Structure of carbohydrate chains of the riboflavin-binding glycoprotein of chicken egg protein. II. 1H-NMR (500 MHz) spectroscopy of the major neutral oligosaccharides

Reductive cleavage of riboflavin-binding glycoprotein from hen egg white (RF-GPw) with LiBH4/tert-BuOH followed by NaBH4/NaOH treatment gave rise to oligosaccharide alditols, fractionated by a successive HPLC on muBondapak C18 and Zorbax NH2 columns. Seven main individual oligosaccharide alditols were isolated and their structure was investigated by 1H NMR 500-MHz spectroscopy. The structure and relative content of the main oligosaccharide chains were proved to be identical in RF-GPw and ovomucoid. Structure of polypeptide chains and their molecular weight, number of glycosylation sites and their structure had little or no effect on the glycosylation pattern in both glycoproteins. HPLC of the oligosaccharide alditols from another egg white glycoprotein, ovotransferrin, also revealed its high microheterogeneity and close resemblance to those of ovomucoid and RF-GPw.     

The structure of carbohydrate chains of riboflavin-binding glycoprotein from chicken egg protein. III. 1H-NMR spectroscopy (500 MHz) of neutral fucosylated oligosaccharides

Using LiBH4/ButOH treatment, oligosaccharides were cleaved off the hen egg white riboflavin-binding glycoprotein. HPLC led to the isolation of four fucose-containing oligosaccharide alditols, whose structure was elucidated by means of 1H NMR 500 MHz spectroscopy. The main fucosylated oligosaccharide, also present in hen ovomucoid, was found to be a biantennary carbohydrate chain of N-acetyllactosamine type.     

Structure of the carbohydrate chain of riboflavin-binding glycoprotein from hen egg white. Neutral oligosaccharides of the hybrid type

Reductive cleavage of the riboflavin-binding glycoprotein from hen egg white with LiBH4/tert-BuOH followed by NaBH4 treatment gave rise to oligosaccharide alditols. After fractionation by HPLC two individual oligosaccharide alditols of a hybrid type were isolated. Their structures were proved by 1H NMR 500 MHz spectroscopy and methylation analysis. One of the oligosaccharides has earlier been found in ovalbumin, whereas the other is identified in glycoproteins for the first time.     

Synthesis of N-glycoproteins with a native type of carbohydrate-peptide bond

Reaction of beta-oligoglycosylamines obtained from carbohydrate chains of N-glycoproteins (ovalbumin, ovomucoid, riboflavin-binding glycoprotein from hen egg white, and asialofetuin) with bovine serum albumin, lysozyme, and poly(L-Asp) in presence of water-soluble carbodiimide gave rise to a series of glycoconjugates, modelling natural N-glycoproteins. Carbohydrate-peptide bond was shown to be of N-glycosylamide type with participation of Asp and Glu residues. The method allows one to obtain synthetic N-glycoproteins from oligomannoside, complex and hybrid oligosaccharide chains, and may find application both in biochemistry and biotechnology for improvement of physico-chemical properties of unglycosylated proteins.     

N-glycan structures from the major glycoproteins of pigeon egg white: predominance of terminal Galalpha(1)Gal

N-Glycans from major glycoproteins of pigeon egg white (ovotransferrin, ovomucoid, and ovalbumins) were enzymatically released and were reductively aminated with 2-aminopyridine, separated, and structurally characterized by mass spectrometry and a three-dimensional mapping technique using three different columns of high performance liquid chromatography (HPLC) (Takahashi, N., Nakagawa, H., Fujikawa, K., Kawamura, Y., and Tomiya, N. (1995) Anal. Biochem. 226, 139-146). Twenty-five major N-glycan structures, all of them hitherto unknown, were identified as pyridylamino derivatives. Of these, 13 were neutral, 10 were monosialyl, and 2 were disialyl oligosaccharides. All N-glycans contain from one to four Galalpha(1,4)Galbeta(1,4) sequences at the nonreducing terminal positions and are devoid of fucose residues. N-Acetylneuraminic acids were alpha(2,6)-linked only to beta-galactose. The HPLC profiles of the N-glycans from four different glycoproteins were qualitatively very similar to each other, but not identical in the peak distributions. Monosialyl glycans were most abundant in all four glycoproteins, followed by neutral glycans. Disialyl glycans were lowest in ovotransferrin, and highest in ovomucoid. Triantennary structures with bisecting GlcNAc were predominant in ovotransferrin, and tetra-antennary (with and without bisecting GlcNAc-containing) structures were predominant in other glycoproteins. Penta-antennary structures (with a sialic acid and without bisecting GlcNAc residue) were also found in small quantities in all four glycoproteins. In contrast to the chicken egg white counterparts, which contain mostly high mannose and hybrid types, all N-glycan structures in the major pigeon egg white glycoproteins are complex type.     

Ovalbumin-related gene Y protein bears carbohydrate chains of the ovomucoid type

We have recently established the monoclonal antibodies (mAbs) specific to the major food allergen, ovomucoid, as mAb 7D, recognizing the carbohydrate moiety of ovomucoid, and mAb 6H, the peptide moiety (Biosci. Biothechnol. Biochem., 68, 2490-2497, (2004)). Using these mAbs, we found commercially available ovalbumin preparations contaminated with a considerable amount of ovomucoid together with other glycoproteins. To examine the contaminants, egg white was subjected to cation-exchange chromatography. An unidentified protein was found in egg white that reacted with mAb 7D but not with mAb 6H, having a molecular size of about 52 kDa and a blocked N-terminus. Two internal amino acid sequences of the fragments obtained after a lysyl endopeptidase and a hydroxylamine treatment revealed the protein to be ovalbumin Y (ovalbumin-related gene Y protein). We conclude that ovalbumin Y is a unique chimeric glycoprotein having an amino acid sequence similar to that of ovalbumin, but having a carbohydrate moiety similar to that of ovomucoid.     

Ovalbumin-Related Gene Y Protein Bears Carbohydrate Chains of the Ovomucoid Type

We have recently established the monoclonal antibodies (mAbs) specific to the major food allergen, ovomucoid, as mAb 7D, recognizing the carbohydrate moiety of ovomucoid, and mAb 6H, the peptide moiety (Biosci. Biothechnol. Biochem., 68, 2490–2497, (2004)). Using these mAbs, we found commercially available ovalbumin preparations contaminated with a considerable amount of ovomucoid together with other glycoproteins. To examine the contaminants, egg white was subjected to cation-exchange chromatography. An unidentified protein was found in egg white that reacted with mAb 7D but not with mAb 6H, having a molecular size of about 52 kDa and a blocked N-terminus. Two internal amino acid sequences of the fragments obtained after a lysyl endopeptidase and a hydroxylamine treatment revealed the protein to be ovalbumin Y (ovalbumin-related gene Y protein). We conclude that ovalbumin Y is a unique chimeric glycoprotein having an amino acid sequence similar to that of ovalbumin, but having a carbohydrate moiety similar to that of ovomucoid.     

Avian egg's white ovomucoid as food-allergen for human

Hen eggs are considered as the most common reason of a food allergy in humans. The most important allergens of egg white proteins are as follows: ovomucoid, lysozyme, ovalbumin and ovomucin. Ovomucoid is a Kazal-type protease inhibitor which accounts for about 10% of avian egg white protein. It is a glycoprotein containing 20 through 25% carbohydrates. The molecule of ovomucoid is composed of three homologous domains. All avian ovomucoid domains contain six cysteines in similar location that form three intradomain disulfide bonds. Ovomucoid (Gal d1) is one of the major allergen in hen's egg. It is a glycoprotein comprising 186 amino acids, and it has a molecular weight of 28000 Da and an isoelectric point of 4.1. Ovomucoid has antibacterial activity resulting from its ability to inhibit bacterial proteolytic enzymes crucial for microbial growth. Many studies reveal that ovomucoid is a thermo stable molecule.     

The carbohydrate units of asialo-ovomucoid: their heterogeneity and enzymic degradation

An ovomucoid variant free from sialic acid has been prepared in a pure state by ion-exchange chromatography on DEAE-cellulose. The purified glycoprotein contained 10-11 residues of mannose, 2-3 residues of galactose, and 21 residues of 2-acetamido-2-deoxyglucose. Glycopeptides have been prepared by exhaustive digestion with Pronase followed by ion-exchange chromatography on Dowex 50 (X2) resin. Three fractions were obtained, all with similar contents of mannose and hexosamine but with various contents of galactose. The sugar-aspartic acid ratios indicated that all of the fractions were heterogeneous, the major fraction having mannose-galactose-hexosamine-aspartic acid ratios of 2.6:0.5:5.8:1.0. Cleavage of asialo-ovomucoid with cyanogen bromide and proteolytic digestion of the isolated fragments gave two heterogeneous glycopeptide fractions of similar composition. Both asialo-ovomucoid and the principal glycopeptide fraction were degraded with beta-D-galactosidase, alpha-D-mannosidase, and beta-N-acetylglucosaminidase singly and in sequence. Removal of much of the carbohydrate from asialo-ovomucoid had no appreciable effect on its anti-tryptic activity. By sequential degradation of the glycopeptide, a pentasaccharide core alpha-D-Man-[alpha-D-Man]-beta-D-Man-beta-D-GlcNAc-beta-D-GlcNAc-Asn was obtained. Other structural features revealed by enzymic degradation are discussed.     

Isolation and characterization of major glycoproteins of pigeon egg white: ubiquitous presence of unique N-glycans containing Galalpha1-4Gal

Ovotransferrin (POT), two ovalbumins (POA(hi) and POA(lo)), and ovomucoid (POM) were isolated from pigeon egg white (PEW). Unlike their chicken egg white counterparts, PEW glycoproteins contain terminal Galalpha1-4Gal, as evidenced by GS-I lectin (specific for terminal alpha-Gal), anti-P(1) (Galalpha1-4Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glcbeta1-1Cer) monoclonal antibody, and P fimbriae on uropathogenic Escherichia coli (specific for Galalpha1-4Gal). Galalpha1-4Gal on PEW glycoproteins were found in N-glycans releasable by treatment with glycoamidase F. The respective contents of N-glycans in each glycoprotein were 3.5%, POT; 17%, POA(hi); and 31-37%, POM. POA(hi) has four N-glycosylation sites, in contrast to chicken ovalbumin, which has only one. High performance liquid chromatography analysis showed that N-glycans on POA(hi) were highly heterogeneous. Mass spectrometric analysis revealed that the major N-glycans were monosialylated tri-, tetra-, and penta-antennary oligosaccharides containing terminal Galalpha1-4Gal with or without bisecting N-acetylglucosamine. Oligosaccharide chains terminating in Galalpha1-4Gal are rare among N-glycans from the mammals and avians that have been studied, and our finding is the first predominant presence of (Galalpha1-4Gal)-terminated N-glycans.     

1H-NMR spectroscopic characterization of dansyl glyco-asparagines derived from hen egg white clycoproteins

Dansyl glyco-asparagines were prepared from a partially fractionated mixture of hen egg white glycoproteins. Reverse-phase high performance liquid chromatography (HPLC) on a silica-based octadecyl column yielded ten such derivatives in a virtually pure state. The detailed structures of the glyco-asparagines were identified by 500-MHz¹H nuclear magnetic resonance (NMR) spectroscopy. Two of them were found to be of the oligomannosideN-type, four were of the intersected-hybridN-type and another four were of the intersected multi-antennaryN-type. In monogalactosylated, intersected structures the galactose residue was proved by¹H-NMR to be attached in (1–4)-linkage to the GlcNAc1-4Man1–3 branch.Dansyl glyco-asparagines turned out to be suitable derivatives for¹H-NMR spectroscopic analysis. The combination of HPLC and high-resolution NMR spectroscopy of such derivatives proved to be a powerful technique in studying the (micro-)heterogeneity of sugar chains in glycoproteins.Abbreviations dns dansyl (5-dimethylaminonaphthalene-1-sulfonyl) - ODS octadecyl-silica - WEFT water-eliminating Fourier transform - DSS sodium 4,4-dimethyl-4-silapentane-1-sulfonate - OVA ovalbumin - OVM ovomucoid - OVT ovotransferrin     

A novel preparative method for the isolation of avidin and riboflavin-binding glycoprotein from chicken egg-white by the use of high-performance liquid chromatography.

A method for the rapid preparative isolation of highly purified avidin using h.p.l.c. on a powerful cation-exchanger TSK SP-5PW column has been developed. The method is based on the following properties of avidin: solubility at a high (NH4)2SO4 concentration, stability and relatively low solubility in organic solvents, as well as the strongly cationic nature of the molecule. Riboflavin-binding glycoprotein may be isolated as by-product.     

Isolation and characterization of thiamin-binding protein from chicken egg white.

A thiamin-binding protein was isolated and characterized from chicken egg white by affinity chromatography on thiamin pyrophosphate coupled to aminoethyl-Sepharose. The high specificity of interaction between the thiamin-binding protein and the riboflavin-binding protein of the egg white, with a protein/protein molar ratio of 1.0, led to the development of an alternative procedure that used the riboflavin-binding protein immobilized on CNBr-activated Sepharose as the affinity matrix. The thiamin-binding protein thus isolated was homogeneous by the criteria of polyacrylamide-gel disc electrophoresis, double immunodiffusion and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, had a mol.wt. of 38,000 +/- 2000 and was not a glycoprotein. The protein bound [14C]thiamin was a molar ratio of 1.0, with dissociation constant (Kd) 0.3 micrometer.     

The influence of some fractions of egg yolk on the survival of ram spermatozoa at 5 degrees C.

Ram spermatozoa were stored at 5 degrees C in diluents containing various fractions of egg yolk prepared by dialysis, ultrafiltration and ion-exchange chromatography. They survived storage best in the presence of components of egg yolk which were non-dialysable and were not filtered through membranes which retained substances of molecular weight greater than 100 000. The substances isolated in peak B of the ion-exchange chromatogram of whole egg yolk described by Seideman et al. (1969) gave greater protection than those from other fractions from this chromatographic system. These data indicate that the low-density lipoprotein fraction of egg yolk is the most likely source of protection to ram spermatozoa against the effects of storage at 5 degrees C.     

Thomsen-Friedenreich (T)-active glycoproteins, blood group N antigen precursor glycoproteins and N antigen precursor glycoproteins with T activity from ascitic fluids of liver cancer patients.

1. Five perchloric acid-soluble fractions (PASFs) obtained from ascitic fluids of three patients with primary hepatocellular carcinoma (HCC), a patient with liver metastatic carcinoma (LUC) from ureteral carcinoma and human normal serum (NS) were subjected to DEAE-cellulose column chromatography to separate seven glycoprotein fractions, respectively. 2. In this chromatography, two HCC-PASFs gave a Thomsen-Friedenreich (T)-active glycoprotein, respectively. 3. Other HCC-PASF gave a T-active glycoprotein, two blood group N antigen precursor glycoproteins and an N antigen precursor glycoprotein with T activity. 4. LUC-PASF gave two T-active glycoproteins and an N antigen precursor glycoprotein with T activity. 5. NS-PASF did not give these serologically active glycoproteins.     

Turtle-dove ovomucoid, a glycoprotein proteinase inhibitor with P1-blood-group antigen activity.

Ovomucoid from the egg white of turtle-dove (Streptopelia risoria) was purified and shown to be a glycoprotein of mol. wt. 29 400, with valine as N-terminal residue. It is an inhibitor of both trypsin and chymotrypsin, but has a lower affinity for trypsin than has hen ovomucoid. Turtle-dove ovomucoid contains antigenic activity cross-reacting with the blood-group-P1 antigen of human erythrocytes. Hen ovomucoid has no detectable blood group-P1 activity. The carbohydrate composition of turtle-dove ovomucoid differs from hen ovomucoid in having substantially higher galactose content. The possible relationship between carbohydrate composition and antigenic activity is discussed.     

Interaction of sialoglycoproteins with wheat germ agglutinin-sepharose of varying ratio of lectin to Sepharose. Use for the purification of mucin glycoproteins from membrane extracts

The influence of varying the amount of wheat germ agglutinin immobilized on Sepharose beads on the binding of glycoproteins to these beads was investigated. A series of wheat germ agglutinin-Sepharose gels containing between 0.10 and 10.0 mg of lectin/ml of gel was prepared, and the actual lectin content was established by acid hydrolysis of the gel followed by analysis of glycine, a major amino acid in wheat germ agglutinin. Affinity chromatography of labeled glycoproteins indicated that glycophorin bound to all the wheat germ agglutinin-Sepharose preparations. Fetuin, ovomucoid, and alpha 1-acid glycoprotein bound not at all or very poorly to gels with a low content of wheat germ agglutinin (less than 0.95 mg/ml). The specific binding of these glycoproteins increased with increasing lectin content on the gels, and on gels of high content (greater than 3 mg/ml) the binding was virtually quantitative. On chromatographing a mixture of glycophorin, alpha 1-acid glycoprotein, fetuin, and ovomucoid on wheat germ agglutinin-Sepharose, containing 0.08 mg of lectin/ml of gel, glycophorin was selectively retained on the gel. It was possible to purify glycophorin from an extract of human erythrocyte membranes in one step by chromatography on the above gel. By using the series of gels, it was demonstrated that Morris hepatoma 7777 membranes contained at least 4-fold more sialoglycoproteins which bound to low density wheat germ agglutinin-Sepharose compared to rat liver membranes. These hepatoma sialoglycoproteins were isolated, purified, and partially characterized as having a high proportion of O-linked sialyloligosaccharides. Our studies illustrate the use of low density wheat germ agglutinin-Sepharose gels both for the detection and for easy isolation of mucin-type glycoproteins from crude extracts of cells or membranes.     

Presence of a Vicia unijuga lectin-binding (Vgu) glycoprotein with Thomsen-Friedenreich (T) activity and Vgu glycoproteins in human primary hepatocellular carcinoma.

1. Twenty perchloric acid-soluble glycoprotein fractions (PASFs) were separated from carcinomatous and non-carcinomatous regions of the livers of 5 patients with primary hepatocellular carcinoma (HCC) and 5 patients with a complication of HCC and hepatocirrhosis (HC). 2. In autoradiography using 125I-labelled Vicia unijuga lectin (VUA) and 125I-labelled Arachis hypogaea anti-T lectin as probes, 10 PASFs from carcinomatous regions of the livers of 10 patients gave 1 Vgu glycoprotein with T activity and/or 1-4 Vgu glycoproteins, respectively, and among 10 PASFs from non-carcinomatous regions of the livers of 10 patients, 7 PASFs gave no glycoproteins, respectively. 3. These results show that a Vgu glycoprotein with T activity and Vgu glycoproteins occur in HCC and in a complication of HCC and HC as oncofetal antigens.     

Purification of the basic protein avidin using Gradiflow technology

The Gradiflow, a preparative electrophoresis instrument, which separates proteins on the basis of charge or size, was used to purify the basic protein avidin, pI 10, from chicken egg white. Using a charge based separation at pH 9.0, the high pI of avidin and lysozyme (pI 10.7) allows them to be easily separated from remaining egg white proteins, as these are the only positively charged proteins. In a second step at pH 10.2, the negatively charged avidin is separated from the positively charged lysozyme. This sequential two-step protocol was complete within 4.5h. Enzyme immunoassay of avidin fractions obtained indicated recoveries of 60-65% from one egg white with minimal lysozyme activity detected.     

Gas chromatography and mass spectrometry of disaccharides from glycoproteins.

Partial acid hydrolysis and methanolysis released disaccharides and disaccharide methylglycosides from the glycoproteins, ovomucoid and porcine gastric mucin in amounts of 0.5--7 microgram disaccharide per mg of glycoprotein. These disaccharides were fractionated by gas chromatography as the trimethylsilyl (Me3Si) derivatives. The composition of recovered disaccharides has been determined by hydrolysis and rechromatography of the Me3Si monosaccharides. The intersaccharide linkages of the disaccharides have been determined by electron impact mass spectrometry. This simple and rapid method can give structural information on small glycoprotein samples.