Ovalbumin Glycopeptides Research Articles

Mucor hiemalis endo-β- N-acetylglucosaminidase can transglycosylate a bisecting hybrid-type oligosaccharide from an ovalbumin glycopeptide

We found that the recombinant endo-β- N-acetylglucosaminidase of Mucor hiemalis (Endo-M) expressed in Candida boidinii had the transglycosylation activity of transferring a bisecting hybrid-type oligosaccharide from an ovalbumin glycopeptide to the acceptor ( p-nitrophenyl 2-acetamido-2-deoxy-β- d-glucopyranoside) in a good yield of 43%.

Carbohydrate-based interactions of oviductal sperm reservoir formation-studies in the pig.

Competitive inhibition of sperm to explants of the oviductal epithelium was used to study the complementary receptor system that may be involved in the establishment of the oviductal sperm reservoir in the pig. Sperm binding to the oviductal explants is expressed as Binding Index (BI = sperm cells/0.01 mm(2)). From a set of glycoproteins with known oligosaccharide structures, only asialofetuin and ovalbumin showed inhibitory activity, indicating that ovalbumin may block high affinity binding sites (IC(50) congruent with 1.3 microM) and asialofetuin low affinity sites (IC(50) congruent with 18 microM) of the complementary receptor systems, whereas fetuin carrying terminal sialic acid has no effect. Ovalbumin glycopeptides were isolated by Con A affinity chromatography and reverse-phase HPLC following tryptic digestion. Glycopeptides and enzymatically released glycans were analyzed by MS, and were shown to represent preferentially the two high mannose type glycans (Man)(5)(GlcNAc)(2) and (Man)(6)(GlcNAc)(2), and as a minor component the hybrid type glycan (Hex)(4)(GlcNAc)(5). Glycopeptides (84% inhibition) and glycans (81% inhibition) significantly reduced sperm-oviduct binding at a concentration of 3 microM, whereas the deglycosylated peptides showed no inhibitory activity. Mannopentaose (IC(50) congruent with 0.8 microM) representing the oligomannose residue of the high mannose glycans of ovalbumin was as effective as ovalbumin. These data indicate that the carbohydrate-based mechanisms underlying the formation of the oviductal sperm reservoir in the pig is the result of the concerted action of at least the high-affinity binding sites for oligomannose or nonreducing terminal mannose residues and low-affinity binding of galactose.

1H NMR Characterization of a Hen Ovalbumin Tyrosinamide N-Linked Oligosaccharide Library

A library of 15 N-linked oligosaccharide structures was prepared from ovalbumin and characterized using high-held NMR and mass spectrometry. The oligosaccharides were enzymatically released from ovalbumin glycopeptides, and the reducing ends were reacted with ammonium bicarbonate to form oligosaccharide-glycosylamines. These reacted with Boc-tyrosine- N-hydroxysuccinimide ester, resulting in a mixture of tyrosinamide-oligosaccharides. The Boc group was removed to expose an amine terminus which enhanced the resolution of tyrosinamide-oligosaccharides when chromatographed on reverse-phase HPLC. Ten major and five minor oligosaccharides were purified on a micromole scale and characterized using 1H NMR and FAB-MS. The structures include high-mannose, hybrid, and complex oligosaccharides possessing from two to five antenna, providing the most complete definition of ovalbumin N-linked oligosaccharides to date. The resulting library is well suited to biological studies due to the presence of a single terminal tyrosine residue on each oligosaccharide that allows radioiodination or the attachment of additional probes to these glycoconjugates prior to biological studies.

Synthesis of Neoglycoproteins Using Oligosaccharide-transfer Activity with Endo-β-N-Acetylglucosaminidase

We describe a novel method for the enzymatic synthesis of neoglycoproteins. Endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) had high levels of transglycosylation activity. The enzyme activity of Endo-A was markedly increased by adding 4-L-aspartyl-glycosylamine (GlcNAc-Asn) to the reaction mixture. Digesting (Man)6(GlcNAc)2 with the enzyme in the presence of GlcNAc-Asn gave a mixture of hydrolytic ((Man)6GlcNAc) and transglycosylic ((Man)6(GlcNAc)2-Asn) products. By means of transglycosylation, (Man)6GlcNAc was transferred en bloc to the partially deglycosylated ovalbumin glycopeptide (EEKYN(GlcNAc)LTSVL) concomitant with the hydrolysis of (Man)6-GlcNAc)2Asn. The structure of the transglycosylation product was designated as (Man)6(GlcNAc)2-peptide by amino acid composition and sequence analysis as well as ion mass spectrometry. The enzyme also transferred oligosaccharide to partially deglycosylated ribonuclease B (GlcNAc-protein) during the hydrolysis of (Man)6-(GlcNAc)2Asn. Native ribonuclease B had (Man)5-9 (GlcNAc)2 as its heterogeneous N-linked sugar chains. High performance liquid chromatography showed that all of the N-linked sugar chains of the synthetic neoribonuclease of the pyridylamino derivatives were modified to (Man)6(GlcNAc)2.

Purification and properties of Acinetobacter sp. endo- β- N-acetylglucosaminidase acting on complex oligosaccharides of glycoproteins

A novel endo- β- N-acetylglucosaminidase capable of acting on complex type sugar chains of glycoproteins was found in the culture broth of a bacterium which was isolated from soil and identified as Acinetobacter sp. The enzyme was purified to homogeneity on polyacrylamide gel electrophoresis by successive purification procedures involving ammonium sulfate fractionation and chromatographies on DEAE-cellulose, hydroxylapatite and Sephadex G-150. Its molecular weight was about 35,000 on gel filtration. The optimum pH was 3.0–3.5, and the enzyme was stable in the pH range from 6–8. The enzyme had high activity on dansyl ovalbumin glycopeptide, and also could hydrolyze dansyl asialotransferrin glycopeptide and dansyl transferrin glycopeptide containing complex type sugar chains. The K m value for dansyl asialotransferrin glycopeptide as the substrate of enzyme assay was 0.68 mM. The enzyme could release complex type sugar chains from intact asialotransferrin without the addition of any detergent.

Purification and characterization of .ALPHA.-mannosidase from kiwifruit.

α-D-Mannosidase of kiwifruit was purified from flesh by ammonium sulfate fractionation and column chromatography on DEAE-cellulose, hydroxylapatite, Sepharose CL-6B and Con A-Sepharose 4B. The purified enzyme was almost homogeneous on disc gel electr phoresis. Its molecular weight was estimated to be 86000 by gel filtration and 83000-85000 by SDS-polyacrylamide gel electrophoresis. The pH optimum was found to be 5.0-5.5, The enzyme was stable in the pH range of 6.5-7.5, and up to 55°C during 10 min incubation. It showed Km value of 6.7mM for p-nitrophenyl α-D-mannoside. Its activity was stimulated by Zn2+. It catalyzed the hydrolysis of ovalbumin glycopeptide and yeast mannan.

Purification and characterization of a novel fungal endo-.BETA.-N-acetylglucosaminidase acting on complex oligosaccharides of glycoproteins.

A novel endo-beta-N-acetylglucosaminidase acting on complex type sugar chains of glycoproteins was found in the culture broth of a fungus isolated from soil and identified as Mucor hiemalis f. hiemalis on the basis of various characteristics. The endo-beta-N-acetylglucosaminidase, named Endo-M, was purified to almost homogeneity by polyacrylamide gel electrophoresis involving ammonium sulfate fractionation, and column chromatographies on DEAE-Sepharose CL-6B, Sephadex G-200, hydroxylapatite, TSK-gel HW-65F and Con A-Sepharose 4B. The molecular weight of the enzyme was estimated to be about 95,000 by gel chromatography. The optimum pH was found to be 6.0-6.5 and the enzyme was stable in the pH range of 7 to 8. The enzyme showed high activity on dansyl ovalbumin glycopeptide, and also could act on dansyl transferrin glycopeptide, and dansyl asialotransferrin glycopeptide containing biantennary complex type sugar chains. The Km value for dansyl asialotransferrin glycopeptide as the substrate was 2.0 x 10(-3) M. The enzyme released complex type sugar chains from intact asialotransferrin without the addition of any detergent and the liberated sugar chains were identified by chromatography on a Bio-Gel P-4 column, calibrated with markers of known structure, and 1H-NMR analysis.

Purification and Characterization of a Novel Fungal Endo-β-N-acetylglucosaminidase Acting on Complex Oligosaccharides of Glycoproteins

A novel endo-β-N-acetylglucosaminidase acting on complex type sugar chains of glycoproteins was found in the culture broth of a fungus isolated from soil and identified as Mucor hiemalis f. hiemalis on the basis of various characteristics. The endo-β-N-acetylglucosaminidase, named Endo-M, was purified to almost homogeneity by polyacrylamide gel electrophoresis involving ammonium sulfate fractionation, and column Chromatographies on DEAE-Sepharose CL-6B, Sephadex G-200, hydroxylapatite, TSK-gel HW-65F and Con A-Sepharose 4B. The molecular weight of the enzyme was estimated to be about 95,000 by gel chromatography. The optimum pH was found to be 6.0–6.5 and the enzyme was stable in the pH range of 7 to 8. The enzyme showed high activity on dansyl ovalbumin glycopeptide, and also could act on dansyl transferrin glycopeptide and dansyl asialotransferrin glycopeptide containing biantennary complex type sugar chains. The Km value for dansyl asialotransferrin glycopeptide as the substrate was 2.0 × 10−3 m. The enzyme released complex type sugar chains from intact asialotransferrin without the addition of any detergent and the liberated sugar chains were identified by chromatography on a Bio-Gel P-4 column, calibrated with markers of known structure, and 1H-NMR analysis.

Induction and Purification of Endo-β- N -Acetylglucosaminidase from Arthrobacter protophormiae Grown in Ovalbumin

Arthrobacter protophormiae produced a high level of extracellular endo-beta-N-acetylglucosaminidase when cells were grown in a medium containing ovalbumin. The enzyme was induced by the glycopeptide fraction of ovalbumin prepared by pronase digestion. Production of the enzyme was also induced by glycoproteins such as yeast invertase and bovine ribonuclease B but not by monosaccharides such as mannose, N-acetylglucosamine, and galactose. The enzyme was purified to homogeneity as demonstrated by polyacrylamide gel electrophoresis and has an apparent molecular weight of about 80,000. The enzyme showed a broad optimum pH in the range of pH 5.0 to 11.0. The enzyme hydrolyzed all heterogeneous ovalbumin glycopeptides, although the hydrolysis rates for hybrid type glycopeptides were very low. The substrate specificity of A. protophormiae endo-beta-N-acetylglucosaminidase was very similar to that of Endo-C(II) from Clostridium perfringens. Therefore, the enzyme induction by A. protophormiae seems to have a close relation to the substrate specificity of the enzyme.

Purification and characterization of an α-1,2-mannosidase involved in processing asparagine-linked oligosaccharides

A calcium-dependent alpha-1,2-mannosidase involved in the processing of asparagine-linked oligosaccharides was purified to homogeneity from rabbit liver microsomes. N-terminal amino acid analysis was consistent with the presence of a homogeneous protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under both reducing and nonreducing conditions, revealed a single protein band with an apparent molecular weight of 52,000. Gel filtration and sedimentation analysis under nondenaturing conditions suggested that the purified enzyme is a monomeric protein. The mannosidase is a glycoprotein based on the presence of protein-linked sugar and specific binding of the enzyme to concanavalin A-Sepharose. Purified mannosidase was optimally active between pH 5.0 and 6.0. The enzyme was inactive with p-nitrophenyl-alpha-D-mannopyranoside and was inhibited by deoxymannojirimycin but not by swainsonine. The enzyme was specifically activated by Ca2+, with half-maximal activation occurring at concentrations of 10 microM or less and was inhibited by Mn2+, Co2+, Ba2+, and Zn2+. Calcium ions protected the enzyme against inactivation by p-chloromercuribenzoate. Rabbit liver mannosidase hydrolyzed alpha-1,2-mannosyl-mannose linkages in a variety of substrates including methyl-2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside (Schutzbach, J. S. (1987) Anal. Biochem. 167, 279-283), ovalbumin glycopeptide IV, and the high mannose chains of thyroglobulin and phytohemagglutinin-P. Approximately 70% of the alpha-1,2-linked mannosyl units in the oligosaccharides of thyroglobulin were accessible to rabbit liver alpha-mannosidase, whereas most of the alpha-1,2-mannosyl units in phytohemagglutinin were resistant to digestion prior to heat denaturation of the plant lectin.

Specificity towards oligomannoside and hybrid type glycans of the endo-?-N-acetylglucosaminidase B from the basidiomy ceteSporotrichum dimorphosporum

We have previously shown that an endo-beta-N-acetylglucosaminidase (EC 3.2.1.96) named "Endo B", isolated from culture filtrates of the basidiomycete Sporotrichum dimorphosporum cleaves asialo-, and to some extent, monosialylated bi-antennary glycans of the N-acetyllactosamine type linked to the asparagine residue of peptide or protein moieties [Bouquelet S, Strecker G, Montreuil J, Spik G (1980) Biochimie 62:43-49]. In the present paper, the substrate specificity of the enzyme towards oligomannoside and hybrid type glycans has been analyzed. The results obtained indicate that ovalbumin glycopeptides containing four to seven mannose residues and bovine lactotransferrin glycopeptides containing four to nine mannose residues were completely hydrolyzed by the enzyme. The degree of cleavage was variable among hybrid type structures, since glycopeptides containing the following glycans: (Gal)1(GlcNAc)3(Man)5(GlcNAc)2; (GlcNAc)3(Man)5(GlcNAc)2;(GlcNAc)3(Man)4(GlcNAc)2 were not hydrolyzed by the enzyme while the percentage of hydrolysis of a glycopeptide containing (GlcNAc)2(Man)5(GlcNAc)2 glycan reached 90%. The bovine lactotransferrin was partially deglycosylated (40%) in the absence of non-ionic detergent while native ovalbumin glycoprotein was not hydrolyzed by the enzyme. The oligomannoside- and the N-acetyllactosamine-type degrading activities present in the culture filtrates were not separated at any step of the purification procedure. Both activities were eluted as a single component with an apparent molecular mass of 89 kDa suggesting that they are located on the same enzyme molecule. Endo B represents a powerful tool for removing oligomannoside- and N-acetyllactosamine-type glycans from N-glycopeptides and N-glycoproteins. Moreover, advantages in the use of Endo B in a soluble form as well as in an immobilized form result in its high activity and in its stability to heat denaturation and storage.

Evidence that serum amyloid P component binds to mannose-terminated sequences of polysaccharides and glycoproteins

Serum amyloid P component (SAP) is a normal human serum protein with pentraxin structure that has morphological and immunochemical identity to the amyloid P component found in normal tissue and amyloid deposits. In the presence of calcium, SAP binds to certain complex polysaccharides, including agarose and zymosan. While the binding of SAP to agarose involves interaction with a galactose pyruvate acetal, the ligand in zymosan has not been defined. In the present study we determined that SAP binds to ligand(s) in a soluble extract of zymosan prepared by alkaline hydrolysis, which contains the mannose oligosaccharide sequences α dManl → 3 dMan and α dManl → 6 dMan. SAP did not bind to the alkaliinsoluble fraction of zymosan, which is predominantly a glucan polymer, and its binding to zymosan extract which had been absorbed with concanavalin A was markedly reduced, suggesting that mannose residues are involved in the binding of SAP to zymosan. We also demonstrated that SAP binds to the glycoproteins ovalbumin, thyroglobulin, β-glucuronidase and C3bi, which contain mannose-terminated sequences, while it did not bind to native and desialized preparations of ovomucoid, α 1-acid glycoprotein and glycophorin, which lack terminal mannose residues. SAP did not bind to pneumococcal C polysaccharide or to N-acetylglucosamine oligosaccharides covalently linked to a protein carrier. The binding of SAP to ligand(s) in zymosan extract or ovalbumin was inhibited by the preincubation of SAP with either zymosan extract or ovalbumin glycopeptides, both of which share similar mannose oligosaccharide sequences. All of the SAP binding reactions required calcium, were maximal at approximately 1 m M calcium, and gave similar results whether purified SAP or SAP in serum was used. These findings indicate that mannose-terminated oligosaccharides of polysaccharides and glycoproteins represent a new class of ligands for SAP and suggest that SAP may function as a mannose-binding protein.

Purification and characterization of .ALPHA.-mannosidase and .BETA.-N-acetylhexosaminidase from watermelon fruit.

Watermelon fruit was found to be rich in α-mannosidase (EC 3.2.1.24) and β-N-acetylhexosaminidase (EC 3.2.1.52). A simple procedure has been devised for the simultaneous isolation of α-mannosidase and β-N-acetylhexosaminidase from watermelon fruit. The purified enzymes were electrophoretically homogeneous and sufficiently free from other glycosidases. The molecular weights of α-mannosidase and β-N-acetylhexosaminidase were estimated to be 270, 000 and 150, 000 by Sephadex G-200 gel filtration. The enzymes had their maximum activities at pHs 4.0-4.5 and 5.0-5.5, and their Km values for the corresponding p-nitrophenyl glycoside were 2.3 and 0.65mM, respectively. α-Mannosidase required Zn2+ to retain its activity and liberated 60% of the mannose from ovalbumin glycopeptide.

Protein-ligand interaction. A calorimetric study of the interaction of oligosaccharides and hen ovalbumin glycopeptides with concanavalin A.

A calorimetric study is reported concerning the interaction between concanavalin A (Con A) and some oligosaccharides and glycopeptides hydrolyzed from hen ovalbumin. The measurements were carried out in acetate buffer, pH 4.5, where, by far, the prevailing form of the protein is the dimeric one [Kalb, A.J., & Lustig, A. (1968) Biochim. Biophys. Acta 168, 366; Dani, M., Manca, F., & Rialdi, G. (1981) Biochim. Biophys. Acta 667, 108]. The calorimetric technique allows the direct determination of the binding enthalpy delta H, degrees B, the evaluation of the apparent association constant K'B, and then the evaluation of the apparent free energy and entropy, delta G degrees' B and delta S degrees' B. Three groups of data have been collected in the present study. The first one concerns the interaction between concanavalin A and some mono- and disaccharides [methyl alpha-glucopyranoside (alpha MGlup), methyl alpha-mannopyranoside (alpha MManp), D-maltose, D-trehalose, and D-cellobiose]. The analysis of the data indicates that in these cases there are small favorable entropic and enthalpic contributions to the affinity. The stoichiometry of the reaction is 2 mol of ligand/mol of Con A dimer, the sites resulting being equivalent and noninteracting. Melezitose, the only trisaccharide studied, shows a different behavior: its affinity for Con A is higher as compared to the other oligosaccharides containing alpha-glucosyl residues and closer to that of methyl alpha-mannopyranoside. However, the stoichiometry is different, namely, 1 mol of ligand/dimer of Con A.(ABSTRACT TRUNCATED AT 250 WORDS)

The release of oligosaccharides from glycoproteins by endo- β- N-acetylglucosaminidase of Flavobacterium sp.

Endo- β- N-acetylglucosaminidase, purified to homogenicity from the cultural filtrate of Flavobacterium sp., liberated oligosaccharides from various glycoproteins. The enzyme could liberate the carbohydrate chain from native ovalbumin. The release of oligosaccharides from ribonuclease B, yeast carboxypeptidase and a Ricinus lectin was also observed. These glycoproteins contain neutral oligosaccharides that are attached to the protein through glycosyl asparagine bonds. The treatment of glycoprotein with SDS and boiling was more effective for removal of oligosaccharides by the enzyme. The enzyme hydrolyzed all five heterogeneous ovalbumin glycopeptides, although the rate of hydrolysis decreased as the size of the sugar moiety increased. Removal of the neutral oligosaccharides did not appear to effect the enzymatic properties of the hemagglutination ability of these glycoproteins.

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.

Effect of alkalis on N-glycosidic linkages of glycoproteins.

Effect of alkalis on N-glycosidic linkages of glycoproteins.

Determination of the structure of ovalbumin glycopeptide AC-B by 1H nuclear magnetic resonance spectroscopy at 500 MHz

Three unique, unmodified ovalbumin glycopeptides were separated to homogeneity by high-pressure liquid chromatography. The nuclear magnetic resonance data, at 500 MHz, confirmed the structure of two of the three species and for the first time established the presence of a Man 8GlcNAc 2Asn glycopeptide in ovalbumin. This compound was a single homogeneous isomeric form out of three possible compounds expected as processing intermediates.

Resolution of some glycopeptides of hen ovalbumin by reverse-phase high-pressure liquid chromatography

Reverse-phase high-pressure liquid chromatography has been shown to be useful in fractionation of oligosaccharides containing N-acetyl amino sugars on the basis of stereochemical differences. Data in this paper show this same stereochemical selectivity can be obtained for glycopeptides. Using dilute aqueous phosphate for elution, three major high-mannose glycopeptides from ovalbumin can be separated on the analytical scale in a few minutes and the same method can also be used for preparation of the glycopeptides on the milligram scale. High-field proton NMR spectroscopy is used to identify two of the compounds as the mannose-5 and -6 glycopeptides (E3 and D3) and the third as the mannose-6 glycopeptide substituted by two N-acetyl glucosamine (GlcNAc) residues (C3B). The NMR spectra and analytical chromatograms of the glycopeptides show high purity and freedom from contaminants previously identified in ovalbumin glycopeptides.

Purification and characterization of an alpha-mannosidase of sea-squirt.

An alpha-mannosidase was isolated from the extract of acetone powder of CHCl3-treated internal organs of the sea-squirt, Styela plicata. The enzyme was purified 4,110-fold in 11% yield. The preparation was fairly homogeneous on disc and SDS-polyacrylamide gel electrophoreses and Sephadex G-200 chromatography. The enzyme had an estimated molecular weight of 275,000 by gel chromatography and 70,000 by SDS-polyacrylamide gel electrophoresis, and was therefore considered to be a tetramer. The optimum pH for the enzyme activity was 3.4 but the stable pH range was from 4 to 6. The isoelectric point was 5.0. The enzyme was activated by Zn2+ but inhibited by Cu2+, Fe2+, Hg2+, and EDTA. The isolated enzyme released mannose not only from stem bromelin glycopeptide and ovalbumin glycopeptide but also from yeast mannan.