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.     

The carbohydrate prosthetic groups of rat fibrinogen plasmic fragment E.

Rat fibrinogen plasmic fragment E was found to contain one oligosaccharide chain per gamma-chain attached by a glycosylamine linkage. The oligosaccharide was composed of 1 sialic acid, 1 galactose, 2 mannose and 2 glucosamine residues. The probable sequence from the nonreducing end was sialic acid leads to galactose beta leads to mannose alpha leads to mannose alpha leads to glucosamine leads to glucosamine. No difference in the rate of clearance from the rat circulation could be detected between native and desialated fragment E. A non-denaturing method for the purification of fragment E is described.     

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

Plasma membranes were isolated from an ascites hepatoma, AH 130, by the fluorescein mercuric acetate (FMA) method. Glycopeptides and mucopolysaccharides were prepared by digesting the membranes with pronase, then by fractionating the digest chromatographically and electrophoretically. Isolated fractions were analyzed for their amino acid and carbohydrate compositions. Results were compared with those for corresponding fractions from AH 66 (J. Biochem. 76, 319-333 (1974)). Mucopolysaccharides and a series of glycopeptides were isolated from the fraction excluded from Sephadex G-50. The mucopolysaccharides were identified as a family of heparan sulfates with different electrophoretic mobilities. The glycopeptides contained serine, threonine, galactose, galactosamine, glucosamine, and sialic acid as the major constituents as aspartic acid and mannose as minor ones. This suggests that most of the carbohydrate moieties are linked to serine or threonine (O-glycosidic), and that some are linked to asparagine (N-glycosidic). No nearly purely O-glycosidic glycopeptides were found in this fraction from AH 130, through they were the major glycopeptides from the AH 66 plasma membranes. In the fraction included in the gel, glycopeptides containing fucose, galactose, mannose, glucosamine, glaactosamine, and sialic acid were found. The presence of galactosamine suggests that some of the glycopeptides are O-glycosidic though most are N-glycosidic. In the corresponding fraction from AH 66, nearly purely N-glycosidic glycopeptides were found.     

Structure of the carbohydrate chain of free secretory component from human milk.

Secretory component from human milk was found to contain 23.4% carbohydrate, which includes galactose, mannose, fucose, glucosamine, and sialic acid. Secretory component could be degraded by pronase or base-borohydride to yield the same, single type of carbohydrate chain. In the glycopeptide produced by pronase digestion, aspartic acid was the only amino acid present in molar quantities after amino acid analysis, which suggests that the carbohydrate moiety is linked to the polypeptide chain at asparagine residues. The positions of links between the various sugar units were studied by methylation analyses of: secretory component, periodate-oxidized and reduced secretory component, the fragment produced by base-borohydride treatment, and the pronase glycopeptide after treatment with specific glycosidases. Sugars released from the glycopeptide by various glycosidases were also quantitated. From the results of these studies a branched chain structure was assigned to the carbohydrate chain of secretory component.     

The carbohydrate moiety of Japanese monkey pepsinogens. Its composition and site of attachment to protein.

Identification and determination of the carbohydrate component of Japanese monkey pepsinogens have been performed, and the amino acid sequence around the carbohydrate chain has been investigated. Glycopeptides were prepared by successive digestion of pepsinogens with thermolysin and aminopeptidases. Analyses of their carbohydrate composition by paper and gas-liquid chromatography showed the presence of 4 glucosamine, 6 galactose, 6–8 mannose, and 8–10 fucose residues per molecule of the carbohydrate chain, among which the high content of fucose is especially unique. The amino acid sequence of a major glycopeptide was deduced to be Ile-Gly-Ile-Gly-Thr-Pro-Gln-Ala-Asn, in which the asparagine residue is the site of attachment of the carbohydrate chain.     

The isolation and partial characterization of the major glycoprotein (LGP-I) from the articular lubricating fraction from bovine synovial fluid.

The articular lubricating fraction from bovine synovial fluid was prepared by repeated fractionation in three consecutive CsCl density gradients to remove completely traces of hyaluronic acid. The major glycoprotein consituent (LGP-I) was then isolated by repeated gel-permeation chromatography. The yield of the LGP-I component was about 20 mg/litre of synovial fluid. Sedimentation-equilibrium measurements showed that this glycoprotein was homogeneous and the mol.wt. was calculated to be 227500. Amino acids represented 43% (w/w) and carbohydrate constituents 44% (w/w) of the molecule. Threonine, glutamic acid, proline and lysine (224, 127, 242 and 128 residues/1000 residues respectively) were the major amino acids. Galactosamine, galactose and N-acetylneuraminic acid (202, 162 and 114 residues/molecule of LGP-I component respectively) accounted for 98% of the total carbohydrate residues present. Small amounts of mannose and glucosamine (1 and 9mol respectively/mol of LGP-I component) were also present. After treatment of LGP-I component with alkali and NaB3H4 radioactivity was incorporated into alpha-aminobutyric acid and alanine in a molar ratio of 4:1, and radioactive galactosaminitol was isolated by ion-exchange chromatography from a cleaved oligosaccharide fraction. These data demonstrate the presence of threonine and serine -O-GalNAc linkages, but only 25% of the theoretical likages involving threonine were cleaved by a beta-elimination reaction. Digestion of LGP-I component with Pronase followed by chromatography on DEAE-cellulose yielded glycopeptide fractions with a similar amino acid and carbohydrate composition to the intact molecule. Treatment of desialylated and intact LGP-I component with galactose oxidase followed by reduction with NaB3H4 revealed the presence of 52mol of terminal galactose in the intact molecule and 153mol of galactose/mol of LGP-I component after treatment with neuraminidase. The data indicate the LGP-I component is composed of a single polypeptide chain containg more than 150 oligaosaccharide side chains composed of O-GaINAc-Gal distributed over the length of the peptide chain and that terminal sialic acid residues are linked to galactose in two-thirds of these side chains.     

The carbohydrate–polypeptide linkages, the amino acid sequences of the peptides adjacent to some of these bonds, and the composition and size of the carbohydrate units of α1-acid glycoprotein

1. The glycopeptides derived from a proteolytic digest of sialic acid-free α₁-acid glycoprotein were separated on a DEAE-cellulose column into five main fractions. 2. The average molecular weight of these glycopeptides was 2400, except for one fraction whose molecular weight was 3100. The average molecular weight of the sialic acid-free carbohydrate units was found to be 2200. From these data and the carbohydrate content of the native protein and the assumed molecular weight of 44000, it was concluded that α₁-acid glycoprotein probably possesses five carbohydrate units. The sialic acid-containing carbohydrate units of this glycoprotein have an average molecular weight of 3000, except for one unit the molecular weight of which is significantly higher. 3. The N-, non-N- and C-terminal amino acids of the main glycopeptides were determined. Aspartic acid and threonine occur in most peptides. Alanine, glycine, proline, serine and lysine were present in varying amounts. Traces of other amino acids were also found. 4. The amino acid sequence of three main glycopeptides was established and indicated that these glycopeptides are located at different positions of the polypeptide chain of the glycoprotein. These sequences are: Asp(NH₂)-Pro-Lys; Thr-Asp(NH₂)-Ala; Asp(NH₂)-Gly-Thr. 5. From the results of a series of chemical reactions (periodate oxidation, hydrazinolysis, dinitrophenylation, mild acid hydrolysis) it was shown that the hydroxyl group of the N-terminal threonine and the -amino group of lysine are free and that the β-carboxyl group of aspartic acid is present as amide. It was concluded that this amide group is involved in the carbohydrate–polypeptide linkages of at least four carbohydrate units of α₁-acid glycoprotein. 6. The carbohydrate composition of the sialic acid-free glycopeptides was determined in terms of moles of neutral hexoses, glucosamine and fucose/mole. 7. Fucose, at least to the larger part, is not linked to sialic acid, and its (glycosidic) linkage is significantly more stable toward acid hydrolysis than the bond of the sialyl residues. 8. Heterogeneity of the carbohydrate units of α₁-acid glycoprotein was found with regard to size and to content of fucose and sialic acid.     

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 structure of a glycopeptide isolated from the yeast cell wall

1. Glycopeptides containing mannose were extracted from isolated yeast cell walls by ethylenediamine and purified by treatment with Pronase and fractionation on a Sephadex column. 2. A glycopeptide that appeared homogeneous on electrophoresis and ultracentrifugation had a molecular weight of 76000, and contained a high-molecular-weight mannan and approx. 4% of amino acids. 3. The amino acid composition of the peptide was determined. It was rich in serine and threonine and also contained glucosamine. No cystine and methionine were detected. 4. The glycopeptide underwent a beta-elimination reaction when treated with dilute alkali at low temperatures. The reaction resulted in the release of mannose, mannose disaccharides and possibly other low-molecular-weight mannose oligosaccharides. During the beta-elimination reaction the dehydro derivatives of serine and threonine were formed. One of the linkages between carbohydrate and amino acids in the glycopeptide is an O-mannosyl bond from mannose and mannose oligosaccharides to serine and threonine. 5. After the beta-elimination reaction the bulk of the mannose in the form of the large mannan component was still covalently linked to the peptide. This polysaccharide was therefore attached to the amino acids by a linkage different from the O-mannosyl bonds to serine and threonine that attach the low-molecular-weight sugars. 6. Mannan was prepared from the glycopeptide and from the yeast cell wall by treatment of the fractions with hot solutions of alkali. The mannan contained aspartic acid and glucosamine and some other amino acids. The aspartic acid and glucosamine were present in equimolar amounts; the aspartic acid was the only amino acid present in an amount equivalent to that of glucosamine. Thus there is the possibility of a linkage between the mannan and the peptide via glucosamine and aspartic acid. 7. Mannose 6-phosphate was shown to be part of the mannan structure. Information about the structure of the mannan and the linkage of the glucosamine was obtained by periodate oxidation studies. 8. The glucosamine present in the glycopeptide could not be released by treatment with an enzyme preparation obtained from the gut of Helix pomatia. This enzyme released glucosamine from the intact cell wall. Thus there are probably at least two polymers containing glucosamine in the cell wall. 9. The biosynthesis of the mannan polymer in the yeast cell wall is discussed with regard to the two types of carbohydrate-amino acid linkages found in the glycoprotein.     

Structural studies on a glycoprotein isolated from alveoli of patients with alveolar proteinosis.

A major glycoprotein 36 000 molecular weight) has been isolated from lung lavage of patients with alveolar proteinosis and found to contain five residues of hydroxyproline, fifty residues of glycine, three residues of methionine, 3 mol of sialic acid, 4.4 mol of mannose, 4.0 mol of galactose, 6.0 mol of glucosamine, and 1 mol of fucose. Cyanogen bromide (CNBr) treatment of the glycoprotein resulted, as expected, in four peptides of apparent molecular weights of 18 000, 12 000, 5000 and 1000, respectively. The chemical compositions of the CNBr peptides indicate the presence of hydroxyproline and high amounts of glycine in all but one of the peptides; two of the four CNBr peptides contain carbohydrate. Gel filtration, acrylamide gel electrophoresis and end-group analyses of the native glycoprotein and its CNBr peptides indicate that the peptides are homogeneous. End-group analyses of the CNBr cleavage products assign the 18 000 molecular weight peptide to the NH2-terminal portion and the 1000 molecular weight peptide to the COOH-terminal portion of the native glycoprotein molecule. Pronase digestion of the 36 000 molecular weight glycoprotein, followed by gel filtration and cation exchange chromatography, resulted in two fractions. One fraction was acidic and contained all the carbohydrate, a high content of aspartic acid and no hydroxyproline. The other fraction was basic and contained 8.4% hydroxyproline, 14% proline, 28% glycine and no carbohydrate, suggesting the presence of collagen-like sequence in the peptide chain. Paper electrophoresis of the basic fraction demonstrated two components, the amino acid compositions of which are identical to those of collagen. Partial amino-terminal sequence analysis of one of the CNBr peptides (18 000 molecular weight) indicated the presence of -Fly-Pro-HyP-Gly-sequence in the peptide chain, which confirms our suggestion that collagen-like regions are present in the native glycoprotein molecule. Limited acid hydrolysis of the acidic fraction and subsequent fractionation of the acid hydrolysate using Dowex column yielded a fraction which produced brown colour with ninhydrin reagent. Paper chromatography of this fraction demonstrated a large component which also stained brown with ninhydrin reagent. After acid hydrolysis, this component was found to consist of equal amounts of asparitic acid and glucosamine, indicating that the N-acetylglucosamine of the oligosaccharides is linked to the asparagine residue of the peptide. No serine or threonine linkages are present.     

Structural characterization of a glycoprotein isolated from alveoli of patients with alveolar proteinosis.

A soluble glycoprotein of Mr = 80,000 has been isolated from lung lavage of patients with alveolar proteinosis and found to contain 5 residues of hydroxyproline, 91 residues of glycine, 3 residues of methionine, 3.8 molecules of sialic acid, 6 molecules of mannose, 5.9 molecules of galactose, 1 molecule of fucose, and 9.1 molecules of glucosamine. Cyanogen bromide (CNBr) treatment of the glycoprotein resulted in four peptides with molecular weights of 36,000, 27,000, 12,000, and 5,000. The chemical compositions of the CNBr peptides indicated the presence of hydroxyproline and high amounts of glycine in all but one of the peptides; two of the four CNBr peptides contained carbohydrate. Limited trypsin digestion of the glycoprotein of Mr = 80,000 resulted in four peptides with molecular weights of 62,000, 36,000, 26,000 and 18,000, the latter being the NH2-terminal peptide of the native glycoprotein molecule. The peptide of Mr = 26,000 was found to be the COOH-terminal peptide.     

Purification and characterization of a prokaryotic glucoprotein from the cell envelope of Halobacterium salinarium.

The glycoprotein which accounts for approximately 50% of the protein and all of the nonlipid carbohydrate of the cell envelope of Halobacterium salinarium (Mescher, M. F., Strominger, J. L., and Watson S. W. (1974) J. Bacteriol. 120, 945-954) has been purified and partially characterized. The glycoprotein has an apparent molecular weight of 200,000, is extremely acidic, and has a carbohydrate content of approximately 10 to 12%. The carbohydrate included neutral hexoses, amino sugar, and uronic acid. Information regarding the number, composition, and mode of attachment of the carbohydrate chains was obtained by isolation and examination of the glycopeptides derived from degradation of cell envelope protein with trypsin and pronase. Trypsin digestion resulted in two glycopeptides. One of these was large (approximately 55,000 daltons) and had most of the neutral hexose linked to it. The carbohydrate moieties consisted of di- and trisaccharides of glucosylgalactose and (uronic acid, glucose)-galactose attached via O-glycosidic linkages between galactose and threonine. The other tryptic glycopeptide had a relatively large heterosaccharide attached to it via an alkaline-stable linkage. The heterosaccharide contained 1 glucose, 8 to 9 galactose, 1 mannose, and 10 to 11 glucosamine residues, and approximately 6 residues of an unidentified amino augar. The alkaline stability of the linkage and the amino acid composition of glycopeptides resulting from Pronase digestion of the tryptic glycopeptide showed that the heterosaccharide was attached to an asparagine residue, presumably via an N-glycosylamine bond to the amide group. The intact glycoprotein has a single N-linked heterosaccharide, 22 to 24 O-linked disaccharides, and 12 to 14 O-linked trisaccharides per molecule. N- and O-glycosidic linkages are the most common carbohydrate-protein linkages in mammalian glycoproteins but, to our knowledge, this is the first report of either type of linkage in a prokaryotic cell envelope protein.     

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.     

Subunit glycosylation of Lupinus angustifolius seed globulins

Reduced polypeptide subunits of α-, β- and γ-conglutins from Lupinus angustifolius seeds were resolved by preparative SDS gel electrophoresis of the fluorescent labelled proteins, into four, six and two major components, respectively. All subunits were glycosylated, to varying degrees, containing mannose, galactose and glucosamine. The major glycopeptides released by pronase digestion of each conglutin had similar galactose/mannose ratios; the MW of the glycopeptide released from α- and β-conglutin was ca 5000. Although on average, each molecule of α-conglutin contains one main oligosaccharide chain, and β-conglutin two, the presence of carbohydrate in all polypeptide subunits suggests that some subunits may arise by proteolytic cleavage of a larger polypeptide after glycosylation. The presence of minor glycopeptide components indicates that modification of carbohydrate chains during seed development may also occur.     

The interaction of α2 macroglobulin with trypsin releases a soluble glycopeptide

When human α₂ macroglobulin (α₂M) or its asialo-[³H]galactose derivative reacts with trypsin, a glycopeptide of molecular weight 3500–4000 is released from the α₂M. The glycopeptide was purified on Biogel P-4 columns and its amino acid and carbohydrate composition were determined. The oligosaccharide contains sialic acid, galactose, mannose and GlcNAc in a ratio of 1.0:0.73:3.85:2.85 and is apparently attached to protein in a GlcNAc→asparagine linkage.     

Carbohydrate compositions of the rabbit plasminogen isozymes.

The carbohydrate compositions of the two affinity-chromatography-resolved isozymes of rabbit plasminogen and plasmin as well as the isoelectric-focusing-resolved subforms of each plasminogen isozyme have been investigated in detail. The first plasminogen isozyme as well as its subforms all possess four to five residues of N-acetylglucosamine, two residues of N-acetylgalactosamine, three residues of mannose and five residues of galactose per molecule of protein. Additionally, we previously reported three residues of sialic acid present on this protein molecule. The corresponding plasmin heavy chain for this isozyme contains essentially all of the carbohydrate, and the plasmin light chain appears devoid of carbohydrate. On the other hand, the second plasminogen isozyme as well as its subforms all possess only trace amounts of N-acetylglucosamine, two residues of N-acetylgalactosamine, less than one residue of mannose and three residues of galactose per molecule of protein. In addition, we have previously reported two residues of sialic acid for this molecule. Here, also, all carbohydrate appears on the heavy chain of the plasmin, which is prepared by activation of this particular plasminogen. Thus, the carbohydrate differences which we reported earlier in rabbit plasminogen isozymes are confirmed and extended.     

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.     

On the carbohydrate composition of bovine colostrum trypsin inhibitor.

The trypsin inhibitor from bovine colostrum has been separated into several forms by CM-Sephadex and DEAE-cellulose chromatography. These forms differ in the amount and composition of the carbohydrate they contain, which has been quantitated for four components by gas-liquid chromatography and standrad colorimetric procedures. The monosaccharides fucose, mannose, galactose, galactosamine, glucosamine and sialic acids have been determined. A microheterogeneity was establish ed in the carbohydrate moiety, which amounts to about 40% of the total molecular weight (Mr 11 000 - 14 000) of bovine colostrum inhibitor.     

Characterization of the oligosaccharide units of the bovine erythrocyte membrane glycoprotein.

The major glycoprotein of the bovine erythrocyte membrane was purified by extraction of the ghosts with lithium 3,5-diiodosalicylate followed by phenol-water extraction and acidification. The glycoprotein contains 20% protein and 80% carbohydrate by weight and gives a single band on sodium dodecyl sulfate-polyacrylamide gels with an estimated molecular weight of 230000 daltons. The carbohydrate composition of the glycoprotein was determined to be (in residues relative to sialic acid): sialic acid, 1.0; fucose, less than 0.01; mannose, 0.1; galactose, 3.3; N-acetylgalactosamine, 0.9; and N-acetylglucosamine, 2.4. Pronase digestion of the isolated glycoprotein followed by Sephadex G-75 gel filtration resulted in the separation of a small pool of glycopeptides (pool III), which included all of the mannose-containing glycopeptides, from the bulk of the glycopeptide material which was in the void fractions of the column (pool I). Alkaline borohydride treatment released over 95% of the oligosaccharide units in pool I and approximately 30% of the oligosaccharide units in pool III. These oligosaccharides were isolated by gel filtration and ion-exchange chromatography. The oligosaccharides released from pool I had molecular weights of 1100-1400 daltons and contained sialic acid, galactose, and N-acetylglucosamine in molar ratios of 0.5-1:3:2 as well as a partial residue of N-acetylgalactosaminitol. The oligosaccharides released from pool III by alkali had molecular weights of 1300-1600 daltons and contained sialic acid, galactose, N-acetylglucosamine, N-acetylgalactosamine and N-ACETYLgalactosaminitol in molar ratios of 1-2:2:1:1:1. These data indicate that the majority of the oligosaccharide units of the bovine erythrocyte glycoprotein are linked O-glycosidically to the peptide backbone of the molecule.     

Studies on the glycopeptides of canine prothrombin]

After degradation of canine prothrombin by the complex of Streptomyces griseus proteases four glycopeptides were obtained. Each of them contained aspartic acid, hexosamines, mannose, galactose and sialic acids. Canine prothrombin contains two or three carbohydrate chanins, which are bound to aspartic (asparagine) residues. Microheterogenity of the carbohydrate chains of canine prothrombin was found.