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.     

The Composition of Saccharogenic Amylase from Rhizopus javanicus and the Isolation of Glycopeptides

Saccharogenic amylase from Rhizopus javanicus sp. 3–46 was known to be a glycoprotein which contained 27 residues of mannose and 4 residues of N-acetylglucosamine per mole of the saccharogenic amylase. Attempts have been made to obtain glycopeptides from the saccharogenic amylase. Three glycopeptides, GP-I-a, GP-I-b and GP-II, were separated from a Pronase digest of heat-denatured saccharogenic amylase by gel filtration on Sephadex G-50 and chromatography on DEAE-Sephadex A-25. GP-I-a contained asparagine, glycine, mannose and N-acetylglucosamine in a molar ratio of 1: 1: 6: 2. GP-I-b contained asparagine, threonine, mannose and N-acetylglucosamine in a molar ratio of 1: 1: 9:2. GP-II consisted of threonine, serine, proline, alanine and mannose in a molar ratio of 6: 2: 2: 2: 12.     

The enzymic degradation of ovalbumin and its glycopeptides

1. Ovalbumin glycopeptides, freed from all amino acids other than aspartic acid and a small proportion of leucine by repeated digestion with Pronase, were hydrolysed by 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (glycoaspartamidase) to the corresponding oligosaccharides. The glycoaspartamidase did not attack ovalbumin itself. 2. Ovalbumin, with mannose/hexosamine ratio 5:4, lost 1.5moles of N-acetylglucosamine and more than 2moles of mannose after incubation with alpha-mannosidase and beta-N-acetylglucosaminidase respectively. 3. In ovalbumin glycopeptides with approximate mannose/hexosamine ratios 5:3 and 5:4, one and two N-acetylglucosamine residues respectively were accessible to the action of beta-N-acetylglucosaminidase. 4. A mixture of alpha-mannosidase and beta-N-acetylglucosaminidase, acting on an ovalbumin glycopeptide with mannose/hexosamine ratio 5:3.7, removed nearly 4moles of mannose and 1.5moles of N-acetylglucosamine. 5. alpha-Mannosidase removed about 1.5moles of mannose from the ovalbumin oligosaccharide with mannose/hexosamine ratio approx. 5:3. The subsequent action of beta-N-acetylglucosaminidase liberated less than 1mole of N-acetylglucosamine and made at least 1mole further of mannose accessible to alpha-mannosidase action. 6. It is concluded that the carbohydrate moiety of ovalbumin is linked through a glycosyl group to asparagine. In a molecule with mannose/hexosamine ratio 5:4, there are two beta-N-acetylglucosamine residues linked together in a terminal position, followed by alpha-mannose. There is also present a side chain containing two alpha-mannose units.     

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.     

Glycopeptides from rat brain glycoproteins

The lipid-free protein residue of rat brain tissue was treated with papain to solubilize the heteropolysaccharide chains of the tissue glycoproteins. The glycopeptides were separated into non-dialyzable and dialyzable glycopeptide preparations. Each preparation was then sorted out into groups of glycopeptides by means of electrophoresis and gel filtration. The quantitatively predominant glycopeptides were the alkali-stable glycopeptides (Group A) which accounted for 64% of the glycopeptide carbohydrate recovered from rat brain. Most of the group A glycopeptides appeared in the non-dialyzable preparation. The molecular weight of the glycopeptides of Group A ranged from approximately 5200–3700. The largest glycopeptide molecule in this mixture possessed the highest electrophoretic mobility and contained one fucose, four N-acetylneuraminic acid (NANA), six N-acetylglucosamine, four galactose, and three mannose residues per molecule. The spectrum of glycopeptides isolated in this group showed a progressive decrease in NANA rsidues, NANA and galactose residues, and NANA, galactose, and N-acetylglucosamine residues which could be correlated with a progressive decline in molecular weight and electrophoretic mobility. Some of the glycopeptides in each fraction recovered from this group of glycopeptides contained sulfate ester groups.A second group of glycopeptides (Group C glycopeptides) accounted for 25% of the total glycoprotein carbohydrate recovered from rat brain. These were recoverd from the dialyzable glycopeptide preparation, and resolved into three fractions by column electrophoresis. These glycopeptides do not contain sulfate, are composed predominately of mannose and N-acetylglucosamine, and possess a molecular weight of approximately 3000.Several minor groups of glycopeptides were detected. Alkali-labile glycopeptides (Group B) appeared in the non-dialyzable glycopeptide preparation. The dialyzable glycopeptide preparation contained glycopeptides (Group E) which contained N-acetylgalactosamine and glucose. These had a molecular weight of approximately 2000. Group D glycopeptides recovered from the dialyzable glycopeptide preparation contained variable amounts of NANA, mannose, galactose, N-acetylglucosamine, and sulfate. These possessed a molecular weight of approximately 2900.     

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

The structure of the carbohydrate moiety of GP–I–a, one of three glycopeptides obtained from Rhizopus saccharogenic amylase, was determined by using enzymatic and chemical techniques.

Six residues (all of the residues in GP–I–a) of mannose and one residue of N-acetylglucosamine were released in that order when GP–I–a was digested successively with purified α-mannosidase and β-N-acetylglucosaminidase.

Exhaustive methylation of GP–I–a 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-methyI and 2,4-di-O-methyl derivatives from the mannose residues in an approximate ratio of 3: 1:2.

After one step of the Smith degradation of GP–I–a, a residual glycopeptide (F–1) consisted of one mole each of asparagine and glycine and two moles each of mannose and N-acetylglucosamine was obtained. Exhaustive methylation of F–1 gave 3,6-di-O-methyl derivative of N-acetylglucosamine, and 2,3,4,6-tetra-O-methyl and 2,3,4-tri-O-methyl derivatives of mannose in a ratio of 1.00: 0.91.

Partial acetolysis of 1→6 linkages in GP–I–a yielded mannose, 3-O-mannosylmannose and a smaller glycopeptide which was resistant to the acetolysis.

From these and other evidences, the following structure was determined for GP–I–a.     

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.     

Glycoprotein enzymes secreted by Aspergillus niger: purification and properties of alpha-glaactosidase.

An alpha-galactosidase (alpha-D-galactoside galactohydrolase [EC 3.2.1.22]) was purified to homogeneity from the culture filtrate of Aspergillus niger. The enzyme had an apparent molecular weight of 45,000 and was a glycoprotein. Radioactive enzyme was prepared by growing cells in [14C]fructose and this enzyme was used to prepare 14C-labeled glycopeptides. The glycopeptides emerged from Sephadex G-50 between stachyose and the glycopeptide from ovalbumin. Based on calibration of the column with various-sized dextran oligosaccharides, the glycopeptides appeared to have a molecular weight of 1,200 to 1,400. Analysis of the glycopeptide(s) indicated that it contained mannose and N-acetylglucosamine (GlcNAc) in an approximate ratio of 3 or 4 to 1. Assuming that there are two GlcNAc residues in the oligosaccharide and based on the molecular weight of the glycopeptide, the oligosaccharide probably contains eight to nine sugar residues. Alks probably attached to the protein by a GlcNAc leads to asparagine linkage. The purified alpha-galactosidase was most active on raffinose (Km = 5 x 10--4 M, Vmax = 3 mumol/min per mg of protein), but also showed good activity on p-nitrophenyl-alpha-D-galactoside ans somewhat less activity on stachyose and melibitol. The enzyme also hydrolyzed guar flour and locust bean gum, but did not attack the p-nitrophenyl glycosides of beta-galactose, alpha- or beta-glucose, or alpha- or beta-mannose.     

Localization and overall structure of a mannose-rich glycopeptide from a pathologic immunoglobulin

The structure of a mannose-rich glycopeptide from a human pathological IgM has been investigated. It belongs to the group I (simple) glycopeptides and contains only mannose and N-acetylglucosamine residues in a molar ratio of 10:2. The structures of its oligosaccharide moiety and peptide chain have been determined: its molecular localization is specified and the relation between its biosynthesis and the oligosaccharide structure determine is discussed. Based on the alpha- and beta-mannosidase digestions and permethylation studies for the oligosaccharide moiety, and on the results obtained after sequential analysis of the peptide chain, the following structure is proposed for the mannose-rich IgM Du glycopeptide: (Formula: see text). The recovery of one molecule of this glycopeptide per molecule of heavy chain and the determination of the amino acid sequence have led us to locate this glycopeptide on asparagine 402 of the Fc portion of the heavy chain mu of IgM Du.     

The oligosaccharide units of a human type L immunoglobulin M (macroglobulin)

1. The carbohydrate composition of the monomeric unit of a type L macroglobulin (immunoglobulin M) was determined as 6 residues of fucose, 35 of mannose, 11 of galactose, 27 of N-acetylglucosamine and 9 of sialic acid. 2. Two types of oligosaccharide unit were present in the protein, one of which (Ca type) contained fucose, mannose, galactose, N-acetylglucosamine and sialic acid in the molar proportions 1:3-4:2:3-5:0-2, and the other (Cb type) contained mannose and N-acetylglucosamine in the proportions 6-8:2-3. 3. A tentative structure is proposed for the Cb type unit. 4. An S-carboxymethylcysteine-containing glycopeptide with a Ca-type unit was isolated after reduction, alkylation and tryptic digestion of the protein. 5. The immunoglobulin monomer appears to contain six oligosaccharide units of the Ca type and two of the Cb type.     

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.     

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.     

Purification, composition, and structure of macrophage adhesion molecule

Macrophage adhesion molecule (MAM) is a surface heterodimer consisting of the trypsin- and plasmin-sensitive glycopeptide gp160 (MAM-alpha) and the glycopeptide gp93 (MAM-beta). MAM, which is the guinea pig analogue of Mo1 and Mac-1, was purified from detergent lysates of peritoneal neutrophils by lentil lectin chromatography and M2-antibody chromatography. The pure heterodimer molecule was dissociated by acidic conditions (pH 3.5), and MAM-alpha and MAM-beta were separated by M7-antibody chromatography. MAM-beta is an approximately 640 amino acid residue polypeptide with exceptionally high cysteine content. At 7.2 residues per 100 amino acids, Cys/2 of MAM-beta is more than 3 times the mean for 200 purified proteins. Reactivity with six beta-subunit-specific monoclonal antibodies recognizing at least four epitopes demonstrated that intrapeptide disulfide bonds are required to maintain the structure of MAM-beta. All six antibodies failed to react when MAM-beta was treated with reducing agents. MAM-beta is 18% carbohydrate; the major monosaccharides are mannose, N-acetylglucosamine, galactose, and sialic acid. MAM-beta is estimated to contain five to six N-linked carbohydrate units. MAM-alpha is an approximately 1100-residue polypeptide with lower Cys/2 content (2.0 residues per 100 amino acid residues). MAM-alpha is 21% carbohydrate. The major monosaccharides are mannose, N-acetylglucosamine, galactose, and sialic acid; the mannose content is higher in MAM-alpha than MAM-beta. MAM-alpha is estimated to contain 12 N-linked carbohydrate units.     

Carbohydrate structure of the major glycopeptide from human cold-insoluble globulin

Cold-insoluble globulin (CIg) is a member of a group of circulating and cell-associated, high-molecular-weight glycoproteins termed fibronectins. CIg was isolated from human plasma by affinity chromatography on gelatin-Sepharose. SDS-polyacrylamide gel electrophoresis of the purified glycoprotein gave a double band that migrated near myosin. The CIg glycopeptides were released by pronase digestion and isolated by chromatography on Sephadex G-50. Affinity chromatography of the major G-50 peak on Con A-Sepharose resulted in two fractions: one-third of the glycopeptides were unbound and two-thirds were weakly bound (WB). Sugar composition analysis of the unbound glycopeptides by GLC of the trimethylsilyl methyl glycosides gave the following molar ratios: sialic acid, 2.5; galactose, 3.0; N-acetylglucosamine, 4.9; and mannose, 3.0. Sugar composition analysis of the WB glycopeptides gave the following molar ratios: sialic acid, 1.7; galactose, 2.0; N-acetylglucosamine, 4.1; and mannose, 3.0. The WB CIg glycopeptides cochromatographed on Sephadex G-50 with WB transferrin glycopeptides giving an estimated molecular weight of 2,800. After degradation with neuraminidase alone or sequentially with β-galactosidase the CIg and transferrin glycopeptides again cochromatographed. Methylation linkage analysis of the intact and the partially degraded glycopeptides indicated that the carbohydrate structure of the major human CIg glycopeptide resembles that of the major glycopeptide from transferrin.     

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.     

The structure of a glycopeptide purified from porcine thyroglobulin

1. The structure of a purified glycopeptide isolated from porcine thyroglobulin was studied by sequential hydrolysis with specific glycosidases, by periodate oxidation and by treatment with galactose oxidase. 2. Sequential hydrolysis with several combinations of neuraminidase, alpha-l-fucosidase, beta-d-galactosidase, beta-N-acetyl-d-glucosaminidase and alpha-d-mannosidase presented the evidence for the following structure. 3. The monosaccharide sequence of the peripheral moiety of the heteropolysaccharide chain was sialic acid-->galactose-->N-acetylglucosamine. Some of the galactose residues were non-reducing end-groups with the sequence galactose-->N-acetylglucosamine. 4. After removal of the peripheral moiety composed of sialic acid, fucose, galactose and N-acetylglucosamine, alpha-mannosidase released 1.4mol of mannose/mol of glycopeptide, indicating that two of the three mannose residues were located between peripheral N-acetylglucosamine and internal N-acetylglucosamine or mannose. 5. Periodate oxidation and sodium borohydride reduction confirmed the results obtained by enzymic degradation and gave information concerning the position of substitution. 6. Based on the results obtained by enzymic hydrolysis and periodate oxidation together with the treatment with galactose oxidase, a structure is proposed for the glycopeptide.     

Carbohydrate Structure of Sindbis Virus Glycoprotein E2 from Virus Grown in Hamster and Chicken Cells

Sindbis virus was used as a probe to examine glycosylation processes in two different species of cultured cells. Parallel studies were carried out analyzing the carbohydrate added to Sindbis glycoprotein E2 when the virus was grown in chicken embryo cells and BHK cells. The Pronase glycopeptides of Sindbis glycoprotein E2 were purified by a combination of ion-exchange and gel filtration chromatography. Four glycopeptides were resolved, ranging in molecular weight from 1,800 to 2,700. Structures are proposed for each of the four glycopeptides, based on data obtained by quantitative composition analyses, methylation analyses, and degradation of the glycopeptides using purified exo- and endoglycosidases. The largest three glycopeptides (S1, S2, and S3) have similar structures but differ in the extent of sialylation. All three contain N-acetylglucosamine, mannose, galactose, and fucose, in a structure similar to oligosaccharides found on other glycoproteins. Glycopeptide S1 has two residues of sialic acid, whereas glycopeptides S2 and S3 contain 1 and 0 residues of sialic acid, respectively. The smallest glycopeptide, S4, contains only N-acetyglucosamine and mannose, and is also similar to mannose-rich oligosaccharides found on other glycoproteins. Each of the complex glycopeptides (S1, S2, or S3) from virus grown in BHK cells is indistinguishable from the corresponding glycopeptides derived from virus grown in chicken cells. Glycopeptide S4 is also very similar in size, composition, and sugar linkages from virus derived from the two hosts. These results suggest that chicken cells and BHK cells have similar glycosylation mechanisms and glycosylate Sindbis glycoprotein E2 in nearly identical ways.     

A structural basis for four distinct elution profiles on concanavalin A--Sepharose affinity chromatography of glycopeptides.

Twelve 14C-acetylated glycopeptides have been subjected to affinity chromatography on concanvalin A (Con A)--Sepharose at pH 7.5. The elution profiles could be classified into four distinct patterns. The first pattern showed no retardation of glycopeptide on the column and was elicited with a glycopeptide having three peripheral oligosaccharide chains: (abstract:see text). Such glycopeptides have only a single mannose residue capable of interacting with Con A--Sepharose; an interacting mannose residue is either an alpha-linked nonreducing terminal residue or an alpha-linked 2-O-substituted residue. The second type of profile showed a retarded elution of glycopeptide with buffer lacking methyl alpha-D-glucopyranoside (indicative of weak interaction with the column) and was given by glycopeptides with the structures: (abstract: see text) where R1 is either H or a sialyl residue. The third profile type showed tight binding of glycopeptide to Con A--Sepharose and elution as a sharp peak with 0.1 M methyl alpha-D-glucopyranoside; glycopeptides giving this pattern had the structures: (abstract: see text) where R2 is either H, glcNAc, Gal-beta 1,4-GlcNAc, or sialyl-Gal-beta 1,4-GlcNAc. These glycopeptides all have two interacting mannose residues, the mimimum required for binding to the column; one of these mannose residues must, however, be a terminal residue to obtain tight binding and sharp elution. The fourth profile type showed tight binding of glycopeptide to the column but elution with 0.1 M methyl alpha-D-glucopyranoside resulted in a broad peak indicating very tight binding; glycopeptides showing this behaviour had the structures: (abstract: see text) where R3 is either GlcNAc,Gal-beta 1,4-GlcNAc, or sialyl-Gal-beta 1,4-GlcNAc.Therefore it can be concluded that although a minimum of two interacting mannose residues is required for binding to Con A--Sepharose, the residues linked to these mannoses can either strengthen or weaken binding to the column.     

A comparison of glycopeptides from the ovotransferrin and serum transferrin of the hen

1. Glycopeptides were prepared from proteolytic digests of ovotransferrin and serum transferrin of the hen. The carbohydrate compositions and amino acid sequences of the peptides were studied. 2. The bulk of the carbohydrate of ovotransferrin is present as a single oligosaccharide composed of 4 residues of mannose and 8 residues of N-acetylglucosamine. Transferrin has most of its carbohydrate in a single unit composed of 2 residues of mannose, 2 residues of galactose, 3 residues of N-acetylglucosamine and either 1 or 2 residues of sialic acid. 3. The amino acid sequences of the glycopeptides carrying these different oligosaccharides are the same in ovotransferrin and serum transferrin, showing that the carbohydrate groups are attached to the same site on the protein molecule.     

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.