Human embryonal prealbumin-1. Chemical and immunochemical characteristics

General chemical and immunochemical characterization of human embryonic prealbumin-1 (EPA-1) isolated from abortive blood is presented. EPA-1 was found to exist as glycosylated and non-glycosylated forms, which are immunochemically identical. Sugar moiety of the glycosylated form contains residues of fucose (3.0%), mannose (3.2%), galactose (7.8%), N-acetylglucosamine (5.4%), N-acetylgalactosamine (1.2%) and N-acetylneuraminic acid. Using methylation studies, types of bonds between the sugar residues were elucidated.     

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.     

Isolation and partial characterization of the major sialoglycoprotein of human T-lymphoblastoid cells of a MOLT-4B cell line.

A sialoglycoprotein with an approx. mol.wt. of 95000 was isolated from human lymphoblastoid cells of a MOLT-4B cell line, which was of human T-lymphocyte origin, by ion-exchange chromatography, affinity chromatography on a column of wheat-germ agglutinin-Sepharose and preparative slab-gel electrophoresis. The localization of this glycoprotein on the cell surface was indicated by surface labelling by the periodate/NaB3H4 and lactoperoxidase-catalysed iodination methods. Carbohydrate analyses of this glycoprotein revealed that its total carbohydrate content is 28% (w/w), and it contains fucose, galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine and sialic acid in molar proportions 1.0:4.0:3.7:3.5:1.2:2.5, suggesting that it has two types of sugar chain, i.e. sugar chains like those of serum glycoproteins and sugar chains of the type found in mucins. Actually, alkaline borohydride treatment of this glycoprotein yielded tri- and tetra-saccharide, the latter containing 1 molecule of fucose in addition to each molecule of galactose, N-acetylgalactosamine and sialic acid. This glycoprotein bound to Ricinus communis agglutinin and concanavalin A as well as to wheat-germ agglutinin.     

Schistosoma mansoni synthesizes novel biantennary Asn-linked oligosaccharides containing terminal beta-linked N-acetylgalactosamine.

This report describes the structure of novel complex-type Asn-linked oligosaccharides in glycoproteins synthesized by the human blood fluke, Schistosoma mansoni. Adult schistosome worm pairs (male and female) isolated from infected hamsters were metabolically radiolabelled with either [3H]glucosamine, [3H]mannose or [3H]galactose. The glycopeptides prepared by pronase digestion of the total glycoprotein fraction were isolated by affinity chromatography on columns of immobilized Concanavalin A (Con A) and Wisteria floribunda agglutinin (WFA). A subset of glycopeptides, designated IIb, that bound to both Con A and WFA was isolated. WFA has been shown to have affinity for oligosaccharides containing beta 1,4-linked N-acetylgalactosamine (GalNAc) at their non-reducing termini. Compositional analysis of IIb glycopeptides demonstrated that they contained N-acetylglucosamine (GlcNAc), GalNAc, mannose (Man) and fucose (Fuc), but no galactose (Gal) or N-acetylneuraminic acid (NeuAc). Methylation analyses and exoglycosidase digestions indicated that IIb glycopeptides were complex-type biantennary structures with branches containing the sequence GalNAc beta 1-4-[+/- Fuc alpha 1-3]GlcNAc beta 1-2Man alpha 1-R. The discovery of these unusual oligosaccharides synthesized by a human parasite, which appear to be similar to some newly discovered mammalian cell-derived oligosaccharides, may shed light on future studies related to the role oligosaccharides may play in host-parasite interactions.     

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.     

Composition en glucides conjugués de l'hémolymphe de Blaberus craniifer: Variations au moment de l'ecdysis imaginale

Analysis of macromolecular carbohydrates performed by gas liquid chromatography on Gregarina blaberae haemolymph showed the presence of hexoses (galactose, mannose, glucose), methyl-pentose (fucose), and hexosamines (N-acetylgalactosamine, N-acetylglucosamine). No trace of pentose or sialic acid or uronic acid was found. Mannose was the main neutral sugar. A change in the molar ratio of mannose consisting of an enrichment of female haemolymph occurred during larval-adult ecdysis. There was a parallel increase in glycoprotein staining with periodate-fuchsin after cellulose acetate electrophoresis of female haemolymph.     

Purification of normal human urinary N-acetyl-beta-hexosaminidase A by affinity chromatography.

N-Acetyl-beta-hexosaminidase A was purified 1000-fold from human urine by chromatography on DEAE-Sephadex followed by concanavalin A--Sepharose affinity chromatography. The optimal pH range was 4.4--4.5 for both the N-acetylglucosamine and N-acetylgalactosamine derivatives. The Km values were 0.51 mM and 0.28 mM respectively for the N-acetylglucosamine and N-acetylgalactosamine derivatives. The glycoprotein nature of the urinary enzyme was established by its affinity towards concanavalin A as well as by the presence of sialic acid, galactose, glucose, mannose and hexosamines in the molecule.     

Purification and partial characterization of the myelin-associated glycoprotein from adult rat brain

The myelin-associated glycoprotein was purified from rat central nervous system myelin by selective extraction with lithium diiodosalicylate-phenol followed by gel filtration on a column of Sepharose CL-6B. Amino acid analysis of the purified glycoprotein revealed an excess of acidic over basic amino acids and a relatively high content of nonpolar residues. On the basis of weight, the molecule is about one-third carbohydrate consisting of 5% fucose, 23% mannose, 20% galactose, 34% N-acetylglucosamine, and 18% N-acetylneuraminic acid.     

Age-related changes in chemical composition and physical properties of mucus glycoproteins from rat small intestine.

Mucus glycoproteins from newborn and adult rat small intestine were radiolabelled in vivo with Na2 35SO4 and isolated from mucosal homogenates by using Sepharose 4B column chromatography followed by CsCl-density-gradient centrifugation. Non-covalently bound proteins, lipids and nucleic acids were not detected in the purified glycoproteins. Amino acid, carbohydrate and sulphate compositions were similar to chemical compositions reported for other intestinal mucus glycoproteins, as were sedimentation properties. There were, however, important differences in the chemical and physical characteristics of the mucus glycoproteins from newborn and adult animals. The buoyant density in CsCl was higher for the glycoproteins from newborn rats (1.55 g/ml versus 1.47 g/ml). On sodium dodecyl sulphate/polyacrylamide/agarose-gel electrophoresis, the glycoprotein from newborn rats had a greater mobility than the adult-rat sample. Although both preparations had similar general amino acid compositions, variations were observed for individual amino acids. The total protein content was greater in the glycoprotein from newborn animals (27%, w/w, versus 18%, w/w). The molar ratio of carbohydrate to protein was less in the newborn, primarily owing to a decreased fucose and N-acetylgalactosamine content. Comparison of the molar ratio of fucose and sialic acid to galactose for both glycoproteins demonstrated a reciprocal relationship similar to that described by Dische [(1963) Ann. N.Y. Acad. Sci. 106, 259-270]. The sulphate content was greater in the glycoprotein from newborn rats (5.5%, w/w, versus 0.9%, w/w). Both had similar sedimentation coefficients in a dissociative solvent. These results suggest an age-related difference in the types of mucus glycoproteins synthesized by small intestine.     

Uridine and dolichyl diphosphate activated oligosaccharides are intermediates in the biosynthesis of the S-layer glycoprotein of Methanothermus fervidus

The surface of the extremely thermophilic archaebacterium Methanothermus fervidus is covered by glycoprotein subunits. The carbohydrate moiety of the surface glycoprtein accounts for about 17 mol%. It is composed of mannose, 3-O-methylglucose, galactose, N-acetylglucosamine and N-acetylgalactosamine. From cell extracts the corresponding surgar-1-phosphates and nucleotide activated derivatives of Man, Gal, GlcNAc and GalNAc were isolated. Furthermore UDP-and dolichyl activated oligosaccharides were obtained. On the basis of the isolated precursors a pathway for the biosynthesis of the oligosaccharide chains is proposed.Abbreviations DNP-Glu N-2,4-dinitrophenyl-glutamic acid - Dol dolichol - Gal galactose - Gal-1-P galactose-1-phosphate - GalNAc N-acetylgalactosamine - GalNAc-1-P N-acetylgalactosamine-1-phosphate - Glc glucose - GlcNAc N-acetylglucosamine - GlcNAc-1-P N-acetylglucosamine-1-phosphate - Man mannose - Man-1-P mannose-1-phosphate - 3-O-MeGlc 3-O-methylglucose - P phosphate - TCA trichloroacetic acid - TLC thin-layer chromatography - Tris tris(hydroxymethyl)aminomethan     

Phenylalanyl-tRNA synthetases from yeast cytoplasm and mitochondria. The presence of a carbohydrate moiety in the mitochondrial enzyme and immunological evidence for structural relationship

Homogeneous yeast cytoplasmic and mitochondrial phenylalanyl-tRNA synthetases (L-phenylalanine:tRNAPhe ligase (AMP-forming), EC 6.1.1.20) are analysed for structural differences. Only the large subunit of the mitochondrial enzyme is a glycoprotein with nearly 3% carbohydrate by weight. The carbohydrates present are: glucose, N-acetylglucosamine, mannose, galactose and N-acetylneuraminic acid. Removal of the sugar moieties yields an activity increase, but no significant change of sensitivity to proteolytic degradation. Antibodies to both homogeneous enzymes demonstrate a structural similarity for both types of subunit using the highly sensitive immunoblotting technique.     

Preparation of glycopeptides and oligosaccharides from thyroxine-binding globulin.

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

Isolation and partial characterization of two glycoproteins from human liver metastases of sigmoid colon carcinoma

1. Perchloric acid-soluble glycoprotein fraction (PASF) extracted from human liver metastases (LM) of sigmoid colon carcinoma was chromatographed on a DEAE-cellulose column. The main fraction (DEAE-nonadsorbed fraction) passed through the column was then subjected to Sephacryl S-200 superfine gel filtration and separated into 12 fractions. 2. Among 12 fractions, only both Fractions 3 and 4 were demonstrated to be chemically and immunologically homogeneous glycoproteins, respectively, by a combination of chemical composition analysis, SDS-PAGE and EITB assay using antisera against the DEAE-nonadsorbed fractions of PASFs from human LMs, normal liver (NL) and normal sigmoid colon (NSC). Each of Fractions 3 and 4 reacted with anti-LM serum to give one immuno complex on a nitrocellulose sheet in EITB assay, but did not react with anti-NL and -NSC sera. 3. Apparent molecular weights of 80,900 and 62,100, respectively, were found for Fractions 3 and 4. Both the fractions, respectively, had abnormal sugar compositions. Fraction 3 contained sialic acid, fucose, galactose, N-acetylglucosamine and N-acetylgalactosamine, but lacked glucose and mannose, and Fraction 4 contained sialic acid, fucose, galactose and N-acetylglucosamine, but lacked glucose, mannose and N-acetylgalactosamine, as sugar components.     

Cytochemical characterization of glycoproteins in the developing acrosome of rats. An ultrastructural study using lectin histochemistry, enzymes and chemical deglycosylation.

The composition and distribution of rat acrosomal glycoproteins during spermiogenesis have been investigated at light and electron microscopic level by means of a variety of morphological techniques including the application of lectins conjugated to peroxidase, digoxigenin and colloidal gold, enzyme and chemical deglycosylation procedures and conventional histochemistry. Results obtained with lectin histochemistry in combination with beta-elimination reaction and endoglucosaminidase F/peptide N-glycosidase F digestion suggest that glycoproteins of mature acrosomes contain both N- and O-linked oligosaccharides. N-linked chains of acrosomal glycoproteins contain mannose and external residues of N-acetylglucosamine and galactose. They also have fucose residues linked to the core region of the oligosaccharide side chains. O-linked oligosaccharide chains contain external residues of both galactose and N-acetylgalactosamine. Mannose, fucose, galactose and N-acetylglucosamine residues were detected in acrosomes at all steps of spermiogenesis. N-acetylgalactosamine residues were only observed in the late steps of the spermiogenesis. N-acetylneuraminic acid residues were not detected throughout the acrosomal development. At initial stages of acrosome formation, glycoproteins were preferentially distributed over the acrosomic granules. In cap phase spermatids, lectin binding sites were homogeneously distributed throughout the acrosomes; however, in mature spermatozoa, glycoproteins were predominantly located over the outer acrosomal membrane.     

Purification method for α-1-acid glycoprotein with subsequent high-performance liquid chromatographic determination of monosaccharides in plasma of healthy subjects and patients with renal insufficiency

A simple purification method for human plasma α-1-acid glycoprotein (AAG) using an ion-exchange and hydroxyapatite column was developed. The recovery of the method was found to be high. We also improved a determination method for N-acetylneuraminic acid and monosaccharides in the carbohydrate moiety of AAG by using an ion-exchange column and pulse-amperometric detection. By this method, a composition analysis of the carbohydrate moiety of AAG (N-acetylneuraminic acid, fucose, N-acetyl glucosamine, galactose and mannose) was possible with 1.0 ml of plasma. We compared these carbohydrate concentrations in the AAG of patients with renal insufficiency with those of healthy subjects. In the AAG of the patients, the concentrations of N-acetylglucosamine, galactose and mannose were significantly higher than those in the AAG of the healthy subjects.     

A novel extracellular membrane elaborated by a mouse embryonal carcinoma-derived cell line

An extracellular membranous structure is synthesized by an embryonal carcinoma-derived cell line, M1536-B3, in suspension cultures. Analysis of the solubilized membranous structure on polyacrylamide gels in sodium dodecyl sulfate yielded two major classes of glycoproteins with molecular weights of approximately 230,000 and 320,000 respectively. The amino acid composition of the purified membranous structures revealed the absence of both hydroxyproline and hydroxylysine. Carbohydrate analysis demonstrated the presence of fucose, xylose, mannose, galactose, glucose, N-acetylglucosamine, N-acetylgalactosamine, and N-acetylneuraminic acid. These carbohydrates represented approximately 9% of the weight of the membrane. A comparison of the electrophoretic patterns of cells grown on monolayers and in suspension revealed a marked accumulation of the glycoproteins under the latter growth conditions. $\text{D}$-[³H]-glucosamine was incorporated into these two and a third major glycoprotein by cells in suspension culture.     

The macromolecular properties of blood-group-specific glycoproteins. Characterization of a series of fractions obtained by density-gradient ultracentrifugation

1. Equilibrium density-gradient ultracentrifugation in caesium salts was used in two stages in the isolation and subfractionation of the glycoprotein component from a human ovarian-cyst fluid. The eight main subfractions thus obtained were the subject of detailed physicochemical characterization. 2. The fractions were unimodal in buoyant-density distribution, but had discrete rho(0) values ranging from 1.31 to 1.35. 3. Weight-average molecular weights and sedimentation coefficients decreased regularly with decreasing density of the fraction, whereas the partial specific volumes and selective solvation parameters increased. The latter behaviour correlates well with the increasing peptide content of the lighter fractions. 4. The fractions exhibited a range of analytical composition, although all were within the limits previously observed for blood-group substances of Le(a) specificity. All fractions had approximately equal Le(a) activity. The peptide content varied systematically from 7% for the densest fraction to 15% for the lightest, but the relative distributions of the amino acids remained essentially constant throughout the series. In particular, serine plus threonine plus proline made up about 50% of the peptide content of all the fractions. Fucose, galactose and N-acetylglucosamine contents decreased with increasing peptide content of the fractions, but N-acetylgalactosamine and sialic acid exhibited the opposite trend. Molar ratios of N-acetylgalactosamine to the sum of serine and threonine remained essentially constant at 0.8-0.9, implying a high degree of glycosylation of all the molecules, but the ratio of N-acetylglucosamine to N-acetylgalactosamine decreased steadily with increasing peptide content, suggesting the presence of oligosaccharide side chains of various lengths. The results are discussed in terms of the accepted structure of glycoprotein molecules. 5. Experiments on the glycoproteins extracted with phenol from the same cyst fluid have confirmed that equilibrium centrifugation in caesium salts does not remove any non-covalently bound protein nor cause any changes in the tertiary structures of these glycoprotein molecules.     

The structure and composition of cartilage keratan sulphate

Keratan sulphate was isolated from bovine intervertebral disc and bovine nasal septum after hydrolysis with proteinases and treatment with dilute alkali. Each preparation was found to contain, per keratan sulphate chain: (a) 1 residue of mannose; (b) 3 residues of N-acetylneuraminic acid (2 residues after alkali treatment); (c) 1 residue of N-acetylgalactosamine (lost after alkali treatment); (d) 1 residue or less of fucose. N-Acetyl-neuraminic acid residues were at non-reducing termini and were bonded to keratan sulphate through galactose residues. Evidence is presented for two different types of linkage between skeletal keratan sulphate and protein. Consideration of molecular parameters and compositions leads to a proposed structure for keratan sulphate-protein as found in skeletal proteoglycans.     

Deglycosylation studies on tracheal mucin glycoproteins

Following several model experiments, conditions were developed for optimal deglycosylation of tracheal mucin glycoproteins. Exposure of rigorously dried material to trifluoromethanesulfonic acid at 0 degree C for up to 8 h results in cleavage of essentially all fucose, galactose, and N-acetylglucosamine, about 80% of the N-acetylneuraminic acid (NeuNAc), and a variable amount of N-acetylgalactosamine (GalNAc), the sugar involved in linkage to protein. Residual N-acetylneuraminic acid is sialidase susceptible and apparently in disaccharide units, presumably NeuNAc2----GalNAc. The remaining N-acetylgalactosamine is mostly present as monosaccharides, and a few Gal beta 1----3GalNAc alpha units are also present; both are cleaved by appropriate enzymatic treatment. The saccharide-free proteins obtained from either human or canine mucin glycoproteins have molecular weights of about 100,000 and require chaotropic agents or detergents for effective solubilization.     

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.