Fetuin Glycopeptides Research Articles

Interaction of human erythrocyte band 3 with Ricinus communis agglutinin and other lectins

Agglutination and competition studies suggest that human erythrocyte Band 3 can interact with both mannose/glucose- and galactose-specific lectins. Purified Band 3 reconstituted into lipid vesicles binds concanavalin A, but the nonspecific binding component, measured in the presence of α-methylmannoside, is very high. This glycoprotein also carries binding sites for the galactose-specific lectin Ricinus communis agglutinin. Binding was inhibited poorly by lactose, but much more effectively by desialylated fetuin glycopeptides, suggesting that the lectin recognizes a complex oligosaccharide sequence on Band 3. The glycoprotein hears two separate classes of binding sites for R. communis agglutinin. High-affinity binding sites exist which show strong positive cooperativity and correspond in number to the outward-facing Band 3 molecules. A low-affinity binding mode is abolished by 40% ethyleneglycol, suggesting the involvement of hydrophobic lectin-glycoprotein interactions. Studies on binding of R. communis agglutin to human erythrocytes indicate positively cooperative binding to 7·10 5 very-high-affinity sites per cell, and lectin binding is completely inhibitable by lactose. Based on its binding characteristics in vesicles, it seems likely that Band 3 forms the major receptor for this lectin in human erythrocytes. Properties such as positive cooperativity thus appear to be a common feature of the interaction of Band 3 with a variety of lectins of different specificity, both in erythrocytes and lipid bilayers.

Glycans of synaptosomal plasma membrane glycoproteins from adult rat forebrain

Glycopeptides obtained by exhaustive proteolytic digestion of synaptosomal plasma membranes from adult rat forebraini were separated by affinity chromatography on concanavalin A-Sepharoe. Concanavalin A-binding glycopeptides are essentially made up of mannose and N-acetylglucosamine in a molar ration of 3.45:1, whereas glycopeptides not bound to concanavalin A have a complex monosaccharide composition. By gel filtration on Bio-Gel P-30, concanavalin A-binding glycopeptides appear as low-molecular-weight glycopeptides (migrating like ovalbumin glycopeptides), whereas glycopeptides not bound to concanavalin A behave as high-molecular-weight glycopeptides (migrating like fetuin glycopeptides). Comparison of concanavalin A-binding glycopeptides from rat brain synaptosomal plasma membranes with concanavalin A-binding glycoproteins isolated from the same membrane fraction shows clear differences in monosccharide composition. We demonstrate here that this discrepancy is due to the presence on most concanavalin A-binding glycoprotein subunits of at least two different types of glycan: in addition to the concanavalin A-binding glycans, these glycoprotein subunits carry other glycans which do not interact with concanavalin A. Biological implications of the presence of two (or more) types of glycan on the same polypeptide are discussed.

The B4 lectin from Vicia villosa seeds interacts with N-acetylgalactosamine residues alpha-linked to serine or threonine residues in cell surface glycoproteins.

We have examined the carbohydrate binding specificity of the B4 lectin from Vicia villosa seeds. The B4 lectin agglutinates Tn-exposed erythrocytes specifically and binds to these erythrocytes (1.4 X 10(6) sites/cell) with an association constant of 4.2 X 10(7) M-1. The concentrations of saccharides and glycopeptides of defined structure which cause 50% inhibition of B4 lectin binding to Tn-exposed erythrocytes were determined. N-Acetylgalactosamine is the best monosaccharide inhibitor, causing 50% inhibition of binding at a concentration of 0.04 mM. Other monosaccharides inhibit lectin binding in the following order of decreasing potency: N-acetylgalactosamine greater than methyl-alpha-galactopyranoside greater than p-nitrophenyl-alpha- or beta-galactopyranoside greater than methyl-beta-galactopyranoside, galactose greater than galactosamine greater than mannose, N-acetylglucosamine. The disaccharide Gal beta 1,3GalNAc causes 50% inhibition of binding at a concentration of 2.8 mM, a concentration similar to that of the p-nitrophenyl-alpha- or beta-galactopyranosides. Glycopeptides containing O-glycosidically linked oligosaccharide units are significantly more potent inhibitors of lectin binding than the oligosaccharide units alone. The most potent glycopeptide inhibitor is a fetuin glycopeptide containing two alpha-linked N-acetylgalactosamine units. This glycopeptide causes 50% inhibition of lectin binding at a concentration of 0.00034 mM and probably closely resembles the B4 lectin binding site on Tn-exposed erythrocytes.

Purification and Characterization of a β-N-Acetylhexosaminidase of Sea-Squirt

A beta-N-acetylhexosaminidase [EC 3.2.1.30] was isolated from internal organs of the sea-squirt, Styela plicata. The enzyme was purified 1,560-fold in 5% yield. The preparation was fairly homogeneous as examined by disc and SDS polyacrylamide gel electrophoresis and Sephadex G-200 chromatography. The molecular weight of the enzyme was estimated to be 132,000 by gel chromatography and 66,000 by SDS polyacrylamide gel electrophoresis. Therefore, this beta-N-acetylhexosaminidase was considered to be a dimer. The optimum pH for activity was 4.0 but the enzyme was stable in the pH range from 5 to 6. The isoelectric point was 4.99. This enzyme was inhibited by Fe2+, Hg2+, Ag+, and PCMB but not by acetate. The isolated enzyme hydrolyzed both p-nitrophenyl-N-acetyl-beta-D-glucosaminide and p-nitrophenyl-N-acetyl-beta-D-galactosaminide. The hydrolysis rate of p-nitrophenyl-N-acetyl-beta-D-galactosaminide was 43% of that of p-nitrophenyl-N-acetyl-beta-D-glucosaminide. The enzyme liberated N-acetylhexosamine from asialodegalactosyl ovomucoid glycopeptide, asialodegalactosyl fetuin glycopeptide and the fragment of hyaluronic acid prepared by hyaluronidase treatment.

Hydrogen-bonded structure of the complex N-linked fetuin glycopeptide.

The conformation of the N-linked complex glycopeptide of fetuin was examined with hydrogen-exchange techniques. The glycopeptide molecule contains eight acetamido hydrogens stemming from five N-acetylglucosamine residues and three N-acetylneuraminic acid residues and also one from the remaining sugar-peptide linkage. The hydrogen-exchange rates of these secondary amides were compared with small molecule model compounds having identical primary structures at their exchangeable hydrogen sites. Differences between the model rates and glycopeptide rates therefore cannot be accounted for by primary structure effects but reflect conformational features of the glycopeptide. Two glycopeptide hydrogens exhibit significantly hindered exchange; the rest exchange at the model rates. Removal of the three N-acetylneuraminic acid residues from terminal positions on the three branches of the glycopeptide removes the slowed hydrogens. The remaining ones continue to exchange at the model rate. These results indicate that two of the eight sugar acetamido hydrogens are involved in intramolecular hydrogen bonds. A likely structure includes two hydrogen bonds between the three N-acetylneuraminic acid residues. These two hydrogens, slowed to a moderate degree, reflect a preferred conformation stabilized by about 1 kcal/mol in free energy. The solution conformation of the glycopeptide suggested by these results is one that is partially ordered and can be easily modulated, owing to the relatively small amount of energy stabilizing the preferred conformation.

Structural analysis of glycopeptides by polyacrylamide gel electrophoresis

This work describes the use of polyacrylamide gel electrophoresis to resolve fluorescein-derivatized asialoglycopeptides. Specific exoglycosidase digestion of pronase glycopeptides of fetuin generates a series of degradation products that are resolved by polyacrylamide gel electrophoresis and are visualized by photography. The logarithm of the relative electrophoretic mobility of each degraded glycopeptide is linearly correlated to the number of monosaccharide residues sequentially removed by exoglycosidases. Such an approach allows the quantitative evaluation of the number of saccharide moieties removed by each enzyme at a detection sensitivity of less than 20 pmol of each compound. Use of specific endoglycosidases results in the direct sizing of oligosaccharide chains.

Use of the smith degradation in the study of the branching pattern in the complex-type carbohydrate units of glycoproteins

The site of substitution of the peripheral branches on the core mannose-residues in the complex-type carbohydrate units of glycoproteins has been studied by Smith degradation. By using glycopeptides of known structure, it is shown that the site of attachment of the (1→4)-linked, third branch to the mannose core in tri- and tetra-antennary structures may be conveniently demonstrated by this technique. A critical factor was found to be the concentration of acid used for cleavage of the periodate-oxidized product. This finding could explain discrepancies concerning the previously published structures of fetuin glycopeptides. The technique was applied for study of the branching pattern in human transferrin, porcine thyroglobulin, and rat plasma and brain glycoproteins. The results support the concept that, in glycoproteins from most sources, the third (1→4)-linked branch is attached to the (1→3)-linked mannose residue of the core portion in the complex-type structures. An indication for a novel type of branching pattern in human transferrin was, however, also obtained.

Conjugation of a fetuin glycopeptide to human follicle-stimulating hormone and its subunits by photoactivation

Human follicle-stimulating hormone (human FSH), dansylated human FSH, human FSH-α and human FSH-β subunits were individually conjugated by photoactivation to an azidobenzoyl derivative of a glycopeptide isolated from fetuin. The conjugates were purified on a column of Sephadex G-100. The carbohydrate content of the conjugated human FSH increased 2.7-fold with a concomitant 1.5- and 2-fold increase in the immunological and biological activity of the human FSH. The glucosamine content of the human FSH-α and human FSH-β increased 6- and 1.4-fold, respectively, after conjugation. Human FSH-α conjugate, when recombined with untreated human FSH-β showed upto 50% increase in the biological activity over the control. When the conjugated human FSH-β was combined with untreated human FSH-α, there was little change in the biological activity. These experiments demonstrate that the photoactivation procedure, although random and site-nonspecific in nature, provides a potential means of attachment of glycopeptides to the protein moiety and enhancement of the hormonal activity of human FSH.

Sialyl- and galactosyltransferases in the human chorionic gonadotropin-treated immature rat ovary.

General properties of glycoprotein sialyl- and galactosyltransferases in control and hCG-primed immature rat ovary were studied by using asialo and agalacto derivatives of fetuin, alpha 1-acid glycoprotein, and purified O-glycosidically linked fetuin glycopeptide as exogenous substrates. Incorporation of the labeled sugars into the derivatives was determined by electrophoresis and precipitation with phosphotugnstic acid and with a rabbit anti-fetuin gamma-globulin fraction. Both enzyme activities were associated with partiulcate fractions, were enhanced by 0.3% Triton X-100, had pH optima between 6.3-6.6, and exhibited apparent Km values of 0.04-0.08 mM for the sugar nucleotides and 0.3-0.6 mM for the asialo- and asialoagalactofetuin derivatives. The galactosyltransferase required exogenous Mn+2 for activity. The specific activities of galactosyltransferase were similar in the control and hCG-primed ovaries, while the specific activity of sialytransferase in the hCG-primed ovaries was consistently twice that in the control.

Structural features of the carbohydrate units of plasma glycoproteins.

For the characterization of the type and relative abundance of different carbohydrate structures in plasma glycoproteins, rat whole plasma was subjected to exhaustive proteolytic digestion and the resulting glycopeptides were fractionated into structurally district groups by chromatography on concanavalin‐A‐Sepharose followed by mild NaOH/NaBH4 treatment and gel filtration. The most abundant fraction was found to be the biantennary transferrin‐type glycopeptides (71% of recovered carbohydrate) followed by triantennary fetuin‐type structures and a smaller proportion of more branched structures (together 24%), neutral mannose‐rich chains (2%) and O‐glycosidic chains (3%). Structural similarity of the two major fractions to the transferrin and fetuin glycopeptides was supported by similar elution volumes in gel filtration, similar behaviour in concanavalin A affinity chromatography and similar sugar composition and substitution pattern of sugars in methylation analysis.The glycopeptide pattern differs significantly from that observed previously for brain, kidney, liver or erythrocytes, where the more branched structures predominate, and which also contain higher amounts of the neutral mannose‐rich chains. As indicated by methylation analysis, the plasma glycoproteins are characterized by carbohydrate units terminating in N‐acetylneuraminic acid. The level of terminal galactose, N‐acetylglucosamine and fucose residues was low. This finding is in correlation with the described occurrence of binding‐activities in liver and other tissues which remove glycoproteins having these sugar terminals from the circulation.

Rosette formation between human lymphocytes and sheep erythrocytes. Inhibition of rosette formation by specific glycopeptides.

Rosette formation with unsensitized sheep erythrocytes is a characteristic of human thymus dependent lymphocytes. Release of glycopeptides from the sheep erythrocyte by trypsin reduces rosette formation. These tryptic glycopeptides inhibit rosette formation by untrypsinized sheep erythrocytes; this suggests that rosetting is mediated by erythrocyte surface glycopeptides. To investigate the molecular nature of this interaction, we examined the abilities of various model compounds to act as haptenic inhibitors of rosette formation. Inhibition is given by glycopeptides bearing oligosaccharide units rich in sialic acid, galactose, N-acetylglucosamine, and mannose linked to asparagine residues through glycosylamine bonds. Among compounds tested, fetuin glycopeptide is most effective, but human transferrin glycopeptide and human erythrocyte glycopeptide I also inhibit rosette formation. Other compounds including human erythrocyte glycopeptide II, human IgG glycopeptide, lacto-N-neotetraose, 3'- and 6'-sialyllactose show no significant inhibition. Neither sialic acid, galactose, manose, nor N-acetyl-glucosamine alone inhibits rosette formation. Stepwise degradation of fetuin glycopeptide established the galactose residues as important determinants of inhibitory activity. Fetuin glycopeptide blocks rosette formation when added to a suspension of human lymphocytes and sheep erythrocytes or when preincubated with human lymphocytes, but not when preincubated with sheep erythrocytes. Studies of the binding of [3H] fetuin glycopeptide to normal lymphocytes demonstrate 7.5 x 10(6) saturable binding sites per cell. No saturable binding of this compound to sheep erythrocyte membranes is observed. Compared to normals, lymphocytes from patients with chronic lymphatic leukemia demonstrate decreased fetuin glycopeptide binding with a mean of 0.9 x 10(6) sites per cell. This decreased binding correlates with the impaired ability of these cells to form rosettes. The data suggest that fetuin glycopeptide inhibits rosette formation by binding to the thymus-dependent cell where competition occurs with sheep erythrocytes for specific lymphocyte surface receptors.

Glycosidases of Aspergillus niger: IV. PURIFICATION AND CHARACTERIZATION OF α-MANNOSIDASE

Abstract A 100-fold purified preparation of Aspergillus niger α-mannosidase free of α- and β-galactosidases, β-N-acetylglucosaminidase, and 1,2-α-mannosidase has been obtained. Unlike many other mannosidases it is quite stable and hydrolyzes specific intersugar linkages involving nonreducing terminal mannose residues in large as well as small molecules. The enzyme hydrolyzes specifically 1,4-α- and 1,6-αmannosidic bonds to the adjoining mannose or N-acetylglucosamine residues. Therefore, 4-O-α- and 6-O-α-mannobioses and 2-acetamido-2-deoxy-4-O-α- and 6-O-α-glucoses are readily hydrolyzed. The β-galactosidase and β-N-acetylglucosaminidase-treated desialyzed glycoproteins or glycopeptides of α1 acid glycoprotein, fetuin, and human chorionic gonadotropin liberate mannose when digested with the enzyme. The enzyme, unlike other mannosidases, hydrolyzes p-nitrophenyl-α-d-mannopyranoside very slowly. It is inhibited strongly by d-mannono-(1→5)-lactone but does not appear to be Zn++-dependent or inactivated by various chelating agents. The pH optimum for the enzyme is 4.2. It has Km and Vmax values of 1.2 x 10-3 and 8.2 x 10-3 m, and 12.3 and 41.3 µmoles per mg per hour for p-nitrophenylα-d-mannopyranoside and methyl 4-O-α-mannopyranosyl-α-d-mannopyranoside as substrates, respectively.

Hepatic uptake of proteins coupled to fetuin glycopeptide

The glycopeptides of fetuin and γ G immunoglobulin have been covalently linked to lysozyme and albumin with the coupling reagent toluene 2,4 diisocyanate. When the fetuin glycopeptide-protein compounds were treated with neuraminidase to remove sialic acid, and then injected into rats, the coupled compounds were rapidly and selectively removed from the circulation by the liver, most likely by the hepatocytes. Removal of the exposed galactose residues of the asialofetuin-proteins with β-galactosidase prevented this clearance. Neither the native proteins nor the γ G glycopeptide-proteins are taken up by the liver, demonstrating the specificity of the phenomenon. Once the asialofetuin-protein complexes enter the hepatocytes, they are degraded within two hours.

Degradation of polysaccharides, mucopolysaccharides, and glycoproteins by lysosomal glycosidases

Isolated rat kidney lysosomes are shown to degrade glycogen, starch, hyaluronic acid, ovomucoid, and fetuin. Studies on the release of N-acetylglucosamine from ovomucoid by lysosomes show that proteolytic activity aids in the degradation of the carbohydrate chain by glycosidases. The results indicate that a large portion of the N-acetylhexosamine residues in ovomucoid are terminally located. The sequential action of sialidase, β-galactosidase, and β- N-acetylglucosaminidase releases monosaccharides from fetuin, fetuin glycopeptides, and modified fetuin. The results of these studies, along with those previously published, show that the lysosomes are the sites of intracellular catabolism of glycoproteins.