The isolation and characterization of the glycopeptides from horseradish peroxidase isoenzyme C.

The purity of horseradish peroxidase isoenzyme C was demonstrated using isoelectric focusing, polyacrylamide gel electrophoresis at two pH values and cellulose acetate electrophoresis at two pH values. The glycopeptides obtained upon trypsin digestion were isolated using the plant lectin, concanavalin A, and were resolved using paper electrophoresis. The carbohydrate content of the native peroxidase was 86% accounted for by the carbohydrate content of the glycopeptides thus suggesting little loss of carbohydrate during glycopeptide isolation and purification. In each of the seven glycopeptides isolated glucosamine was associated with asparagine, thus suggesting the carbohydrate chains are covalently bound to the peptide chain through N-glycosidic linkages. The purity of each glycopeptide was demonstrated by the sequential release of single amino acid residues by Edman degradation. As six glycopeptides had unique amino acid sequences, it was concluded that the carbohydrate prosthetic group was distributed in at least six units along the protein backbone. Five glycopeptides possessed the amino acid sequence about the point of carbohydrate attachment of Asn-X-(Ser, Thr) where X is any amino acid. The size of the carbohydrate units ranged from 1600 to 3000 daltons. The predominant carbohydrate residues in each glycopeptide were mannose and glucosamine with lesser and varying amounts of fucose, xylose, and arabinose. There was no apparent correlation of the carbohydrate composition with the amino acid sequence.     

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.     

Location of the carbohydrate groups of ovomucoid.

Tryptic glycopeptides were purified from the sialic acid-free variant of ovomucoid, O1, and its CNBr fragments. The amino acid sequences adjacent to the four major sites of carbohydrate (Carb.) attachment were: (1), Phe-Pro-Asn(Carb.)-Ala-Thr-Asp-Lys-Glu-Gly-Lys; (2), Ala-Try-Ser-Ile-Glu-Phe-Gly-Thr-Asn (Carb.)-Ile-Ser-Lys; (3), Glu, Thr-Val-Pro-Met-Asn(Carb.)-cys-Ser; (4), Ser-Ser-Tyr-Ala-Asn (Carb.)-Thr-Thr-Ser-Glu-Asp-Gly-Lys, Glycosylated Asn residues were located at position 10, between residues 49 and 60, and at positions 69 and 75, in the primary sequence. All of these carbohydrate groups contained GlcNAc, Man and Gal in the approximate molar proprotions 5:3:0.5. A further glycopeptide containing His was isolated in low yield, suggesting that some carbohydrate is attached at a fifth site. Two of the carbohydrate-attachment sites (Asn-10 and Asn-75) occur in sequences that show internal homologies. These are presumed to have evolved as a consequence of partial gene duplication. Three of the carbohydrate-attachment sites occur in similar positions to the carbohydrate groups in quail ovomucoid [Laskowski (1976) Protides Biol. Fluids Proc. Colloq. 23, in the press]. Prediction of peptide conformation from the sequence data by the method of Chou & Fasman [(1974) Biochemistry 13, 222-225] indicated that four glycosylated Asn residues in hen ovomucoid are very close to groups of amino acids that occur with high frequency in beta-turns. The possible significance of peptide-chain conformation in the attachment of carbohydrate to glycoproteins is briefly discussed.     

Studies on Egg White Protein

The carbohydrate of ovomucoid was analyzed for components I, II, III and IV which were, fractionated by CMC-column chromatography. The total hexose content and the molar ratio of d-mannose to d-galactose (4:1) were identical in each component, but the d-glucosamine and sialic acid contents were found to be higher in components I and II (both are trypsin inhibitors) compared with components III and IV (both are apo-proteins of flavomucoid). The amino acid composition of each component of ovomucoid varied considerably. There were remarkable differences in the amino acid composition between components I and II, both had an antitryptic activity. The N-terminal amino acid of components I and II was alanine and in the case of components III and IV, threonine was found on the N-terminal. The free carboxylic residue of sialic acid was found to be responsible for the negative charge of ovomucoid, and its electrophoretic heterogeneity was reaffirmed by paper electrophoresis. It is evident from the ultracentrifugal analysis that the four components of ovomucoid have a similar molecular size.     

A glycopeptide isolated from human gastric juice.

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

The covalent structure of individual N-linked glycopeptides from ovomucoid and asialofetuin

In order to explore whether individual N-linked glycans in a given glycoprotein may be processed to different end products and at the same time prepare a number of well characterized glycopeptides as substrates for glycopeptide hydrolases, we have prepared the individual glycopeptides representing the four major glycosylation sites in ovomucoid and the three sites in asialofetuin. The individual glycopeptides were characterized by amino acid sequence determination before and after removal of the glycan by peptide:N-glycanase (amidase), and the liberated glycans were subjected to mass spectrometric analysis. As expected from available sugar analyses of the individual glycans in ovomucoid, no major differences were detected between the four glycosylation sites in this glycoprotein, but a definite trend toward less processed (less extensively branched) species was observed in going from site 1 to 4. In fetuin, for which the glycan pool is known to be made up of about two-thirds triantennary and one-third biantennary structures, the analysis of the three glycopeptides gave triantennary to biantennary ratios of 75/25, 67/33, and 70/30, respectively, demonstrating that the three sites are processed to a very similar, albeit perhaps not identical, extent. All the glycopeptides obtained in these studies, including the CNBr-produced glycopeptide from ovalbumin, were purified by a set series of steps, gel filtration on Sephadex G-50 followed by ion-exchange chromatography on DE52 and/or reverse phase high performance liquid chromatography. Based on the results, these procedures appear to have general application for the preparation of glycopeptides.     

A new neuraminic acid derivative and three types of glycopeptides isolated from the Cuvierian tubules of the sea cucumber Holothuria forskali

The Cuvierian tubules of Holothuria forskali Della Chiaje, a sea cucumber found in the Adriatic Sea, were investigated with regard to their carbohydrate moieties. From a Pronase digest of these tubules three types of carbohydrate units were isolated and characterized. 1. A high-molecular-weight glycopeptide fraction was shown to contain sulphated polyfucose, galactosamine, a uronic acid and a previously unknown neuraminic acid derivative. The sulphate was shown by i.r. analysis to be present as an O-ester. The carbohydrate unit was linked O-glycosidically to threonine and serine residues in the polypeptide chain. The hitherto unknown neuraminic acid derivative (Hf-neuraminic acid) was resistant to enzymic cleavage by neuraminidase, even after mild alkaline hydrolysis for the removal of O-acyl residues. However, the glycosidic linkage of this compound to the other part of the carbohydrate moiety was readily cleaved by mild acid hydrolysis. Its chromatographic properties distinguished Hf-neuraminic acid from other known neuraminic acid derivatives (N-acetyl-, NO-diacetyl-, NOO-triacetyl- and N-glycollyl-neuraminic acid). Further, this acidic sugar was shown to possess neuraminic acid as its basic structure. Thus, an as yet unknown substituent lends the distinct properties to Hf-neuraminic acid. 2. The carbohydrate composition of a second glycopeptide fraction consisting of a derivative of neuraminic acid, galactose, mannose and glucosamine was similar to that of the well-known carbohydrate groups of the globular glycoproteins. 3. The third fraction contained two glycopeptides containing the disaccharide, glucosylgalactose, which was shown to be linked to the hydroxyl group of hydroxylysine residues of a collagen-like protein. Approximately half of these residues were glycosylated. In addition to these glycopeptides, a small amount of a third glycopeptide that carried only a galactosyl residue was detected. The amino acid sequence of the two major compounds were found to be Gly-Ala-Hyl*-Gly-Ser and Gly-Pro-Hyl*-Gly-Asp, where Hyl* represents a glycosylated amino acid residue.     

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.     

Evidence for unique homologous peptide sequences around the glycosylated seryl and threonyl residues in polysialoglycoproteins isolated from the unfertilized eggs of the Pacific salmon Oncorhynchus keta

Structures of glycopeptides obtained by exhaustive Pronase digestion of high molecular weight (1.7 X 10(5)) salmon egg polysialoglycoprotein have been elucidated. Six principal glycopeptides isolated by gel chromatography and DEAE-Sephadex A-25 chromatography in the absence or presence of borate ion were analyzed for their carbohydrate and amino acid composition, as well as amino acid sequence, and found to be of two distinct types: glycotripeptides, Thr*-Ser*-Glu, and glycotetrapeptides, Thr*-Gly-Pro-Ser, where an asterisk indicates the amino acid residues to which either the Gal beta 1----3GalNAc or Fuc alpha 1----3GalNAc beta 1----3Gal beta 1----4Gal beta 1----3GalNAc chain is attached. Their final yield corresponds to 64% of the original desialylated glycoprotein. In view of the simple amino acid composition of salmon egg polysialoglycoprotein (molar ratio Asp2Thr2Ser3Glu1Pro1Gly1Ala3) and the result of alkaline beta-elimination indicating three carbohydrate units linked to two of two threonine and one of three serine residues, a unique primary structure comprising repetitive sequences of the above two types of glycopeptides, which are interspersed by short nonglycosylated peptides consisting of alanine and aspartic acid, has been proposed for the core protein. The molecular secondary ion mass spectra of underivatized glycopeptides were used to obtain their structural information. The anomeric configuration of the proximal sugar-peptide linkages was proven to be alpha by proton nuclear magnetic resonance spectroscopy. This is the first systematic reported study of O-glycosidically linked glycopeptides by these instrumental methods.     

Methods for the identification of N-asparaginyl and O-seryl/threonyl glycosidic linkages to aminosugars in glycoproteins

Methods are presented for the identification of certain glycopeptide bonds in glycoproteins. Mucin-type linkages are determined following treatment of glycoproteins with alkaline sodium [³H]borohydride. Such treatment cleaves O-glycosidic bonds to serine and threonine and simultaneously labels the sugar and amino acid components of the linkage. Following acid hydrolysis and dansylation, the sugar component of the linkage is identified as its corresponding dansyl-hexosaminitol by fluorographic techniques. A method is described for the separation of dansyl-galactosaminitol and dansyl-glucosaminitol by thin-layer electrophoresis in borate buffers. The amino acid component of the glycopeptide linkage is identified by fluorography following two-dimensional thin-layer chromatography of its dansyl derivative on polyamide plates. For the analysis of plasma-type glycoproteins, glycopeptides are prepared by exhaustive pronase digestion and purified by gel filtration chromatography. Final purification is effected by dansylation and thin-layer electrophoresis. The linkage compound 2-acetamido-1-N-β-l-aspartyl-2-deoxy-β-d-glucopyranosylamine is isolated from such glycopeptides as its dansyl derivative following partial acid hydrolysis. Its identity is confirmed by comparison of its properties with those of the synthetic compound. Thus the components of the glycosylamine linkage are identified following complete acid hydrolysis, redansylation, and separation by thin-layer electrophoresis.     

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

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

Attachment Sites of Carbohydrate Portions to Peptide Chain of κ-Casein from Bovine Colostrum

Short glycopeptides were prepared from bovine colostral κ-casein treated with cyanogen bromide and proteases (pronase P and thermolysin), followed by gel filtrations and ion exchange chromatography. It was confirmed by Edman degradation that glycopeptide I among short glycopeptides obtained was homogeneous. From the effect of alkali treatment, it was assumed that three polysaccharide chains of glycopeptide I were attached to the peptide chain through OH groups of threonines. By chemical procedures and carboxypeptidase P treatment, the amino acid sequence of glycopeptide I was established to be Ser-Gly-Glu-Pro-Thr-Ser-Thr-Pro-Thr-Thr-Glu-Ala-Val. Threonine residues of No. 5, 7 and 9 were bound to the carbohydrate chains through galactosamine. The sugar chain bound to the threonine residue at No. 7 contained glucosamine. Glycopeptide I corresponded to residues of No. 127–139 in κ-casein A from normal milk.     

The amino acid sequences of two large glycopeptides derived from the carbohydrate-carrying region of α1-acid glycoprotein

Specific fragmentation with cyanogen bromide and subsequent reduction and carboxymethylation of α₁-acid glycoprotein, a normal human plasma globulin, permitted isolation of a large fragment which was shown to represent the amino-terminal half and to contain the total carbohydrate moiety of this protein. The amino acid sequences of two large glycopeptides derived from this fragment were established. One glycopeptide was composed of 22 amino acid residues and one carbohydrate unit, and the other consisted of 65 amino acid residues and carried four carbohydrate units.     

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.     

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.     

Isolation and characterization of human and canine gastric mucosal glycoproteins and their degradation by proteases and acid hydrolases

High molecular weight glycoproteins were isolated and purified from canine antral and fundic mucosal tissue by means of non-degrading techniques. The results disclosed the advantage of urea extraction technique over the culture method in isolating the native glycoproteins. The glycoproteins were susceptible to degradation by protease, thus yielding low molecular weight glycopeptides. Chemical analysis of these glycopeptides and their parent macromolecules revealed that the oligosaccharide residues are attached to threonine, serine and proline residues of the protein chains. Similarly, high molecular weight glycoproteins isolated from human gastric gel mucin showed the same characteristics of canine gastric glycoproteins. Canine fundic glycoprotein or glycopeptide released their prosthetic carbohydrate groups under the lytic effect of fundic acid hydrolases.     

Incompatible blood-group A determinants in tumoral mucins. Isolation of oligosaccharides having a 2-acetamido-2-deoxy-alpha-D-galactopyranosyl group at the non-reducing end

Two glycopeptide fractions were obtained from pseudomyxomatous mucins secreted by an ovarian cystadenocarcinoma from a female having blood-group B, and by an appendix tumor from a male having blood-group O. The carbohydrate and amino acid content of these fractions suggests the presence of numerous carbohydrate side-chains linked through O-glycosyl bonds to a peptide core rich in threonine and proline. The two glycopeptide fractions exhibit compatible B- and H-blood-group activities. They are reactive towards Dolichos biflorus lectin and human anti-A agglutinins, and so exhibit an incompatible A activity. Alkali-borohydride degradation of Pronase-digested glycopeptides gave dialyzable oligosaccharides that were purified and shown to possess 2-acetamido-2-deoxygalactitol at the terminal reducing-end. 2-Acetamido-2-deoxyglucose, galactose, fucose, and neuraminic acid were absent, or present, in variable proportions. Four oligosaccharides containing 2-acetamido-2-deoxy-D-galactose residues were reactive towards Dolichos biflorus lectin and human anti-A agglutinins, indicating the presence, at the nonreducing end, of a 2-acetamido-2-deoxy-alpha-D-galactopyranosyl group, responsible for blood-group A activity.     

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.     

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.     

The comparison of an acidic glycopeptide from the ascitic tumor fluid and of the glycopeptide from Ehrlich cells

An acidic glycopeptide of molecular weight of 12000 daltons and of pI 5.6 from the ascitic tumor fluid of Ehrlich cells was isolated and characterized with regard to its physico-chemical properties and compared with an acidic glycopeptide of Ehrlich cells. Both glycopeptides have the same carbohydrate and amino acid components but differ in respect of quantity. The origin of an acidic glycopeptide from the ascitic tumor fluid of Ehrlich cells is briefly discussed.