The isolation and characterization of glycopeptides and mucopolysaccharides from plasma membranes of an ascites hepatoma, AH 130 FN.

Plasma membranes were isolated from an ascites hepatoma, AH 130 FN, a free-cell type subline of AH 130, by the fluorescein mercuric acetate (FMA) method. Glycopeptides and mucopolysaccharides were prepared from the membranes by pronase digestion then fractionated chromatographically and electrophoretically. Isolated fractions were analyzed for amino acid and carbohydrate compositions. The results were compared with those for corresponding fractions from AH 66 and AH 130 ((1974) J. Biochem. 76, 319-333; (1975) ibid., 78, 863-872). The fraction excluded from Sephadex G-50 contained mucopolysaccharides and a series of glycopeptides. The mucopolysaccharides were identified as chondroitin sulfate A on the basis of their chemical composition, electrophoretic behavior on cellulose acetate and digestibility with chondroitinase AC [EC 4.2.2.5]. This contrasts with previous findings that mucopolysaccharides from the corresponding fractions from AH 130 and AH 66 were heparan sulfate. The chemical composition of the glycopeptides, which showed high contents of threonine, serine, galactose, galactosamine, glucosamine, and sialic acid, indicated the presence of glycopeptides with O-glycosidic linkages. The glycopeptides also contained a small but significant amount of aspartic acid, suggesting that N-glycosidic glycopeptides were also contained in this fraction. The fraction included in Sepnadex G-50 contaoned N-glycosidic glycopeptides as major components, since the carbohydrate moieties were composed of fucose, galactose, mannose, glucosamine, sialic acid, and a smaller amount of galactosamine. The presence of galactosamine suggested that O-glycosidic glycopeptides were present as minor components. Glycopeptides with both O- and N-glycosidic linkages were isolated from AH 130, but not from AH 66.     

Nature of the Carbohydrate Side Chains and Their Linkage to the Protein in Chicken Egg White Ovomucin

Some proteolytic digests of chicken egg white ovomucin were fractionated and characterized. It was shown that there are at least three types of carbohydrate side chains in ovomucin; a chain composed of galactose, galactosamine, sialic acid and sulfate in a molar ratio of about 1: 1: 1: 1, a chain composed of galactose and glucosamine in a molar ratio of about 1:1, and a chain composed of mannose and glucosamine in a molar ratio of about 1:1. It was also shown that the carbohydrate side chain composed of galactose, galactosamine, sialic acid and sulfate is linked O-glycosidically to serine or threonine in the protein core of ovomucin.     

A glycopeptide isolated from human gastric juice.

A glycopeptide containing 69% carbohydrate was isolated from human gastric juice. The complex was found to be homogeneous and to have mol.wt. 9600. The glycopeptide consisted of a protein core to which were linked, by O-glycosidic linkages to threonine and N-glycosidic linkages, carbohydrate side chains composed of N-acetylgalactosamine, N-acetylglucosamine, galactose, mannose, fucose and sialic acid, in the proportions 2:10:7:4:12:1.     

Purification and chemical study of a Collocalia glycoprotein]

A glycoprotein was purified from the aqueous extract of "edible bird's nest" (Collocalia) using free flow preparative electrophoresis and represented the main fraction of Collocalia glycoproteins. This glycoprotein is homogeneous upon agarose electrophoresis and slightly polydisperse upon ultracentrifugation (S So 20w = 3,0). The carbohydrate moiety contains galactose, mannose, glucosamine, galactosamine and sialic acid, which is completely released by Clostridium perfringens or Diplococcus pneumoniae neuraminidases and has the same chromatographic behaviour as N-acetyl-neuraminic acid. The peptide part of the glycoprotein is rich in serine, threonine and proline. About 40 p. cent of the hydroxyaminoacids are involved in carbohydrate-peptide linkages.     

The oligosaccharide units of rabbit immunoglobulin G. Multiple carbohydrate attachment sites

The carbohydrate structure of rabbit immunoglobulin G isolated from pooled sera was investigated. Amino sugar analysis of fragments of the molecule allowed three oligosaccharides to be located at separate sites on the H-chain. The corresponding glycopeptides were isolated. The average composition of the C1-oligosaccharide was 2 glucosamine, 1 mannose and 2 galactose residues. It appeared to be present in approx. 15% of the H-chains; the carbohydrate was coupled through the amide group of asparagine in a peptide containing asparagine, glycine and threonine within the Fd fragment of the molecule. The average composition of the C2-oligosaccharide was 1 galactosamine, 1 galactose and either 1 or 2 sialic acid residues. It was present on approx. 40% of the H-chains and was attached glycosidically to the OH group of threonine in a peptide Ser-Lys-Pro-Thr-Cys-Pro-Pro-Glu-Leu in the hinge region of the molecule. The average composition of the C3-oligosaccharide was 5 glucosamine, 2 galactose, 5 mannose, 1 fucose and 1 sialic acid residue. It appeared to be present in all the H-chains and was linked through the amide group of asparagine in a peptide Gln-Gln-Phe-Asn-Ser-Thr-Ile-Arg within the Fc fragment of the molecule.     

Comparison of the properties of glucoamylases from Rhizopus niveus and Aspergillus niger

Some properties of the glucoamylase from Rhizopus niveus have been determined and compared with the comparable properties of the glucoamylase from Aspergillus niger. The enzymes from these organisms possess the following common properties: quantitative conversion of starch to glucose, molecular weights in the range 95,500 to 97,500, and glycoprotein structures with many oligosaccharide side chains attached to the protein moieties of the enzymes. Differences in the glucoamylases exist in electrophoretic mobility, amino acid composition, nature of carbohydrate units, and types of glycosidic linkages. Lysine, threonine, serine, glutamic acid, tyrosine, and phenylalanine differ in the two glucoamylases by 25 to 50%. Whereas the enzyme from R. niveus contains mannose and glucosamine, in the N-acetyl form, as the carbohydrate constituents, the enzyme from A. niger contains mannose, glucose, and galactose. The carbohydrate chains of the R. niveus enzyme are linked by O-glycosidic and N-glycosidic linkages to the protein, while those of the A. niger enzyme are linked by O-glycosidic linkages only. Antibodies directed against the two glucosamylases have been isolated by affinity chromatography and found to be specific for the carbohydrate units of the glucoamylases. Cross reactions did not occur between the glucoamylases and the purified antibodies.     

Epithelial basement membrane of bovine renal tubuli. Isolation and analysis of the carbohydrate chains.

The carbohydrate chains present in the tubular basement membrane of bovine kidney were studied. Digestion with collagenase followed with pronase resulted in a complete solubilization of the basement membrane. The different glycopeptides were purified by gel filtration and ion-exchange chromatography. Two kinds of carbohydrate chains could be characterized: oligosaccharides composed of glucosamine, mannose, galactose, fucose and sialic acid, and glucosylgalactose disaccharides. A very small portion of the oligosaccharide chains (ca. 4%) appeared to be free of sialic acid. The bulk of these chains contained sialic acid and fucose, although in small amounts. Only traces of galactosamine were found.     

Carbohydrates of influenza virus. III. Nature of oligosaccharide-protein linkage in viral glycoproteins.

Four different glycopeptides can be distinguished after pronase digestion of influenza A virus glycoproteins: Ia and Ib, containing N-acetylglucosamine, mannose, galactose, and fucose, and IIa and IIb, containing mannose and N-acetylglucosamine. All glycopeptides yielded N-acetylglucosaminyl-asparagine after mild acid hydrolysis. There was no evidence for O-glycosidic bonds. Thus, the carbohydrate complement is linked to the polypeptide exclusively by N-glycosidic linkages between N-acetylglucosamine and asparagine.     

Biochemical study of cardiac valvular tissue. Biosynthesis in vitro of hexosamine-containing substances in bovine heart valve

1. Two distinct hexosamine-containing substances have been obtained from bovine cardiac valvular tissue which was incubated with labelled glucosamine. These were identified as mucopolysaccharides and glycopeptides respectively, both by the elution pattern from a Sephadex G-50 column and by chemical analysis. 2. In the mucopolysaccharide fraction about 80% of both the total hexosamine and total radioactivity were present; the galactosamine/glucosamine ratio of the amounts was about 1.36. 3. The glycopeptide fractions had about 20% of both total hexosamine and total radioactivity; the galactosamine/glucosamine ratio of amounts was about 0.44. In this fraction, over 15% of radioactivity was present in sialic acid. 4. In contrast with the concentrations of the several chemical components, there were remarkable differences in the biosynthetic activities among the four valves; the tricuspid valve had highest specific radioactivity in all components of both substances, followed then in turn by mitral, aortic and pulmonary valves. 5. Glucosamine in mucopolysaccharides had a rapid rate of turnover, followed then in turn by turnover rates of both glucosamine and galactosamine in glycopeptides, and of galactosamine in mucopolysaccharides. The possible significance of these findings is discussed.     

The carbohydrate components of hydrolysates of gastric secretion and extracts from mucous glands of the gastric body mucosa and antrum

1. The sugars and amino sugars of hydrolysates of gastric secretion were determined by gas-liquid chromatography. 2. All the gastric aspirations examined showed on hydrolysis the presence of fucose, galactose, mannose, glucose, galactosamine, glucosamine, N-acetylneuraminic acid and sulphate. 3. Galactose and glucosamine were always found in equimolar amounts, but the galactose/galactosamine ratio in different aspirations was 2:1, 3:1, 4:1 or 5:1. Repeated gastric aspirations of each subject examined showed constant ratios of these carbohydrate components. 4. Fucose and sialic acid appear to be related to glucosamine and galactosamine respectively. 5. The carbohydrate components of extracts from the mucous glands of the body mucosa and antrum did not differ from those of gastric secretion.     

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.     

Glycoproteines sulfates des membranes de l'oeuf de poule et de l'oviducte Isolement et caracterisation de glycopeptides sulfatesSulfated glycoproteins form egg shell membranes and hen oviduct. Isolation and characterization of sulfated glycopeptides

Sulfated glycopeptides were isolated from pronaisc and tryptic digests of egg shell membranes and hen oviduct. They were precipitated by cationic detergents and separated by preparative electrophoresis, after removal of small quantities of glucuronoglycosaminoglycans detected only in the oviduct (isthmus and magnum). The principal isolated sulfated glycopeptides were divided according to increasing electrophoretic mobilities into two groups A and B. The homogeneity of the purified glycopeptides was confirmed by gel filtration and polyacrylamide gel electrophoresis.Glycopeptides from pool preparation of tissue are analysed and carbohydrate and amino acids average values are estimated. Hexosamines (mainly N-acetylglucosamine), hexoses (galactose, glucose, mannose) and fucose were found in Glycopeptides A. The molar ratio of hexose/hexosamine was 0.4. N-Acetylneuraminic acid and sulfate were also present in Glycopeptides A. The molar ratio of sulfate/hexosamine ranged from 0.1 to 0.25. The Glycopeptides A composition indicated the presence of chains with many glycosyl groups and a few of amino acids residues. The carbohydrate components of Glycopeptides B from egg shell membranes and magnum were found to be hexosamines (N-acetylgalactosamine and N-acetylglucosamine in equimolar proportions), hexoses (galactose mainly and glucose), N-acetylneuraminic acid, and fucose. The molar ratio of hexose/hexosamine was 1. Sulfate was also present and the molar ratio of N-acetylneuraminic acid and sulfate to hexosamine was ranged from 0.8 to 1. The main amino acid residues in these glycopeptides were serine and threonine with destruction of these hydroxyamino acids during alkali treatment. Glycopeptides B probably consist of short carbohydrate chains, linked to the polypeptide through O-glycosidic bonds involving N-acetylgalactosamine and serine and threonine. Approximately 40% of the amino acid residues were linked to carbohydrate chains.Glycopeptides B from egg shell membranes magnum and egg white were very similar in their carbohydrate and amino acid composition and in their properties.Gylcopeptides A from egg shell membranes, isthmus and magnum showed similarities and divergences especially in the amino acid composition. These results suggest that magnum and isthmus in oviduct are both concerned with the synthesis of egg shell membrane glycoproteins.     

Carbohydrate components of the glycopeptides of ovine lutropin

The three tryptic glycopeptides from ovine lutropin, in which two were from the α-subunit (α-56 and α-82 glycopeptides) and one from the β-subunit (β-13 glycopeptide), have been isolated and their carbohydrate compositions analyzed. The results indicate that the α-56 glycopeptide has the highest amount of carbohydrate, whereas the β-13 glycopeptide has the least. In general, each of the glycopeptides has similar distribution of various sugars, i.e. high in mannose and glucosamine and low in fucose, sialic acid, galactose and galactosamine. Within the limit of experimental error, the sum of their carbohydrate composition is in agreement with the published data on the intact hormone or separated subunits.     

Identification of the milk fat globule membrane proteins: I. Isolation and partial characterization of glycoprotein B

The salt soluble proteins from the fat globule membrane of cow's milk were resolved into three fractions by Sephadex column chromatography in sodium dodecyl sulfate. One of the fractions, termed glycoprotein B, was purified by rechromatography to essentially one band on sodium dodecyl sulfate gel electrophoresis. It was found to contain 14% carbohydrate including sialic acid, mannose, galactose, glucose, glucosamine and galactosamine. The amino acid composition of glycoprotein B was determined; it has amino terminal serine and carboxyl terminal leucine. The molecular weight of this glycoprotein as estimated by sodium dodecyl sulfate gel electrophoresis is 49 500.     

Isolation and fractionation of glycopeptides from porcine thyroglobulin

1. Glycopeptides were isolated by gel filtration on Sephadex G-25 and Sephadex G-50 from a Pronase digest of porcine thyroglobulin. 2. Isolated glycopeptides were separated into five main fractions on a column of DEAE-Sephadex A-25. Of these fractions I to III were further purified by SE-Sephadex C-25 or DEAE-Sephadex A-25 column chromatography. Several of the purified glycopeptides were homogeneous on paper electrophoresis. 3. Based on the chemical composition and molecular weight of the fractionated glycopeptides, two distinct types of heterosaccharide chain were demonstrated. 4. One type of the heterosaccharide unit consisted of four to eight residues of mannose and two residues of glucosamine and had a molecular weight of 1000-1700. The other type of unit contained sialic acid, fucose and galactose in addition to mannose and glucosamine and had a molecular weight of about 3600. 5. Mild alkaline treatment of the glycopeptide did not result in the destruction of threonine and serine. 2-Acetamido-1-N-(4-l-aspartyl)-2-deoxy-beta-d-glucopyranosylamine was isolated from partial acid hydrolysates.     

Isolation of the major glycoprotein from plasma membranes of an ascites hepatoma AH 66.

Plasma membranes were isolated from AH 66 cells, some of which had been labeled with [14C]glucosamine, by the following procedure: homogenization of cells which had been hardened by treatment with Zn ions, fractionation of the homogenate by sucrose density gradient centrifugation and purification of the membranes by partition in an aqueous two-phase polymer system. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) of the plasma membranes and subsequent staining of the gel for protein and carbohydrate, and determination of radioactivity on the gel eluates indicated the presence of at least 10 bands of glycoproteins. The major band contained 27% of the total radioactivity incorporated into the plasma membranes and was most heavily stained with the periodate-Schiff reagent. To isolate the major glycoprotein, the membranes were solubilized with 0.6 M lithium diiodosalicylate containing 0.5% Triton X-100, then the solution was treated with phenol. The major glycoprotein, obtained in the aqueous phase, was further purified mainly by repeated chromatographies on Sepharose 6B. The purified preparation was practically homogeneous on SDS-polyacrylamide gel electrophoresis, as judged by radioactivity determination and by carbohydrate staining, but contained small amounts of carbohydrate-free proteins. The major glycoprotein had an apparent molecular weight of 160,000, as determined by SDS-polyacrylamide gel electrophoresis. The final preparation contained about 44% carbohydrate on a weight basis, and the carbohydrate moiety was composed of glucosamine, galactosamine, galactose, mannose, fucose, and sialic acid. This composition indicates that the major glycoprotein contains both N- and O-glycosidically linked oligosaccharide moieties.     

Release of glycopeptides and mucopolysaccharides from ascites hepatoma cells by tryptic treatment.

Two types of ascites hepatoma cells, AH 66 and AH 130 FN, were treated with trypsin to observe the release of complex carbohydrates constituting the plasma membranes. From AH 66 cells, mucopolysaccharide (heparan sulfate) was preferentially released. From AH 130 FN cells, N-glycosidic glycopeptides were preferentially released whereas no mucopolysaccharide (chondroitin sulfate A) was released.     

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.     

Comparison of glycopeptides from control and virus-transformed baby hamster kidney fibroblasts

Glucosamine-labeled glycopeptides from control and virus-transformed BHK fibroblasts were characterized by size, lectin affinity, charge, and composition. As already demonstrated, on the basis of elution position on a column of Sephadex G-50, transformed cells contained a greater proportion of large glycopeptides than did control cells. Transformed cells also contained a larger proportion of glycopeptides which do not bind to Con A-Sepharose. By sequential chromatography on Sephadex G-50, Con A-Sepharose, and DEAE-Sephadex, approximately 40 individual peaks were partially or completely resolved. If sialic acid was removed from the glycopeptides prior to analysis by ion-exchange chromatography, 95% of the glycopeptides from control cells and 85% of the glycopeptides from transformed cells were no longer bound by DEAE-Sephadex. It was concluded that the DEAE-Sephadex elution properties of the glycopeptides are determined almost entirely by the sialic acid content of the molecules. A comparison of the profiles of control and transformed cell glycopeptides simultaneously eluting from columns of DEAE-Sephadex revealed that the differences between the two cells were largely quantitative; however, the possibility of the existence of qualitative differences as well cannot be excluded. In particular, there was one component present on the surface of transformed cells that was virtually absent in control cells. It was degraded by nitrous acid hydrolysis and heparinase and appeared to be heparan sulfate like material. After fractionation, each isolated glycopeptide population was analyzed for carbohydrate and, in some cases, amino acid content. The apparently larger glycopeptides, group A, the dominant population in transformed cells, were found to contain 3 to 4 mannose residues/glycopeptide when the sugars were normalized to sialic acid content. On the basis of the same criteria, group B glycopeptides contained 4-6 mannose residues/glycopeptide. The carbohydrate and amino acid compositions of the glycopeptides from transformed cells were, with a few exceptions, similar to those from control cells. Some isolated glycopeptides appeared to contain both O-glycosidic anad N-glycosidic linkages on the same oligopeptide.     

Glycosylation of vesicular stomatitis virus glycoprotein in virus-infected HeLa cells.

Glycosylation of the envelope glycoprotein of vesicular stomatitis virus was examined using virus-infected HeLa cells that were pulse-labeled with radioactive sugar precursors. The intracellular sites of glycosylation and the stepwise elongation of the carbohydrate side chains of the G protein were monitored by membrane fractionation and gel filtration of Pronase-digested glycopeptides. The results with short pulses of sugar label (5 to 10 mtein linkage (glucosamine and mannose) are added to G which was associated with the rough endoplasmic reticulum-enriched membrane fraction, whereas the more distal sugars (galactose, sialic acid, fucose, and possibly more glucosamine) are added in the light-density internal membrane fraction. Accumulation of mature G was observed in the plasma membrane-enriched fraction. The gel filtration studies indicated that the initial glycosylation event may be the en bloc addition of a mannose and glucosamine oligomer, followed by the stepwise addition of the more distal sugars.