The monosaccharide composition and sequence of the carbohydrate moiety of human serum low density lipoproteins.

Human serum low density lipoprotein (d = 1.027-1.045) was delipidated with organic solvents and the apoprotein digested with thermolysin. The digest was fractionated by gel filtration and DEAE-cellulose chromatography. Two glycopeptides were obtained. One of the glycopeptides (GP-I) contained 2 residues of N-acetylglucosamine and 6 residues of mannose per mole of the glycopeptide, while the other contained 2 sialic acid, 5 mannose, 2 galactose, and 3 N-acetylglucosamine residues per mole of glycopeptide. The results of sequential enzymatic digestion with purified glycosidases, periodate oxidation, and partial acid hydrolysis lead us to propose the following sturctures for the two glycopeptides: (see article). These glycopeptides represent at least 50% of the carbohydrate moiety of LDL.     

Preparation of glycopeptides and oligosaccharides from thyroxine-binding globulin.

Over 99% of thyroxine (T4), the major form of thyroid hormone in plasma, is bound to the plasma glycoprotein thyroxine-binding globulin (TBG). The carbohydrate composition of TBG (14.6% by weight) consists of mannose, galactose, N-acetylglucosamine, and N-acetylneuraminic acid in the molar ratios of 11:9:16:10 per mol of glycoprotein. No fucose or N-acetylgalactosamine were detected. Amino acid analyses were performed. Glycopeptides, prepared by exhaustive pronase treatment of the glycoprotein, were separated by gel filtration and ion exchange chromatography. All glycopeptides contained the four sugars present in the native glycoprotein. One-fourth of the glycopeptide fraction was resolved into a discrete component, glycopeptide I. The remaining glycopeptides were a mixture termed glycopeptides II and III. Glycopeptides II and III were resolved into two discrete carbohydrate units, termed oligosaccharides A and B, by alkaline-borohydride treatment and DEAE-cellulose chromatography. We propose that TBG contains four oligosaccharide chains as calculated from the molecular weights of the glycopeptides and from compositional data assuming 1 asparagine residue/glycopeptide. The carbohydrate structures of the glycopeptides and relative affinities of TBG, glycopeptides and oligosaccharides for hepatocyte plasma membrane binding are presented in the accompanying paper (Zinn, A.B., Marshall, J.S., and Carlson, D.M. (1978) J. Biol. Chem. 253, 6768-6773.     

Isolation and Properties of Carbohydrate Rich Fraction of Wheat Gluten

Two carbohydrate rich fractions A and B were isolated from wheat gluten. Fraction B contained more lipid than fraction A. Lipid portion of fraction B consisted mainly of glycolipid and was fractionated into five fractions by thin-layer chromatography. The two main fractions were extracted and determined to be galactolipid and glucolipid, respectively, by the analyses of fatty acid and sugar components by gas chromatography. Defatted fraction A was assumed to consist of glycoprotein. After complete pronase digestion of defatted fraction A, the remaining glycopeptide moiety was isolated by column chromatography on DEAE-cellulose followed by gel filtration through Sephadex G–25. The amino acid and sugar components of the glycopeptide were investigated.     

The carbohydrate units of asialo-ovomucoid: their heterogeneity and enzymic degradation

An ovomucoid variant free from sialic acid has been prepared in a pure state by ion-exchange chromatography on DEAE-cellulose. The purified glycoprotein contained 10-11 residues of mannose, 2-3 residues of galactose, and 21 residues of 2-acetamido-2-deoxyglucose. Glycopeptides have been prepared by exhaustive digestion with Pronase followed by ion-exchange chromatography on Dowex 50 (X2) resin. Three fractions were obtained, all with similar contents of mannose and hexosamine but with various contents of galactose. The sugar-aspartic acid ratios indicated that all of the fractions were heterogeneous, the major fraction having mannose-galactose-hexosamine-aspartic acid ratios of 2.6:0.5:5.8:1.0. Cleavage of asialo-ovomucoid with cyanogen bromide and proteolytic digestion of the isolated fragments gave two heterogeneous glycopeptide fractions of similar composition. Both asialo-ovomucoid and the principal glycopeptide fraction were degraded with beta-D-galactosidase, alpha-D-mannosidase, and beta-N-acetylglucosaminidase singly and in sequence. Removal of much of the carbohydrate from asialo-ovomucoid had no appreciable effect on its anti-tryptic activity. By sequential degradation of the glycopeptide, a pentasaccharide core alpha-D-Man-[alpha-D-Man]-beta-D-Man-beta-D-GlcNAc-beta-D-GlcNAc-Asn was obtained. Other structural features revealed by enzymic degradation are discussed.     

Isolation and characterization of a blood group active glycopeptide from porcine aorta

A fucose-rich glycopeptide was prepared from the pronase digest of porcine thoracic aorta by gel-filtration through Sephadex G-100, DEAE-Sephadex A-25 column chromatography and alpha-amylase digestion. This glycopeptide was electrophoretically homogeneous. The large molecular size and chemical composition suggested that this glycopeptide was derived from mucin-type glycoprotein. The results of the beta-elimination reaction indicated that this glycopeptide contained the O-glycosidic linkages between galactosamine and serine/threonine. This glycopeptide exhibited blood group A and H activities. The present study revealed that the porcine thoracic aorta contains a blood group antigen of mucin-type glycoprotein nature.     

Resolution of the charge forms and amino acid sequence and location of a tryptic glycopeptide in rat alpha-lactalbumin.

Three charge forms of rat alpha-lactalbumin were separated by ion exchange chromatography on DEAE-cellulose. The amino acid composition of each form was similar but they differed in carbohydrate composition. Each form contained a tryptic glycopeptide having a common polypeptide and heteropolysaccharide unit. The tryptic glycopeptide was sequenced and positioned in rat alpha-lactalbumin, which was partially sequenced from residues 1 to 50. The carbohydrate attachment site was at Asn45. Secondary structure calculations predicted that Asn45 is in a beta bend conformation whereas Asn45 in bovine alpha-lactalbumin, a poorly glycosylated protein, is not in a bend conformation.     

Partial deglycosylation of blood-group-specific glycoproteins.

The time course for the partial deglycosylation of blood-group-specific glycoproteins from human ovarian-cyst fluids with 0.25 M-H2SO4/acetic acid and 6 M-HCl in methanol was studied. Either reagent readily removed about 80% of the carbohydrate from the glycoproteins to leave non-diffusible glycopeptides that contain N-acetylgalactosamine as the predominant sugar. Some changes in amino acid distribution were observed during the deglycosylation, which were attributed to an accelerated break-up of the nonglycosylated regions of the parent glycoprotein. The N-acetylgalactosaminyl-peptides isolated were judged to be polydisperse by gel filtration, and ion-exchange chromatography divided the glycopeptide population into several fractions with differing amino acid compositions. A Lumbricus terrestris hexosaminidase preparation was successful in removing almost all the remaining sugar from the glycopeptides, but caused further rupture of the peptide. When a per O-acetylated glycoprotein was treated with the H2SO4/acetic acid reagent the glycopeptide contained, in addition to N-acetylgalactosamine, about 50% of the sialic acid present in the parent glycoprotein, indicating that most of this sugar is located near the peptide end of the carbohydrate chains.     

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.     

Structure of carbohydrate chains of a high-molecular-weight pulmonary glycoprotein

A human, alveolar glycoprotein having an apparent mol. wt. of 250 000 gave two major glycopeptide fractions (I and II) by Pronase digestion, followed by gel filtration, DEAE-cellulose column chromatography, paper chromatography, and paper electrophoresis. Glycopeptide I contained d-galactose, d-mannose, 2-acetamido-2-deoxy-d-glucose, and N-acetylneuraminic acid in the molar ratio of 2:3:4:1, whereas these sugars were present in Glycopeptide II in the molar ratio of 2:3:4:2.l-Fucose was present only in Glycopeptide II at a concentration of one l-fucose per three d-mannose residues. In both glycopeptides, 2-acetamido-2-deoxy-d-glucose was linked to an asparagine residue of the peptide chain. Based on the results of alkaline borohydride treatment, periodate oxidation, methlylation analysis, and sequential glycosidase degradation of the glycopeptides, tentative structures are proposed for both glycopeptides.     

Sialoglycopeptides isolated from bovine aorta.

Intima-media of bovine aorta was digested with pronase, after preliminary extraction of saline (1%)-soluble substances and fat. Crude glycopeptide fraction was then obtained from the resulting complex carbohydrate fraction by fractionation with CPC (cetylpyridinium chloride). Complete separation of sialoglycopeptides was achieved by chromatography on a DEAE-cellulose column at pH 7.2 followed by repeated chromatography on a DEAE-Sephadex A-25 column at pH 5.2. Nine sialoglycopeptides (SGP 1-SGP 9) thus obtained were homogeneous on high-voltage paper electrophoresis at pH 3.5 and pH 5.2. The analytical data showed great heterogeneity of the carbohydrate chains of these preparations, although they consisted of the same monosaccharides (galactose, glucose, mannose, glucosamine, galactosamine, fucose, and sialic acid), except that SGP 1 lacked galactosamine. Heterogeneity was also observed in their peptide chains. It was noticed, however, that the contents of hexose, hexosamine, and aspartic acid of the fractions (SGP 3, SGP 4, and SGP 5) which eluted from the DEAE-Sephadex A-25 column at lower molarity of the eluting salt were higher than those of the fractions (SGP 7, SGP 8, and SGP 9) which eluted at higher molarity, while the contents of sialic acid and hydroxyamino acids were in an opposite relationship. Representative fractions (SGP 7 and SGP 9) of the latter contained many more alkali-sensitive linkages than those (SGP 3 and SGP 5) of the former, indicating the presence of many more O-glycosidic linkages between hydroxyamino acid(s) and sugar(s) in the latter than in the former. The sialoglycopeptides contained significant amounts of sialic acid, ranging from 10% (sgp 1) to 32.4% (SGP 8). The highest contents were in SGP 8 and SGP 9, which contained equimolar amounts of sialic acid and hexosamine. Furthermore, infrared spectra indicated the presence of sulfate groups in most of the sialoglycopeptides.     

Guinea-pig kidney beta-N-acetylgalactosaminyltransferase towards Tamm-Horsfall glycoprotein. Requirement of sialic acid in the acceptor for transferase activity.

A beta-N-acetylgalactosaminyltransferase that preferentially transferred N-acetylgalactosamine to Sd(a-) Tamm-Horsfall glycoprotein was found in guinea-pig kidney microsomal preparations. This enzyme was kidney-specific and was able to transfer the sugar to other glycoproteins, such as fetuin and alpha 1-acidic glycoprotein. The presence of sialic acid in the acceptors was essential for the transferase activity when either glycoproteins or their Pronase glycopeptides were used as acceptors. Two glycopeptides (Tamm-Horsfall glycopeptides I and II) with a different carbohydrate composition were separated by DEAE-Sephacel chromatography from Pronase-digested Tamm-Horsfall glycoprotein. The amount of N-acetylgalactosamine transferred to glycopeptides by the enzyme correlated with their degree of sialylation. Enzymic digestion of N-[14C]acetylgalactosamine-labelled Tamm-Horsfall glycopeptide II showed that the transferred sugar was susceptible to beta-N-hexosaminidase. The amount of sugar cleaved by beta-hexosaminidase was strongly increased when the labelled Tamm-Horsfall glycopeptide II was pretreated with mild acid hydrolysis, a procedure that removed the sialic acid residues. Alkaline borohydride treatment of the labelled Tamm-Horsfall glycopeptide II did not release radioactivity, thus indicating that enzymic glycosylation took place at the N-asparagine-linked oligosaccharide units of Tamm-Horsfall glycoprotein.     

Prominent 85-kDa oligomannosidic glycoproteins of rat brain are signal regulatory proteins and include the SHP substrate-1 for tyrosine phosphatases

The glycoprotein component in rat brain reacting most strongly with Galanthus nivalis agglutinin (GNA) on western blots migrates as an 85-kDa band. GNA identifies mannose-rich oligosaccharides because it is highly specific for terminal alpha-mannose residues. After purification of this 85-kDa glycoprotein band by chromatography on GNA-agarose and preparative gel electrophoresis, binding of other lectins demonstrated the presence of fucose and a trace of galactose, but no sialic acid. Treatment with N-Glycanase or endoglycosidase H produced a 65-kDa band, indicating that it consisted of about one-fourth N-linked oligomannosidic carbohydrate moieties. High-performance anion-exchange chromatography and fluorescence-assisted carbohydrate electrophoresis indicated that the major carbohydrate moiety is a heptasaccharide with the structure Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) Manbeta1-4Glc-NAcbeta1-4GlcNAc (Man5GlcNAc2). Determination of amino acid sequences of peptides produced by endoproteinase digestion demonstrated that this 85-kDa mannose-rich glycoprotein component contained the SHP substrate-1 for phosphotyrosine phosphatases and at least one other member of the signal-regulatory protein (SIRP) family. The unusually high content of oligomannosidic carbohydrate moieties on these receptor-like members of the immunoglobulin superfamily in neural tissue could be of functional significance for intercellular adhesion or signaling.     

Studies on the structure of the oligosaccharide chains of alpha 1-acid glycoprotein associated with rough membranes from rat liver

The high mannose form of rat alpha 1-acid glycoprotein was isolated from rough membranes of rat liver using methods described previously. The high mannose glycopeptides were prepared by Pronase digestion, and oligosaccharides were isolated following digestion with endohexosaminidase-H. The structure of the carbohydrate chains of the high mannose glycopeptide and the oligosaccharides was examined by 300 MHz nuclear magnetic resonance spectroscopy. The glycopeptide contained a mixture of about equal amounts of AsnGlcNAc2Man9 and AsnGlcNAc2Man8. Analysis of the oligosaccharide fraction showed that it consisted of about equal amounts of GlcNAc Man9 and GlcNAc Man8; the GlcNAc Man8 fraction contained 85% of the "A" isomer (which was missing the terminal mannose from the middle antenna). The results suggested that mannose processing of alpha 1-acid glycoprotein in rough membranes of rat liver in vivo occurred only as far as the Man8 structure and that the "A" isomer was the main isomer formed.     

Purification and characterization of a lectin (plant hemagglutinin) with N blood group specificity from Vicia graminea seeds.

A lectin with N blood group specificity was isolated from Vicia graminea seeds. This lectin was purified from a crude extract by precipitation with ammonium sulfate, DEAE-cellulose chromatography and Sephadex G-150 gel filtration. Purification steps were followed by increase of specific activity. Its homogeneity was demonstrated by polyacrylamide gel electrophoresis, immunoelectrophoresis, electrofocusing and ultracentrifugation. This lectin is an acid glycoprotein with 7.3% carbohydrate, a high percentage of serine and contains no sialic acid. The native lectin has a molecular weight about 100 000 and dissociates into four subunits of 25 000 as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Preliminary hemagglutination inhibition has shown that the lectin was not inhibited by any of the monosaccharides contained in N blood group substances; however it was inhibited by the erythrocyte membrane major glycoprotein and the tryptic fragments obtained from erythrocytes.     

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.     

Glycopeptides in pus of acute pleurisy patients

Glycopeptides were isolated from tryptic digests of pus from acute pleurisy patients. The hexose, hexosamine, and sialic acid contents rose over the first 2-4 days after admission to the hospital, continued at high levels for 5-8 hospital days, and fell to low levels after 9 hospital days. The course of duration after admission to the hospital was divided into three stages: 1-4 days after admission to the hospital, 5-8 hospital days, and 9-21 hospital days. Materials corresponding to these three stages were then collected for fractionation by DEAE-cellulose column chromatography. Fractionation of glycopeptides by DEAE-cellulose column chromatography yielded three glycopeptide fractions at 0.05 to 0.2 M NaCl. Column chromatography of crude material on DEAE-cellulose showed an increase in the 0.2 M NaCl fraction from the concentrate over a period of 4-8 hospital days due to a large increase in sialic acid-rich glycopeptide fraction.     

Use of radioactive glucosamine in the perfused rat liver to prepare alpha 1-acid glycoprotein (orosomucoid) with 3H- or 14C-labelled sialic acid and N-acetylglucosamine residues.

1. A method was developed whereby [1-14C]glucosamine was used in a perfused rat liver system to prepare over 2 mg of alpha 1-acid glycoprotein with highly radioactive sialic acid and glucosamine residues. 2. The liver secreted radioactive alpha 1-acid glycoprotein over a 4-6 h period, and this glycoprotein was purified from the perfusate by chromatography on DEAE-cellulose at pH 3.6. 3. The sialic acid on the isolated glycoprotein had a specific radioactivity of 3.1 Ci/mol, whereas the glucosamine-specific radioactivity was 4.3 Ci/mole. The latter amino-sugar residues on the isolated protein were only 13-fold less radioactive than the initially added [1-14C]glucosamine. Orosomucoid with a specific radioactivity of 31.3 microCi/mg of protein was obtainable by using [6-3H]glucosamine. 4. The amino acid composition of the purified orosomucoid was comparable with that found by others for the same glycoprotein isolated from rat serum. A partial characterization of the carbohydrate structure was done by sequential digestion with neuraminidase, beta-D-galactosidase and beta-D-hexosaminidase. 5. Many other radioactive glycoproteins were found to be secreted into the perfusate by the liver. Thus this experimental system should prove useful for obtaining other serum glycoprotein with highly radioactive sugar moieties.     

Isolation and characterization of cyanogen bromide fragments and a glycopeptide from the Dolichos biflorus lectin.

The 110000 molecular weight Dolichos biflorus lectin is a glycoprotein composed of four subunits of approximately 27000 molecular weight with one methionine residue per subunit (Carter and Etzler, 1975b). Cyanogen bromide cleavage of the lectin yielded two fragments with approximate molecular weights of 15000 and 12000 as determined by electrophoresis on sodium dodecyl sulfate gels. Only the 15000 molecular weight fragment stained for carbohydrate with the periodic acid-Schiff stain. The two fragments were isolated, and their amino acid compositions were determined. The 15000 molecular weight fragment was identified as the amino terminal segment of the lectin subunits by NH2-terminal amino acid analysis. A glycopeptide with a minimum molecular weight of 1100 was isolated from the lectin by exhaustive Pronase digestion. Complete acid hydrolysis of the glycopeptide yielded aspartic acid, mannose, and N-acetylglucosamine in the ratio of 1:4-5:1-2. Partial acid hydrolysis of the glycopeptide produced a component which had an identical mobility with commercial N-acetylglucosaminylasparagine in high voltage paper electrophoresis. The data indicate that the carbohydrate unit of the lectin is bound to the amino terminal half of the subunits by a glycosylamine linkage between N-acetylglucosamine and asparagine.     

The carbohydrate moiety of band-3 glycoprotein of human erythrocyte membranes.

Band-3 glycoprotein was purified from human blood-group-A erythrocyte membranes by selective solubilization and gel chromatography on Sepharose 6B in the presence of sodium dodecyl sulphate. The purified glycoprotein was subjected to hydrazinolysis in order to release the carbohydrate moiety. The released oligosaccharides were N-acetylated and applied to a column of DEAE-cellulose. Most of the band-3 oligosaccharides obtained were found to be free of sialic acids. When this neutral fraction was subjected to gel chromatography on a column of Sephadex G-50, two broad peaks were observed indicating that the band-3 glycoprotein was heterogeneous in the size of the oligosaccharide moieties. All fractions from gel chromatography were found to contain galactose, mannose, N-acetylglucosamine and fucose. The higher-molecular-weight (mol.wt. 3000-8000) peak consisted of fucose, mannose, galactose, N-acetylglucosamine and N-acetylgalactosamine in a molar proportion of 1.6:3.0:8.4:10.5:0.2. Most of these oligosaccharides were digested with a mixture of beta-galactosidase and beta-N-acetylhexosaminidase after alpha-L-fucosidase treatment to give a small oligosaccharide with the structure alpha Man2-beta Man-beta GlcNAc-GlcNAc. Methylation studies and limited degradation by nitrous acid deamination showed that the oligosaccharides contained the repeating disaccharide Gal beta 1----4GlcNAc beta 1----3, with branching points at C-6 of some of the galactose residues. These results indicate that a major portion of the band-3 oligosaccharide has a common core structure, with heterogeneity in the numbers of the repeating disaccharides, and contains fucose residues both in the peripheral portion and in the core portion. Haemagglutination tests were also carried out to determine the blood-group specificities of the glycoprotein and the results demonstrated the presence of both blood-group-H and I antigenic activities.     

Grucuronic acid-containing glycopeptide from squid cartilage

A glycopeptide fraction containing glucuronic acid as a component sugar was extracted and purified from squid cartilage to give a single band migrating much slower than hyaluronic acid in cellulose acetate electrophoresis. The molecular weight of the glycopeptide was fairly large since its K_av value in Sephadex G-200 chromatography was 0.18; however, it was soluble in 66% ethanol. This glycopeptide contained glucuronic acid, glucosamine, galactosamine, galactose, and fucose. The total amino acid content was 1.87 μmol of amino acid per mg of the glycopeptide. Threonine, serine and proline represented 80% of the amino acids. Digestion with chondroitinase ABC or reaction with nitrous acid did not result in degradation of the glycopeptide; however, it was completely degraded by reaction with 0.5 M KOH at 37°C. Two hexasaccharides were separated from the alkaline degradation products, and they both contained glucuronic acid, fucose, galactosamine, and reducing terminal glucosamine in the molar ratio, 2:1:2:1. These results indicated that the glycopeptide contains glucuronic acid-containing sugar chains that are distinct from any known glycosaminoglycan.