Characterization of macrophage adhesion molecule

Macrophage adhesion molecule (MAM), an abundant surface molecule which functions in the adhesion and spreading of guinea pig macrophages on surfaces, is characterized as a heterodimer of the trypsin- and plasmin-sensitive glycopeptide gp160 (MAM-alpha) and the glycopeptide gp93 (MAM-beta). The density of MAM molecules is estimated at 630,000 per macrophage on the basis of quantitative binding of 125I-labeled monoclonal antibody. The glycopeptide subunits display microheterogeneity on isoelectrofocusing; the pI is 5.8-6.3 for gp160 (MAM-alpha) and 6.4-7.0 for gp93 (MAM-beta). A neutrophil gp160, gp93 molecule was shown to be indistinguishable from macrophage MAM on the basis of electrophoresis, isoelectrofocusing, and reactivity with 10 monoclonal antibodies. A related heterodimer of gp93 associated with a larger, antigenically different glycopeptide (gp180,gp93) was identified on circulating lymphocytes. Cumulative properties indicate that MAM is the guinea pig analogue of human Mo1 and mouse Mac-1.     

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

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

Preparation 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 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.     

Study of the glycosylation of apolipoprotein H

Apolipoprotein H is a single chain polypeptide composed of 326 amino acids highly glycosylated. Its carbohydrate content is approximately 19% of the molecular weight. We show that it is rich in sialic acid linked alpha (2-6) to galactose or N-acetylgalactosamine. Sialic acid is not alpha (2-3) linked to galactose. Galactose is beta (1-4) linked to N-acetylglucosamine and beta (1-3) linked to N-acetylgalactosamine. Carbohydrate O-linked chains (mainly sialic acid) are alpha (2-6) linked to galactose or N-acetylgalactosamine. Galactose is also organised in O-linked chains and beta (1-4) linked to N-acetylglucosamine and beta (1-3) linked to acetylgalactosamine. Concanavalin A lectin was used to isolate two groups of apolipoprotein H molecules bearing biantennary and truncated hybrids and high mannose and hybrid oligosaccharides. Apolipoprotein H fails to bind lysine-Sepharose. Our results thus show that it presents truncated hybrid or hybrid-type carbohydrate chains which bear few unmasked mannose residues as a terminal sugar. Biochemical analysis of carbohydrate structures conducted on single isoforms separated through IEF revealed that no specific carbohydrate complex is bound to a single isoform.     

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.     

Methylation analysis of the carbohydrate portion of fibronectin isolated from human plasma

Fibronectin was isolated from pooled human plasma by affinity chromatography on Sepharose-coupled gelatin and subjected to methylation analysis. The results indicate that the carbohydrate chains in fibronectin are very similar to those found in other serum glycoproteins, i.e. a common inner core structure consisting of two N-acetylglucosamine and three mannose residues attached to the polypeptide backbone via a N-acetylglucosamine-asparagine linkage. To this inner core are attached two side chains made up of N-acetylglucosamine, galactose and terminated by sialic acid.     

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.     

Characterization of collagenous and non-collagenous peptides of a glycoprotein isolated from alveoli of patients with alveolar proteinosis.

A glycoprotein of Mr 62 000 was isolated from lung lavage material of patients with alveolar proteinosis. The glycoprotein was found to contain (per molecule) 72 residues of glycine, 5 residues of hydroxyproline, 3 molecules of sialic acid, 4.9 molecules of mannose, 4.0 molecules of galactose, 0.9 molecule of fucose and 7.0 molecules of N-acetylglucosamine. Limited pepsin digestion of the glycoprotein resulted in six peptides, three of which contained hydroxyproline and nearly 30% glycine, and two of which contained all the carbohydrate present in the glycoprotein of Mr 62 000. The three peptides containing hydroxyproline and with high content of glycine contained a repeating -Gly-X-Y-sequence in the peptide chain. Partial amino acid-sequence analyses on the peptides derived from the digestion of the alveolar glycoprotein with various proteolytic enzymes indicate that this glycoprotein is characterized by the presence of alternating collagenous and non-collagenous regions in the same polypeptide chain.     

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.     

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.     

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.     

Isolation and characterization of the two glycosylation isoforms of low molecular weight mannose 6-phosphate receptor from bovine testis. Effect of carbohydrate components on ligand binding.

Low molecular weight mannose 6-phosphate receptor from bovine testis exhibits two isoforms on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Mr values of 45,000 (MPR-2A) and 41,000 (MPR-2B), respectively. Each isoform was purified to near homogeneity by the sequential application of differential centrifugation and affinity chromatography. The isoforms contain a common polypeptide core, but differ in their carbohydrate content. Treatment with specific endoglycosidases demonstrated that each isoform contains two high mannose and/or hybrid and two complex N-linked oligosaccharide chains. The results obtained from treatment of each isoform with endo-beta-galactosidase and neuraminidases and from lectin affinity chromatography reveal that MPR-2A contains a linear polylactosamine sequence(s) comprised of approximately 5 lactosamine units. A majority of the outer branches of the complex chains associated with MPR-2A are terminated with sialic acid residues. In contrast, MPR-2B lacks a polylactosamine sequence and a majority of the outer branches of the complex chains are terminated with galactose residues. MPR-2A exhibited a lower affinity than MPR-2B for mannose 6-phosphate-containing ligands. Treatment of MPR-2A with endo-beta-galactosidase and/or neuraminidases followed by affinity chromatography revealed that polylactosamine and sialic acid residues impair the ability of MPR-2A to bind ligands.     

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

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

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

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

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

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

Structural studies on carcinoembryonic antigen. Periodate oxidation.

Periodate oxidation has been applied to examine the carbohydrate structure of carcinoembryonic antigen (CEA) and the possible role of the carbohydrate residues in its antigenic activity. Sialic acid (N-acetylneuraminic acid) and fucose were completely destroyed, and galactose and mannose were partially destroyed by a single periodate treatment. Serial periodate treatment (Smith degradation) destroyed additional amounts of galactose and mannose as well as significant amounts of N-acetylglucosamine. Prior removal of sialic acid by neuraminidase treatment led to increased destruction of galactose by periodate. Antigenic activity persisted indicating that the residues destroyed played little, if any, part in the antigenicity of CEA. These results yield an initial view of the structural arrangement of the carbohydrate residues in the CEA molecule.     

Isolation and characterization of a major glycoprotein from milk-fat-globule membrane of human breast milk.

A major periodate--Schiff-positive component from milk-fat-globule membrane of human breast milk has been purified by selectively extracting the membrane glycoproteins, followed by lectin affinity chromatography and gel filtration on Sephadex G-200 in the presence of protein-dissociating agents. The purified glycoprotein, termed epithelial membrane glycoprotein (EMGP-70), has an estimated mol.wt. of 70 000 and yields a single band under reducing conditions on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The glycoprotein contains 13.5% carbohydrate by weight, with fucose, mannose, galactose, N-acetylglucosamine and sialic acid 17.2, 17.0, 21.1, 7.9 and 36.6% respectively of the carbohydrate moiety. Aspartic and glutamic acid and serine are the major amino acid residues.     

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.     

Preparation and chemical characterisation of calf Tamm-Horsfall glycoprotein.

Tamm-Horsfall glycoprotein preparations were obtained from calf urine by 1.0 M NaCl precipitation followed by 4 M urea/Sepharose 4B chromatography. By using 0.1% sodium dodecyl sulfate polyacrylamide gel electrophoresis a molecular weight of 86 500 +/- 4500 (n = 12) was calculated for the glycoprotein. Amino acid and carbohydrate analyses were performed, the carbohydrate composition being (in residues per 100 amino acid residues in the glycoprotein): fucose, 0.90; galactose, 4.82; mannose, 4.63;N-acetylglucosamine, 7.36; N-acetylgalactosamine, 1.38; sialic acid, 2.93. Under conditions of mild acid hydrolysis (0.05 M H2SO4, 80 degrees C, 1 h) the calf Tamm-Horsfall glycoprotein preparations were degraded partially into two lower molecular weight fragments (approximate Mr 66 000 and 51 000), as shown by polyacrylamide gel electrophoresis, both fragments being periodic acid-Schiff reagent positive.