Preparation and properties of concanavalin A-binding glycopeptides derived from rat brain glycoproteins

Mannose-rich glycopeptides derived from brain glycoproteins were recovered by affinity chromatography on Concanavalin A-Sepharose. These glycopeptides, which adsorb to the lectin and are eluted with α-methylmannoside, constitute about 25–30% of the total glycopeptide material recovered from rat brain glycoproteins. They contain predominately mannose and N-acetylglucosamine (mannose/N-acetylglucosamine = 3), as well as small amounts of galactose and fucose. Approx. 65% of the Concanavalin A-binding glycopeptide carbohydrate was recovered after treatment with leucine aminopeptidase, gel filtration on Biogel P-4, and ion-exchange chromatography on coupled Dowex 50-hydrogen and Dowex 1-chrolide columns. The purified glycopeptide fraction contained six mannose and two N-acetylglucosamine residues per aspartic acid and possessed an apparent molecular weight of about 2000 as assessed by gel filtration and amino acid analysis. Galactose and fucose were absent. Treatment of the purified glycopeptides with α-mannosidase drastically reduced their affinity for Concanavalin A, suggesting the presence of one or more terminal mannose residues.     

Subcellular and anatomical distribution in rat brain of glycoproteins that contain mannose-rich heteropolysaccharide chains

Mannose-rich glycopeptides derived from brain glycoproteins were obtained by proteolysis of bovine brain tissue or subcellular fractions derived from rat brain tissue. The dialyzable mannose-rich glycopeptides were isolated by colum electrophoresis and gel flitration. These glycopeptides contained, on the average, six mannose and two N-acetylglucosamine residues with variable amounts of fucose and galactose. Over 50% of the mannose-rich glycopeptides of rat brain were localized in the microsomal and synaptosomal fractions; myelin and the soluble fraction contained lesser amounts. None was recovered from the mitochondria. The amount, per mg protein, of mannose-rich oligosaccharide chains in the myelin exceeded the concentration found in the microsomal and synaptosomal fractions. The concentration of mannose-rich glycopeptides derived from glycoproteins was 50% higher in white matter than in gray. On the other hand, the non-dialyzable and acidic sialoglycopeptides showed a three-fold enrichment in gray matter compared to white. The relatively lower ratio of sialoglycopeptides to mannose-rich glycopeptides observed in white matter (2.5) compared to gray matter (6.9) is reflected in the lower value for the ratio in myelin (1.1) compared to synpatosomes (2.1). Although glycoproteins that contain mannose-rich oligosaccharide chains are present in the nerve cell and its terminals, these glycoproteins appear to be relatively enriched in myelin and/or glial membranes.     

Preparation and properties of concanavalin A-binding glycopeptides derived from rat brain glycoproteins.

Mannose-rich glycopeptides derived from brain glycoproteins were recovered by affinity chromatography on Concanavalin A-Sepharose. These glycopeptides, which adsorb to the lectin and are eluted with alpha-methylmannoside, constitute about 25--30% of the total glycopeptide material recovered from rat brain glycoproteins. They contain predominately mannose and N-acetylglucosamine (mannose/N-acetylglucosamine = 3), as well as small amounts of galactose and fucose. Approx. 65% of the Concanavalin A-binding glycopeptide carbohydrate was recovered after treatment with leucine aminopeptidase, gel filtration on Biogel P-4, and ion-exchange chromatography on coupled Dowex 50-hydrogen and Dowex 1-chloride columns. The purified glycopeptide fraction contained six mannose and two N-acetylglucosamine residues per aspartic acid and possessed an apparent molecular weight of about 2000 as assessed by gel filtration and amino acid analysis. Galactose and fucose were absent. Treatment of the purified glycopeptides with alpha-mannosidase drastically reduced their affinity for Concanavalin A, suggesting the presence of one or more terminal mannose residues.     

Characterization of glycopeptides isolated from membranes of F9 embryonal carcinoma cells

From cells of a nullipotential line of embryonal carcinoma was isolated a membrane fraction enriched in the cell surface F9 antigen. More than 40% of the radioactive fucose and galactose incorporated by cells into nondialyzable material was recovered in this membrane preparation, corresponding to an approximately 10-fold purification of the labeled material. Extreme heterogeneity of membrane glycoproteins labeled with these sugars was revealed by sodium dodecyl sulfate gel electrophoresis. Glycopeptides prepared by extensive pronase digestion of membranes labeled with fucose or galactose showed properties similar to those already described for fucose-labeled glycopeptides from whole cells. Namely, large glycopeptides eluted near the excluded volume of Sephadex G-50 column were the predominant glycopeptide species, while complex glycopeptides of molecular weight around 2500 were minor components. Therefore, these large glycopeptides, characteristic of embryonal carcinoma cells, are derived mainly from a variety of glycoproteins closely associated with the membrane system, most probably cell-surface membrane of the cells. The large glycopeptides were also significantly labeled with glucosamine, but only slightly with mannose; major components of mannose-labeled glycopeptides from the membranes were high-mannose glycopeptides of low molecular weight. Several experiments excluded the possibility that the larg glycopeptides are mucopolysaccharides, glycolipids or mucin-type glycoproteins with short oligosaccharide chains.     

Isolation and carbohydrate composition of glycopeptides of human apo low-density lipoprotein from normal and type II hyperlipoproteinemic subjects.

Glycopeptides were prepared from the delipidized protein of low-density lipoprotein (LDL, d=1.019-1.063) of three normal and three familial heterozygous type II hyperlipoproteinemic (HLP) subjects. The glycopeptides of all subjects were resolved into three groups by gel filtration on Bio-Gel P6 following papain (EC 3.4.22.2) digestion and initial purification on Bio-Gel P2.In normal individuals the component of largest molecular weight (F-1) contained mannose (Man), N-acetyl glucosamine (GlcNAc) galactose (Gal), and N-acetyl neuraminic acid (NANA) in the respective amounts of 45.9 +/- 6.7, 37.3 +/- 5.9, 28.6 +/- 3.4, and 27.0 +/- 3.9 nmol/mg original apoprotein. The group of smallest molecular weight (F-3) contained essentially only Man (25.8 +/- 1.5 nmol/mg protein) and GlcNac (3.0 +/- 0.4 nmol/mg protein) with traces of Gal and NANA. A group of intermediate molecular weight (F-2) exhibited considerable heterogeneity and contained Man, GlcNAc, Gal, and NANA in the amounts of 45.9 +/- 5.1, 18.3 +/- 1.7, 11.0 %/- 1.7, and 7.7 %/- 1.2 nmol/mg protein. While the major portion of NANA (78%), Gal (71%), and GlcNAc (64%) was present in F-1, approximately 22% of the total Man was in F-3. No major differences were detected in the carbohydrate composition of the three glycopeptide fractions of LDL apoptotein from normal and Type II subjects.     

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.     

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.     

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.     

On the oligosaccharide moiety of angiotensin-converting enzyme.

Two glycopeptide fractions in a pronase digest of rabbit pulmonary angiotensin-converting enzyme were resolved by gel filtration. GP-I, the minor component (～1 mole/mol enzyme) contained mannose, galactose, glucose $\text{N}$-acetylglucosamine, $\text{N}$-acetylgalactosamine and sialic acid in an approximate molar ratio of 1:5:3:4:1:2 and molar equivalents of aspartic acid, threonine and serine. GP-II, the major oligosaccharide unit (～ 12 moles/mol enzyme, ～ 90% of total carbohydrate), contained fucose, mannose, galactose, $\text{N}$-acetylglucosamine, sialic acid and aspartic acid in a molar ratio of 1:4:4:4:1:1. Although accounting for about one-quarter of the weight of the enzyme, GP-II did not compete with the intact glycoprotein for binding to goat antienzyme antibodies. Some structural features of GP-II were deduced by periodate oxidation and digestion with various glycosidases.     

Studies on phytohemagglutinins XXIII. A receptor which does not conform to the so called sugar specificity of the pea phytohemagglutinin

A receptor glycopeptide for hemagglutinin of the pea (Pisum sativum L.) was isolated from rabbit erythrocytes by the method originally applied for isolation of a receptor glycopeptide from human erythrocytes (Kubánek, J., Entlicher, G. and Kocourek, J. (1973) Biochim. Biophys. Acta 304, 93–102). The receptor isolated from rabbit erythrocytes contains galactose, mannose, N-acetylglucosamine, asparic acid, threonine, serine glutamic acid and glycine in approximate molar ratios of 2:1:3:3:1:1:1:1. It has a minimum molecular weight of about 2000. According to the sequence analysis the following structure has been proposed for the receptor:

Full-size image (1K)

About 90% of the receptor-site activity for the pea hemagglutinin is due to the presence of two galactose residues on non-reducing terminals of the saccharide chain in spite of the fact that the pea haemagglutinin is not inhibited by this sugar.     

Structure of the "keratosulfate-like" material in liver from a patient with G M1 -gangliosidosis ( -D-galactosidase deficiency)

Large amounts of a glycopeptide containing galactose, $\text{N}$-acetylglucosamine, $\text{N}$-acetylgalactosamine and threonine in the ratio 4:3:1:1, together with smaller amounts of mannose, fucose, sialic acid, sulfate, serine, and other amino acids were isolated from the liver of a patient with G_M1-gangliosidosis. Treatment with mild alkali and sodium borohydride indicated an $\text{O}$-glycosidic linkage between $\text{N}$-acetylgalactosamine and threonine. All the hexosamine residues were resistant to sodium metaperiodate whereas 2 out of 4 D-galactose residues were destroyed. Further studies indicated that one of the galactose residues was 1→3 linked to $\text{N}$-acetylgalactosamine (as in G_M1) and the other 1→4 linked to $\text{N}$-acetylglucosamine as found in skeletal keratosulfate.     

The Structure of the Carbohydrate Moiety of a Glycopeptide GP-I-b from Rhizopus Saccharogenic Amylase

The structure of the carbohydrate moiety of GP–I–b which is one out of three glycopeptides isolated from a Pronase digest of the saccharogenic amylase of Rhizopus javanicus sp. 3–46, was investigated by enzymatic and chemical techniques.

Nine moles of mannose followed by one mole of N-acetylglucosamine were released per mole of GP–I–b when it was treated sequentially with purified jack bean α-mannosidase and β-N-acetylglucosaminidase.

Methylation of GP–I–b gave 3, 6-di-O-methyl derivative from the N-acetylglucosamine residues, and 2, 3, 4, 6-tetra-O-methyl, 3, 4, 6-tri-O-methyl and 2, 4-di-O-methyl derivatives from the mannose residues in an approximate ratio of 3: 4: 2.

A smaller glycopeptide (F–l) containing two moles each of mannose and N-acetylglucosamine per mole of asparagine was obtained when GP–I–b was subjected to one step of the Smith degradation. Exhaustive methylation of F–l gave 3, 6-di-O-methyl derivative of Nacetylglucosamine, and 2, 3, 4, 6-tetra-O-methyl and 2, 3, 4-tri-O-methyl derivatives of mannose in a ratio of 1.00: 0.85.

Controlled acetolysis of GP–I–b yielded mannose, O-α-mannosyl-(l→2)-O-α-mannosyl-(l→3)-mannose and a smaller glycopeptide which was resistant to the acetolysis.

From these and previous evidences, the following structure was determined for GP–I–b.     

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.     

Induction and Purification of Endo-β-N-Acetylglucosaminidase from Arthrobacter protophormiae Grown in Ovalbumin

Arthrobacter protophormiae produced a high level of extracellular endo-β-N-acetylglucosaminidase when cells were grown in a medium containing ovalbumin. The enzyme was induced by the glycopeptide fraction of ovalbumin prepared by pronase digestion. Production of the enzyme was also induced by glycoproteins such as yeast invertase and bovine ribonuclease B but not by monosaccharides such as mannose, N-acetylglucosamine, and galactose. The enzyme was purified to homogeneity as demonstrated by polyacrylamide gel electrophoresis and has an apparent molecular weight of about 80,000. The enzyme showed a broad optimum pH in the range of pH 5.0 to 11.0. The enzyme hydrolyzed all heterogeneous ovalbumin glycopeptides, although the hydrolysis rates for hybrid type glycopeptides were very low. The substrate specificity of A. protophormiae endo-β-N-acetylglucosaminidase was very similar to that of Endo-C_II from Clostridium perfringens. Therefore, the enzyme induction by A. protophormiae seems to have a close relation to the substrate specificity of the enzyme.     

A third glycopeptide (nephritogenoside) isolated from the glomerular basement membrane

A new glycopeptide was isolated from the glomerular basement membrane (GBM) of normal rats. Unlike already known glycopeptides, this glycopeptide has biological activity (nephritogenic activity) to induce glomerulonephritis when injected once into the footpads of homologous animals. A close relationship was found between the nephritogenic activity and the non-dialyzable glucose content of this glycopeptide. Thus the nephritogenic activity can be assessed quantitatively by estimating the content of "non-dialyzable glucose." Chemical purification of the nephritogenic glycopeptide involved the selective removal of inactive glycopeptide containing galactose, mannose, and N-acetyl-glucosamine (but no glucose). Trichloroacetic acid (TCA) treatment was a simple but highly effective procedure for selective removal of this inactive glycopeptide. The non-reducing terminus of the nephritogenic glycopeptide is alpha-D-glucopyranoside, and the glycopeptide reacts specifically with concanavalin A, even in the crude state. We propose that the nephritogenic glycopeptide is not an artifact produced during exhaustive proteolytic digestion, but a natural substance having a fixed molecular shape, even in the crude state, and whose union with GBM-proper can be easily broken by proteolytic digestion.     

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.     

Glycopeptide fractions prepared from purified central and peripheral rat myelin

Myelin was purified from rat brain and sciatic nerve after invivo labeling with [³H]fucose and [¹⁴C]glucosamine to provide a radioactive marker for glycoproteins. The glycoproteins in the isolated myelin were digested exhaustively with pronase, and glycopeptides were isolated from the digest by gel filtration on Bio-Gel P-10. The glycopeptides from brain myelin separated into large and small molecular weight fractions, whereas the glycopeptides of sciatic nerve myelin eluted as a single symmetrical peak. The large and small glycopeptide fractions from central myelin and the single glycopeptide fraction from peripheral myelin were analyzed for carbohydrate by colorimetric and gas liquid chromatographic techniques. The glycopeptides from brain myelin contained 2.4 μg of neutral sugar and 0.59 μg of sialic acid per mg total myelin protein, whereas sciatic nerve myelin glycopeptides contained 10 μg of neutral sugar and 3.8 μg of sialic acid per mg total protein. Similarly, the gas-liquid chromatographic analyses showed that the glycopeptides from peripheral myelin contained 4- to 7-fold more of each individual per mg total myelin protein than those from central myelin. Most of the sialic acid and galactose in the glycopeptides from central myelin were in the large molecular weight fraction, and the small molecular weight glycopeptides contained primarily mannose and $\text{N-}\text{acetylglucosamine}$. The considerably higher content of glycoprotein-carbohydrate in peripheral myelin supports the results of gel electrophoretic studies, which indicate that the major protein in peripheral myelin in glycosylated while the glycoproteins in purified central myelin are quantitatevely minor components.     

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.     

Studies on pig serum lipoproteins. IV. Isolation and characterization of glycopeptides from pig serum low density lipoprotein.

Three glycopeptides were isolated from the pronase digest of the protein moiety of pig serum low density lipoprotein. The isolation procedure consisted of pronase digestion, gel filtration on Sephadex G-25 and G-50 columns, paper chromatography and DEAE-Sephadex A-50 column chromatography. Based on the carbohydrate analysis, the isolated glycopeptides were classified into two types. One type (GDI) consisted of mannose and N-acetylglucosamine residues in the molar ratio of 6:2 and had a molecular weight of about 2,300. The other type (GDII and GDIII) consisted of sialic acid, mannose, galactose, fucose, and N-acetylglucosamine residues in the molar ratio of 1:4:2:1:3 and 2:4:3:1:3, respectively. The molecular weights of GDII and GDIII were about 2,100 and 3,100, respectively. The results on the strong alkaline treatment of these glycopeptides suggested that all carbohydrate chains were linked to the peptide chains through N-acetylglucosaminyl-asparagine linkages. Of these glycopeptides and pig serum lipoproteins, only glycopeptide GDI and native LDL strongly interacted with concanavalin A.     

Some properties of the lectin from Datura stramonium (thorn-apple) and the nature of its glycoprotein linkages

The lectin from Datura stramonium (thorn-apple; Solanaceae) has been purified by affinity chromatography and shown to be a glycoprotein containing about 40% (w/w) of carbohydrate. The most abundant amino acids are hydroxyproline, cystine, glycine and serine. Results obtained by gel filtration in 6m-guanidinium chloride on Sepharose 4B suggest that it has a subunit mol.wt. of about 30000 and that it probably associates into dimers. The lectin is inhibited specifically by chitin oligosaccharides and bacterial-cell-wall oligosaccharides, but only weakly by N-acetylglucosamine. Glycopeptides from soya-bean (Glycine max) lectin and fetuin are also strong inhibitors of Datura lectin, indicating that it interacts with internal N-acetylglucosamine residues. Its specificity is similar to, but not identical with, that of potato (Solanum tuberosum) lectin. After prolonged proteolytic digestion of reduced and S-carboxymethylated or S-aminoethylated derivatives of the lectin, glycopeptides of mol.wt. of about 18000 were isolated. The glycopeptides contained all the carbohydrate and hydroxyproline of the original glycoprotein, and lesser amounts of serine, S-carboxymethylcysteine and other amino acids. The arabinose residues of the glycoprotein are present as β-l-arabinofuranosides linked to the polypeptide chain through the hydroxyproline residues, and can be removed by mild acid treatment; the ratio of arabinose to hydroxyproline is 3.4:1. Some of the serine residues of the polypeptide chain are substituted with one or two α-galactopyranoside residues, most of which can be removed by the action of α-galactosidase. The galactose residues are more easily removed from the acid-treated glycopeptide (from which arabinose has been removed) than from the complete glycopeptide, indicating a steric hindrance of the galactosidase action by the adjacent chains of arabinosides. There is a slow release of galactose residues by a process of β-elimination in 0.5m-NaOH (pH13.7) from the complete glycopeptide, and a fairly rapid release of galactose by this process from the acid-treated glycopeptide, which lacks arabinose. This is probably due to the inhibitory effect of the negative charge on the adjacent arabinofuranoside residues. The similarities and differences between the lectins from Datura and potato are discussed, as are their structural resemblance to glycopeptides that have been isolated from plant cell walls.