Comparative study of chicken ovalbumin subfractions having different carbohydrate chain from each other by high performance anion exchange chromatography

1. Ovalbumin was fractionated to six fractions according to their phosphate content by high performance anion exchange chromatography. 2. This method was applied to analyze the phosphate content of ovalbumin subfractions having different carbohydrate chain from each other which were prepared by concanavalin A/Sepharose chromatography from oviduct slices incubated with [2-3H]mannose. 3. Most biosynthetic intermediates bearing a carbohydrate chain of Man8 or 9 GlcNAc2 was not phosphorylated while other fractions bearing differently processed carbohydrate chains such as Man5 or 6 GlcNAc2 or hybrid type carbohydrate chain was phosphorylated at their peptide portion.     

Degradation of heparin in mouse mastocytoma tissue

1. Heparin was prepared from mouse mastocytoma tissue by mild procedures, including extraction of mast-cell granules with 2m-potassium chloride, precipitation of the extracted polysaccharide with cetylpyridinium chloride from 0.8m-potassium chloride and finally digestion of the isolated material with testicular hyaluronidase. The resulting product (fraction GE(H)) represented approx. 40% of the total heparin content of the tissue. 2. Fraction GE(H) was fractionated by gel chromatography on Sepharose 4B into three subfractions, with average molecular weights ( M(w)) of approx. 60000-70000 (highly polydisperse material), 26000 and 9000 respectively. Treatment of each of the subfractions with alkali or with papain did not affect their behaviour on gel chromatography. Amino acid and neutral sugar analyses indicated that the two low-molecular-weight fractions consisted largely of single polysaccharide chains lacking the carbohydrate-protein linkage region. It was suggested that these heparin molecules had been degraded by an endopolysaccharidase. 3. Pulse labelling in vivo of mastocytoma heparin with [(35)S]sulphate showed initial labelling of large molecules followed by a progressive shift of radioactivity toward fractions of lower molecular weight. Further, heparin-depolymerizing activity was demonstrated by incubating (35)S-labelled heparin in vitro with a mastocytoma 10000g-supernatant fraction. Appreciable degradation of the polysaccharide occurred, as demonstrated by gel chromatography. In contrast, no depolymerization was observed on subjecting (14)C-labelled chondroitin sulphate to the same procedure.     

Interactions between different corneal proteoglycans.

Proteoglycans were extracted from bovine cornea with 4M-guanidinium chloride and purified by CsCl-density-gradient centrifugation. Under associative conditions two fractions were found: one capable of forming assemblies of high molecular weight and another lacking this property. The heavier fraction (density 1.59 g/ml) was eluted as a single retarded peak from Sepharose 2B, but on DEAE-Sephadex chromatography, gave two peaks: the first (eluted with 0.75 M-NaCl) contained mainly proteochondroitin sulphate and the second (eluted with 1.25 M-NaCl) mainly proteokeratan sulphate. Each of these proteoglycans was more retarded on Sepharose 2B than was the original sample from density-gradient centrifugation. Re-aggregation was obtained by recombination of the two fractions. The lighter fraction (density 1.44 g/ml), containing predominantly keratan sulphate chains, was eluted from DEAE-Sephadex as a single peak with 1.25 M-NaCl and was retarded on Sepharose 2B: this fraction was not able to form aggregates with proteochondroitin sulphate. Chemical analyses of the carbohydrate and protein moieties of the proteoglycans from DEAE-Sephadex confirmed that, in the cornea, different subunits are present with characteristic aggregation properties and hydrodynamic volumes.     

Outer Cell Envelope Glycoprotein from Two Strains of Serratia marcescens

Cell envelope glycoproteins were extracted with sodium dodecyl sulfate from isolated cell walls of two strains of Serratia marcescens and purified by gel filtration column chromatography on Sepharose 4B. There was no significant difference in the chemical composition. Both fractions contained approximately 50% proteins and 10% carbohydrates. Glucosamine and glucose were identified as the only sugar components in the carbohydrate moiety. Immunochemically they shared at least one common antigenic component with each other and possibly with their corresponding lipopolysaccharide fractions.     

Deglycosylation of ovalbumin prohibits formation of a heat-stable conformer

To study the influence of the carbohydrate-moiety of ovalbumin on the formation of the heat-stable conformer S-ovalbumin, ovalbumin is deglycosylated with PNGase-F under native conditions. Although the enzymatic deglycosylation procedure resulted in a complete loss of the ability to bind to Concavalin A column-material, only in about 50% the proteins lost their complete carbohydrate moiety, as demonstrated by mass spectrometry and size exclusion chromatography. Thermal stability and conformational changes were determined using circular dichroism and differential scanning calorimetry and demonstrated at ambient temperature no conformational changes due to the deglycosylation. Also the denaturation temperature of the processed proteins remained the same (77.4 +/- 0.4 degrees C). After heat treatment of the processed protein at 55 degrees C and pH 9.9 for 72 h, the condition that converts native ovalbumin into the heat-stable conformer (S-ovalbumin), only the material with the intact carbohydrate moiety forms this heat-stable conformer. The material that effectively lost its carbohydrate moiety appeared fully denatured and aggregated due to these processing conditions. These results indicate that the PNGase-F treatment of ovalbumin prohibits the formation and stabilization of the heat-stable conformer S-ovalbumin. Since S-ovalbumin in egg protein samples is known to affect functional properties, this work illustrates a potential route to control the quality of egg protein ingredients.     

Role of the Carbohydrate Chain and Two Phosphate Moieties in the Heat-Induced Aggregation of Hen Ovalbumin

We investigated the effect of the carbohydrate chain and two phosphate moieties on heat-induced aggregation of hen ovalbumin. The dephosphorylated form of ovalbumin was obtained by treating the original protein with acid phosphatase. The single carbohydrate chain was removed by digestion of heat-denatured ovalbumin with glycopeptidase F, and the resulting polypeptide without this carbohydrate chain was correctly refolded to acquire protease-resistance. Thermal unfolding can be approximated by a mechanism involving a two-state transition between the folded and unfolded states with a midpoint temperature of 76 °C for the original form, of 74 °C for the dephosphorylated form, and of 71 °C for the carbohydrate-free form. The conformational stability of the original form was higher than that of the carbohydrate-free form. When the three forms of ovalbumin were heated to 80 °C and then cooled rapidly in an ice bath, the polypeptide chains were compactly collapsed to metastable intermediates with secondary structures whose properties were indistinguishable. Upon incubation at 60 °C, renaturation was possible for a large portion of the intermediates of the original form, but for only a small portion of those of the carbohydrate-free form. Light scattering experiments showed that in the presence of sulfate anions, the intermediates of the carbohydrate-free form aggregated to a greater extent than did those of the original form. The intermediates of the carbohydrate-free form bound to the chaperonin GroEL with about 10-fold higher affinity than those of the original form. It follows that the carbohydrate chain and the two phosphate moieties do not affect hydrophobic collapse in the kinetic refolding of hen ovalbumin but play an important role in the slow rearrangement. They block the off-pathway reaction that competes with correct refolding by effectively decreasing surface hydrophobicity.     

The vitelline envelope of eggs from the giant keyhole limpet Megathura crenulata. II. Products formed by lysis with sperm enzymes and dithiothreitol.

The egg vitelline envelope of the marine invertebrate, Megathura crenulata, was lyzed either by sperm lysins A, B, C or by dithiothreitol. In each case the lysis mixture consisted of two major fractions, I and II, that could be separated by hydroxylapatite chromatography and had different electrophoretic mobilities on cellulose acetate strips. The amino acid, amino sugar, and neutral sugar compositions of fractions I and II were similar and resembled that of the intact vitelline envelope. Fractions I and II of each lysis mixture emerged in the exclusion volume of a Sepharose 6B column. A vitelline envelope fragment enzymatically formed by lysin was further degraded by dithiothreitol to form smaller fragments. A model of the vitelline envelope of the Megathura crenulata egg is suggested whereby the envelope is composed of polypeptide chains cross-linked by disulfide bonds and built to a large extent of closely spaced threonine residues. Most of the threonine residues are linked to carbohydrate units. Dithiothreitol dissolves the envelope by reducing disulfide bonds, whereas lysins most likely dissolve the envelope by degrading polypeptide chains.     

New GlcNAc/GalNAc-specific lectin from the ascidian Didemnum ternatanum

Previously we isolated GlcNAc-specific lectin (DTL) from the ascidian Didemnum ternatanum by affinity chromatography on cross-linked ovalbumin. Here we report the purification and characterization of new D-GlcNAc/D-GalNAc-specific lectin DTL-A from the same ascidian. This lectin was isolated from non-bound cross-linked ovalbumin fraction and further was purified by gel filtration on Sepharose CL-4B, affinity chromatography on GlcNAc-agarose and gel filtration on Superdex 200. SDS-polyacrylamide gel electrophoresis and gel filtration of purified lectin on Sepharose CL-4B indicates that it exists as large aggregates in the native state. Investigations of the carbohydrate specificity of DTL-A by enzyme-linked lectin assay suggest the multi-specificity of this lectin. DTL-A binds BSM, asialo-BSM as well as heparin and dextran sulfate. The binding of DTL-A to BSM was inhibited by monosaccharides D-GlcNAc and D-GalNAc, their alpha- but not beta-anomers. Among polysaccharides and glycoconjugates, DTL-A binding to BSM was effectively inhibited by BSM, asialo-BSM, pronase-treated BSM and synthetic alpha-D-GalNAc-PAA. Fetuin and asialofetuin showed a much lower inhibitory potency, heparin and dextran sulfate were noninhibitory. On the other hand, DTL-A binding to heparin was effectively inhibited by dextran sulfate, fucoidan, whereas BSM showed insignificantly inhibitory effect. DTL-A binding to heparin was not inhibited by D-GlcNAc and D-GalNAc.     

Frontal affinity chromatography of ovalbumin glycoasparagines on a concanavalin A-sepharose column. A quantitative study of the binding specificity of the lectin

The interactions of Sepharose 4B-immobilized concanavalin A (ConA) with 10 glycoasparagines derived from ovalbumin were investigated quantitatively by frontal affinity chromatography. In this method, a carbohydrate solution is applied continuously to a ConA-Sepharose column and the retardation of the elution front is measured as a parameter of the strength of the interaction. The dissociation constant (Kd) for each saccharide with ConA can be determined. An analysis of the binding of p-nitrophenyl-alpha,D-mannoside has shown that the binding properties of ConA do not change essentially after immobilization on Sepharose 4B. Each of the ovalbumin glycoasparagines was labeled with tritium by the reductive methylation method for analysis. A comparison of the Kd values obtained showed that the binding of ConA varies considerably with very slight structural differences of the glycosyl chain. The results suggest that ConA recognizes a specific glycosyl chain structure, Man alpha 1-6(Man alpha 1-3)Man, in which at least one hydroxyl group at the C-3 position of C-6-linked mannose should be free. The glycoasparagines containing this structure bound strongly to ConA-Sepharose with dissociation constants below 3.4 X 10(-7) M.     

Structures of the carbohydrate chains of membrane glycoproteins IIb and IIIa of human platelets

Human platelet membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa), which have been proposed to be subunits of a receptor for fibrinogen, were purified from Triton X-100-solubilized platelet membranes by affinity chromatography on a concanavalin A (Con A)-Sepharose column followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Compositional analyses of the purified glycoproteins showed that GPIIb and GPIIIa contain 15% and 18% carbohydrate by weight, respectively, which consists of galactose, mannose, glucosamine, fucose, and sialic acid. This suggested that these glycoproteins contained N-linked carbohydrate chains. The carbohydrate chains were released from each glycoprotein by hydrazinolysis and then fractionated by ion-exchange chromatography on a Mono Q column. From each glycoprotein, mono-, di-, and trisialylated and neutral oligosaccharide fractions were obtained. The structures of these oligosaccharides were investigated by means of compositional and methylation analyses and digestion by exoglycosidase, and their reactivities to immobilized lectins were also examined. The neutral oligosaccharides, which comprised about 14% of the total oligosaccharides released from GPIIb and about 52% of that from GPIIIa, were found to be of the high mannose-type, in that they contained 5 or 6 mannose residues. On the other hand, a major part of the acidic oligosaccharides was found to consist of typical bi- and triantennary complex-type sugar chains, and much smaller amounts of tetraantennary complex-type sugar chains, and complex-type sugar chains with a fucosyl residue at a N-acetylglucosamine residue in the peripheral portion or a bisecting N-acetylglucosamine at a beta-mannosyl residue in the core portion were also detected. In conclusion, we found that GPIIb contained mainly complex-type sugar chains, whereas high mannose-type sugar chains were the predominant carbohydrate units in GPIIIa, and that the detected differences in the carbohydrate moieties of GPIIb and GPIIIa were quantitative but not qualitative.     

Microheterogeneity of cross-linked gamma dimers isolated from bovine stabilized fibrin

Bovine stabilized fibrin was reduced, carboxymethylated and separated by chromatography on a Sepharose 4B column. The fraction containing cross-linked γ dimers was then subjected to linear gradient chromatography on a CM-52 column. On this column, the γ dimers were separated into an adsorbed and unadsorbed fraction. The components in these fractions were designated as the γ-1 and γ-2 dimers. They each gave a single band on SDS-polyacrylamide gel electrophoresis and both had a molecular weight of 90,000 (±2,000). The identities of the γ-1 and γ-2 dimers were also shown by their amino acid compositions, terminal residues and tryptic and plasmic maps. However, they differed in electrophoretic mobilities on gels at pH 8.3 and pH 3.6 and in carbohydrate composition. The γ-1 dimer was slightly acidic and contained more hexoses and glucosamine than the γ-2 dimer. These results indicate that the characteristics of the bovine monomeric γ chains, γ-1 and γ-2, previously reported by Gerbeck $\text{et al}$., are transferred to their corresponding cross-linked γ dimers, formed in the stabilization of fibrin.     

Uniformity of carbohydrate chains within molecular variants of rat alpha 1-fetoprotein with distinct affinity for concanavalin A

Three rat alpha 1-fetoprotein fractions were obtained by chromatography on concanavalin-A--Sepharose: one non-reactive, one weakly reactive and one reactive to concanavalin A. The non-reactive and reactive variants were found to vary in the structure of their carbohydrate chains while the conformation of the weakly reactive form may modulate the accessibility of these chains to the lectin. N-Glycosidically linked glycans from unfractionated alpha 1-fetoprotein were isolated and chemically characterized. Particular attention was paid to develop sensitive methods based upon hydrazinolysis, quantitative re-N-acetylation of glycans with [14C]acetic anhydride and thin-layer chromatography of labeled compounds. With the aid of these methods two main kinds of glycans (1a and 2a) were obtained and fractionated on concanavalin-A--Sepharose into non-reactive (1a) and reactive (2a) molecules. Moreover it was demonstrated that each alpha 1-fetoprotein variant contained either two glycans 1a or two glycans 2a, not randomly, but a pair of the identical carbohydrate chains at the two glycosylation sites.     

Separation of oligo(adenosine diphosphate ribose) fractions with various chain lengths and terminal structures.

Oligo(adenosine diphosphate ribose) preparations with chain lengths of 3 to 10 adenosine diphosphate ribose units were fractionated according to their chain lengths and their terminal structures by hydroxyapatite column chromatography and then polyacrylamide gel electrophoresis. The peak fractions from the hydroxyapatite column were each separated into two distinct subfractions by gel electrophoresis. The two subfractions were found to differ in chain length and terminal structure. A linear correlation was observed between the mobility and the logarithm of the chain length of oligo(adenosine diphosphate ribose) on gel electrophoresis, irrespective of the terminal structure.     

Inhibition of lymphocyte proliferation by uterine fluid from the pregnant ewe

Immunosuppressive molecules in uterine fluid from the nonpregnant uterine horn of unilaterally pregnant ewes at Days 60, 100, and 140 of gestation were examined. Uterine fluid from all days inhibited [3H]thymidine incorporation into phytohemagglutinin-stimulated lymphocytes. Inhibitory activity increased with advancing gestation. Uterine fluid also inhibited lymphocyte proliferation caused by other mitogens or by mixed lymphocyte reactions. Inhibitory activity was found in both salt volume (Mr less than 1000) and void volume (Mr greater than 5000) fractions of uterine fluid resolved by Sephadex G-25 desalting columns. Only activity in the void volume was sensitive to pronase. Several fractions containing inhibitory activity were resolved when dialyzed uterine fluid was fractionated into acidic and basic components by cation-exchange chromatography and further resolved by gel filtration using Sepharose CL-6B. The most active fractions (inhibition/micrograms protein) for both acidic and basic components eluted at the void volume of Sepharose CL-6B (Mr greater than 4 x 10(6). The inhibitory factor in the basic component that eluted at the void volume of Sepharose CL-6B was rich in carbohydrate, slightly cytotoxic, and partially sensitive to digestion with trypsin or oxidation with periodate. In conclusion, uterine fluid of unilaterally pregnant ewes is enriched in molecules that inhibit lymphocyte proliferation in vitro. Among these are low molecular weight, non proteinaceous factors and a very high molecular weight (Mr greater than 4 x 10(6) fraction that contains protein and carbohydrate.     

Synthesis of Arabinose-Containing Cell Wall Precursors in Suspension-Cultured Tobacco Cells: IV. Differential Subfractionation and Comparison of the Acidic Precursors

A series of ^l4C-arabinose-labelled cell wall precursors weredifferentially subfractionated to compare their properties andanalyze their sugar side chains. The ratio of radioactivity/proteinof the subfractions increased 100- to 5,000-fold from that ofthe starting homogenate. The less acidic fractions showed higherratio of radioactivity/protein. Molecular weights of these fractions'constituents were distributed mostly between 130,000 and 150,000and their dispersion in each fraction was small. The chemicalcomposition of the fractions, which was rather invariable, wasthat of the typical arabinogalactan protein, however, a lowcontent of protein and uronic acid was noticed in the less acidicfractions which showed the highest radioactivity/protein ratio. Gel chromatography of an alkaline hydrolysate of these subfractionsindicated that the precursors consisted of two kinds of molecules:1) a less acidic polysaccharide of high radioactivity/proteinratio, probably arabinogalactan, and 2) a more acidic arabinogalactanprotein of low specific radioactivity/protein ratio. The averagesize of the side chains of the arabinogalactan protein was about10,000 and linked to about 70% of the hydroxyproline residues.No small oligosaccharide side chain (DP<13) was found. Also,no precursor-product relationship was observed between thesesubfractions. (Received September 1, 1986; Accepted May 7, 1987)     

Analysis of the multiple forms of Gaucher spleen sphingolipid activator protein 2.

Gaucher spleen sphingolipid activator protein 2 was fractionated into concanavalin A binding- and non-binding fractions. These fractions each contained several bands on non-denaturing polyacrylamide gel electrophoresis (PAGE). The two fractions were further fractionated by electroblotting the proteins from preparative gels onto nitrocellulose, staining with Ponceau S to locate the bands of protein and then eluting the protein components from the nitrocellulose. A total of ten fractions, each containing only one or two major components, was collected. All of these subfractions activated beta-glucocerebrosidase and sphingomyelinase and most subfractions also activated beta-galactocerebrosidase. The structural relationship of the bands was investigated using endoglycosidase digestions. The results indicated that the two bands with the fastest mobility on non-denaturing PAGE did not contain any carbohydrate. The remaining bands showed only limited or partial digestion with endoglycosidase H and endoglycosidase D, but were readily hydrolysed with endoglycosidase F. The products of these digestions included bands with similar mobilities to the non-carbohydrate containing bands.     

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.     

Lactoferrin Is the Major Deoxyribonuclease of Human Milk

Lactoferrin is the major iron-transferring protein of human barrier fluids such as blood and milk. It is a polyfunctional protein capable of binding DNA exposed on the surface of various cells. Electrophoretically homogenous lactoferrin was prepared by sequential chromatography of human milk proteins on DEAE-cellulose, heparin-Sepharose, and Sepharose containing immobilized anti-lactoferrin antibodies. By subsequent chromatography on Blue Sepharose the resulting lactoferrin was fractionated into several subfractions with different affinity for the sorbent, and this was associated with separation of additional lactoferrin peaks with DNase activity from the main peak. By various techniques, in particular, by in situ testing the DNase activity of lactoferrin in a DNA-containing gel after SDS-electrophoresis, hydrolysis of DNA was for the first time shown to be an intrinsic property of lactoferrin. The substrate specificity of lactoferrin in hydrolysis of DNA was different from specificities of known human DNases. Hydrolysis of DNA was activated by bivalent metal ions and also by ATP and NAD. Unlike the main fraction of lactoferrin with the highest affinity for Blue Sepharose, all protein subfractions with DNase activity were cytotoxic and suppressed growth of human and mouse tumor cell lines.     

13C-n.m.r.-spectral study of galactosyltransferase specificity with regard to the carbohydrate side-chain of native ovalbumin

As a prelude to studies using bovine N-acetylglucosaminide-β-(1→4)-galactosyltransferase to label membrane-surface glycoproteins with isotopically enriched d-galactose, the structural specificity of the enzymic reaction with water-soluble, hen ovalbumin has been examined. The enzyme-catalyzed transfer of d-galactose from UDP-d-galactose requires a (nonreducing) terminal 2-acetamido-2-deoxy-d-glucosyl group and exhibits selectivity towards saccharide chains containing d-mannose. This study considers the structural specificity of the enzyme with regard to the anomeric linkage between 2-acetamido-2-deoxy-d-glucose and d-mannose in the carbohydrate chains of hen ovalbumin. Uniformly ¹³C-enriched d-galactose was enzymically attached to the ovalbumin carbohydrate chain (which exhibits microheterogeneity in its structure), the protein was hydrolyzed, and separate glycopeptide fractions were chromatographically isolated. The ¹³C-n.m.r. spectra (60.5 MHz) of the fractions revealed two peaks for the anomeric carbon atom of d-galactose. The two peaks, at 104.20 and 104.39 p.p.m., were ascribed to d-galactosyl groups attached to 2-acetamido-2-deoxy-d-glucose respectively linked β-(1→4) and β-(1→2), to d-mannose in the glycopeptide chains. Quantifying of the spectral data revealed no specificity of d-galactosyltransferase towards the linkage from the terminal 2-acetamido-2-deoxy-d-glucosyl group to the penultimate d-mannosyl residue.     

Primary structure of the major glycans of the N-acetyllactosamine type derived from the human immunoglobulins M from two patients with Waldenström's macroglobulinemia

The carbohydrate chains of the pathological human immunoglobulins M from two patients with Waldenström's macroglobulinemia were released by hydrazinolysis. The N-acetyllactosamine-type glycans were obtained by affinity chromatography on concanavalin A and fractionated by high-voltage paper electrophoresis. The primary structure of the major compounds was elucidated on the basis of carbohydrate analysis, methylation analysis, including mass-spectrometry, and 500 MHz ¹H-NMR spectroscopy. For both patients, this appeared to be a monosialyl monofucosyl biantennary structure; the compounds differed by the presence of an intersecting N-acetylglucosamine residue.