glycoproteins from purified Rauscher murine leukemia virus (R-MuLV) and from AKR thymic lymphoblastoid cell membranes. In addition to gp70, a minor glycoprotein of approximately 52,000 daltons (gp52) was demonstrated in purified R-MuLV preparations, which was antigenically related to gp70. Analysis of R-MuLV glycopeptides obtained after exhaustive Pronase digestion showed that gp70 has at least two different glycopeptide size classes with molecular weights of 5,100 and 2,900, respectively. gp52, however, contained only a single glycopeptide size class of approximately 5,100 daltons, indicating that the two glycoproteins contain distinct carbohydrate components. Trypsin treatment of R-MuLV converted gp70 into a product with a molecular mass of approximately 52,000 daltons as well as a 45,000-dalton minor product, with little effect on virus infectivity. Similarly, trypsin treatment of 125I-labeled glycoproteins derived from AKR mouse lymphoblastoid cell membranes generated fragments antigenically related to gp70 and similar in size to those obtained by trypsin treatment of R-MuLV. In both cases, the appearance of cleavage products was accompanied by a decrease in gp70 during trypsin treatment. The occurrence of glycosylated components antigenically related to gp70 in AKR membrane glycoprotein preparations and in purified R-MuLV preparations which were similar to those generated by trypsin treatment supports the concept that these minor components arise from proteolytic cleavage of gp70.     

Further Studies of Pollen Wall Glycoproteins in Cucurbita pepo L.

Samples of pollen wall protein of Cucurbita pepo were prepared as reported in previous paper. Gas chromoatographic analyses snowed that the carbohydrate fraction of the pollen wall glycoprotcin contained 20.4% rhamnose, 15.3% fucose, 11% mannose, 11% galactose, 31% glucose, 4% arabinose and traces of xylose. The glycoproteins were further purified by Con. A affinity chromatography, Isoelectric focussing electrophoresis of the purified sample showed 3 PAS-positive bands, with respective PI 5.2, 6.0 and 6.3. The glycoprotein samples were subjected to hydrolysis with 6N HC1. After hydrolysis, the mixture was analyzed for amino acid composition with Backman 121-MB automatic amino acid analyzer, Results show the amino acid composition of the 3 glycoprotein was very similar, They all have glycine, glutamic acid and serine as their major component (these three amino acids constitute 50–60% of the total amino acids); and they all contain very small amount of methionine, phenylalanine, isoleucine and tyrosine. The lysine content of each glycoprotein is consistent with its respective PI, the glycoprotein which contains more lysine has higher PI.     

Preferential degradation of the terminal carbohydrate moiety of membrane glycoproteins in rat hepatoma cells and after transfer to the membranes of mouse fibroblasts

Glycoproteins in the plasma membrane of rat hepatoma cells were labeled at their externally exposed tyrosine residues with 131I and at their galactose and sialic acid residues with 3H. The degradation of both isotopes in the total cell protein fraction, in glycoproteins purified by concanavalin A, and in glycoproteins separated on two-dimensional gels was determined. Similarly, the total cellular membrane glycoproteins were metabolically labeled with [35S]methionine and [3H]fucose. The fate of both incorporated labels was followed by lectin chromatography or by precipitation of the proteins with specific antibodies followed by electrophoretic gel separation. In both labeling experiments, the carbohydrate markers were lost from the ligand- recognized fraction with similar kinetics as from the total cell protein fraction. In some glycoprotein species which were separated by two-dimensional gel electrophoresis, the polypeptide portion exhibited up to a twofold slower rate of degradation relative to that of the carbohydrate moiety. This difference is most pronounced in carbohydrate- rich glycoproteins. To corroborate this finding, double-labeled membrane glycoproteins were incorporated into reconstituted phospholipid vesicles which were then transferred via fusion into the plasma membrane of mouse fibroblasts. Both the polypeptide and carbohydrate moieties of the transferred membrane glycoproteins were degraded with the same relative kinetics as in the original hepatoma cells. The rate of degradation is mostly a function of the structural properties of the membrane components as shown by the preservation of metabolically stable fucogangliosides of Reuber H-35 hepatoma cells transferred onto the fibroblasts. The technique of insertion of membrane components into the plasma membrane of another cell should assist in the elucidation of the exact route and mechanism of membrane protein destruction.     

EVIDENCE FOR THE CLOSE ASSOCIATION OF A GLYCOPROTEIN WITH MYELIN IN RAT BRAIN

Abstract— Myelin was purified from rats which had been injected intracerebrally with radioactive fucose in order to label specifically the glycoproteins. Myelin contained a small amount of fucose-labelled glycoproteins in comparison to that in other subcellular fractions, but polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate revealed a unique pattern of radioactive glycoproteins dominated by a major peak. The same glycoprotein was not prominent in the other subcellular fractions which were examined. This major glycoprotein in the myelin fraction was also labelled after injection with [³H]glucosamine or N -[³H]acetylmannosamine. It was the most intensely staining myelin protein when gels were treated with periodic acid-Schiff reagents, an indication that, in terms of protein-bound carbohydrate, it is the major glycoprotein in the myelin fraction. The glycoprotein was present in myelin purified from rats ranging in age from 14 days to 14 months. Extensive recycling of the myelin through the purification procedures did not significantly reduce the amount of glycoprotein in the myelin. Double label experiments with [³H]fucose and [¹⁴C]fucose were used to compare glycoproteins in myelin purified from white and grey matter, respectively, and from mixed homogenates of myelinated and unmyelinated brain. The results obtained from these experiments suggested that the glycoprotein is closely associated with myelin and that it is not in an unrelated contaminating structure. Possible locations of the glycoprotein are discussed. They include the myelin membrane itself, the oligodendroglial plasma membrane, and the axolemma of myelinated axons.     

Glycosidase inhibitors: inhibitors of N-linked oligosaccharide processing.

The biosynthesis of the various types of N-linked oligosaccharide structures involves two series of reactions: 1) the formation of the lipid-linked saccharide precursor, Glc3Man9(GlcNAc)2-pyrophosphoryl-dolichol, by the stepwise addition of GlcNAc, mannose and glucose to dolichyl-P, and 2) the removal of glucose and mannose by membrane-bound glycosidases and the addition of GlcNAc, galactose, sialic acid, and fucose by Golgi-localized glycosyltransferases to produce different complex oligosaccharide structures. For most glycoproteins, the precise role of the carbohydrate is still not known, but specific N-linked oligosaccharide structures are key players in targeting of lysosomal hydrolases to the lysosomes, in the clearance of asialoglycoproteins from the serum, and in some cases of cell:cell adhesion. Furthermore, many glycoproteins have more than one N-linked oligosaccharide, and these oligosaccharides on the same protein frequently have different structures. Thus, one oligosaccharide may be of the high-mannose type whereas another may be a complex chain. One approach to determining the role of specific structures in glycoprotein function is to use inhibitors that block the modification reactions at different steps, causing the cell to produce glycoproteins with altered carbohydrate structures. The function of these glycoproteins can then be assessed. A number of alkaloid-like compounds have been identified that are specific inhibitors of the glucosidases and mannosidases involved in glycoprotein processing. These compounds cause the formation of glycoproteins with glucose-containing high mannose structures, or various high-mannose or hybrid chains, depending on the site of inhibition. These inhibitors have also been useful for studying the processing pathway and for comparing processing enzymes from different organisms.     

Studies on Stigma Pellicle Glycoproteins of Raphanus sativus L.

Stigma surface diffusates of Raphanus sativus were subjected to polyacrylamide gel electrophoresis. There appeared protein bands, one of which gave positive PAS reaction, indicating it was glycoprotein. SDS polyacrylamide gel electrophoresis showed that the stigma diffusates contained many protein bands. By comparing their mobilities with those of standard proteins of known molecular weights, the molecular weights of some of the major fractions were estimated to be 15000, 30000—46000 and 70000 daltons. After Schiff reagent staining two main glycoprotein fractions appeared on the SDS gel electrophoretic pattern. Their molecular weights were estimated to be lower than 15000 and higher than 100000 daltons. By using acrylamide gel isoelectric focusing method, it was found that the stigma surface diffusates contained an acidic glycoprotein with pH of about 3.7. The amino acid composition of the purified stigma glycoprotein was determined with amino acid analyzer. Glycine, glutamic acid, serine, aspartic acid were some of the predominant amino acids. The diffusates were analysed by gasliquid chromatography for sugars. Results showed that the carbohydrate fraction of the glycoprotein consisted of arabinose 17.3%; galactose 19.1%, xylose 8.1%, mannose 5.4%, glucose 23.7%, rhamnose and/or fucose 26.4%. In the stigma surface diffusates of Raphanus sativus, the content of protein was estimated to be 16% and that carbohydrate was 11%.