Identification of Peptides That Mimic the Pertussis Toxin Binding Site on Bovine Fetuin

The introduction of acellular pertussis vaccines has greatly enhanced the safety profile of vaccines to prevent whooping cough. Pertussis toxin (Ptx) is one component produced by Bordetella pertussis that is contained in all of these vaccines, either in combination with other known pertussis virulence factors or as the sole pertussis component, combined with tetanus and diphtheria toxoids. A hydrogen peroxide toxoid of Ptx has been shown to be efficacious in preventing pertussis infections in a mass vaccination trial and is presently licensed in the United States and Europe (B. Trollfors, J. Taranger, T. Lagergard, L. Lind, V. Sundh, G. Zackrisson, C. U. Lowe, W. Blackwelder, and J. B. Robbins, N. Engl. J. Med. 333:1045-1050, 1995). The industrial production of Ptx can be performed through the cultivation of B. pertussis in well-defined growth media, in which the components can be well characterized and their origins can be documented. Once the bacteria are removed from the culture, Ptx can be isolated from the supernatant and purified by using the technique described by Sekura et al. (R. D. Sekura, F. Fish, C. R. Manclark, B. Meade, and Y. L. Zhang, J. Biol. Chem. 258:14647-14651, 1983). The only drawback of this procedure, which combines two affinity chromatography steps, one with Blue Sepharose and a second with matrix-bound bovine fetuin (BF), is the source and purity of the BF. Concern about vaccine preparations that may possibly risk contamination by material associated with bovine spongioform encephalopathy has continued to increase. We thus sought a replacement for the BF affinity chromatography and, more specifically, for the glycosidic moiety on BF. We describe here the identification of a seven-amino-acid peptide that mimics the glycosidic moiety on BF to which Ptx binds. Furthermore, we have constructed an affinity column containing this peptide that can be used to replace BF in Ptx purification. Finally, we used the X-ray crystallographic structure of Ptx bound to the oligosaccharide moiety of BF as a scaffold and replaced the oligosaccharide with the peptide.     

Role of pertussigen (pertussis toxin) on the mouse protective activity of vaccines made from Bordetella species

Pertussigen [pertussis toxin (Ptx)] from Bordetella pertussis, when detoxified, induces protection in mice to intracerebral challenge (ic) with virulent B. pertussis. In its native form, minute nonprotective doses promote the development of immunity induced by other antigens of B. pertussis. As little as 4 ng of Ptx, given with a nonprotective dose of 8 X 10(7) killed cells of the phase III Sakairi strain, promoted detectable protection to ic challenge. Native Ptx in doses of 0.4 to 400 ng did not protect mice, and vaccines made from strains not producing Ptx induced only weak protection. The marked enhancing action of Ptx was also observed with 5 micrograms of purified filamentous hemagglutinin and with vaccines made from other species of the Bordetella genus, such as B. parapertussis and B. bronchiseptica, but it was not observed with B. pertussis endotoxin. In addition, Ptx was still effective when given as late as 7 days after the vaccine. Antibodies to surface antigens of the challenge strain were demonstrated in sera of mice immunized with vaccines prepared with the different Bordetella species tested, but antibodies to Ptx were detected only in the sera of mice immunized with the wild-type B. pertussis strains. Glutaraldehyde detoxified Ptx does not have this action. Pretreatment of normal mice with Ptx, also enhanced the protective action of a mouse antiserum to a wild-type strain of B. pertussis. These observations show that antigens other than Ptx are responsible for the protection, and that Ptx acts non-specifically to enhance the mouse protective action of those antigens.     

Isolation of specific receptors from Xanthomonas albilineans cell walls for lectins-like glycoproteins of sugarcane

Abstract

Two families of sugarcane glycoproteins differing in their molecular mass have been isolated from sugarcane stalks. These glycoproteins specifically bind to cell wall receptors of Xanthomonas albilineans, a sugarcane pathogen, producing bacterial agglutination. Bound glycoproteins can be desorbed from bacterial cell walls by galactitol, a component of the glycosidic moiety of the sugarcane protein. This indicates that sugarcane glycoproteins bind through their glycosidic rest to the peptide moiety of the bacterial receptor. Several cell wall receptors have been isolated by affinity chromatography and separated by capillary electrophoresis.     

Immune responses to the pertussis toxin in Balb/c and C57B1/6 mice

Abstract Immunization of Balb/c and C57B1/6 mice with the pertussis toxin (Ptx), purified from the culture supernatant of Bordetella pertussis (the whooping cough bacillus) resulted in different immune reactions in these genetically different strains of mice. Antibody responses to Ptx were detected only in Balb/c, whereas both Balb/c and C57B1/6 produced anti-Ptx antibodies when immunized with detoxified Ptx. Also, delayed-type hypersensitivity reactions differ strongly according to the use of Ptx or detoxified Ptx as eliciting antigen.     

Assessment of glycosylation-site heterogeneity using plasma desorption mass spectrometry

Plasma desorption mass spectrometry (PD-MS) was used to assess the molecular weight heterogeneity of glycopeptides (6-12 amino acids) from each of the three N-linked glycosylation sites of bovine fetuin (R.G. Spiro (1962) J. Biol. Chem. 237, 382-388). The glycopeptides were purified by a combination of anion exchange chromatography and reverse-phase HPLC. Since no detectable fragmentation was observed in the PD-MS of these asialoglycopeptides, the observation of multiple molecular ions could be attributed to either carbohydrate or peptide heterogeneity. Assignment of molecular ions, within 3 to 5 amu of the theoretical mass, of glycopeptides from each glycosylation site was made from amino acid composition, peptide sequence around the glycosylation sites, and previously reported triantennary oligosaccharide structures (B. Nilsson, N.E. Nordén, and S. Svensson (1979) J. Biol. Chem. 254, 4545-4553). Ion groups differing in mass by one N-acetyllactosamine unit were observed in glycopeptides from the Asn-Asp and Asn-Cys sites, localizing these previously observed biantennary oligosaccharide structures (R.R. Townsend, M.R. Hardy, T.C. Wong, and Y.C. Lee (1986) Biochemistry 25, 5716-5725; S. Takasaki and A. Kobata (1986) Biochemistry 25, 5709-5715) to these two sites. The presence of biantennary oligosaccharides at the Asn-Asp sites could be substantiated using 1H NMR but were not detected in the Asn-Cys glycopeptides. PD-MS was also implemented in the purification protocol for these glycopeptides and proved to be useful in assessing purity of chromatographic fractions which were mixtures of glycopeptides displaying both carbohydrate and peptide heterogeneity. A preparation scheme was developed to obtain molecular ions of desialylated glycopeptides by PD-MS.     

Differences between the carbohydrate units of cell-surface glycoproteins of moust B- and T-lymphocytes.

Carbohydrate units of cell-surface glycoproteins of mouse B- and T-lymphocytes, labelled in their sialic acid residues by the periodate/NaB3H4 method and in their galactose residues by the galactose oxidase/NaB3H4 method after neuraminidase treatment, have been studied. Glycopeptides were prepared from the labelled cells by Pronase digestion and fractionated by concanavalin A affinity chromatography into two fractions (A and B). Alkali-labile oligosaccharides were isolated after mild NaOH/NaBH4 treatment by gel filtration. The alkali-labile oligosaccharides were further analysed by t.l.c. To study the relative proportion of neutral mannose-rich carbohydrate units (fraction C) in lymphocyte glycoproteins, glycopeptides were also prepared from unlabelled cells and subjected to concanavalin A affinity chromatography after N-[3H]acetylation of their peptide moiety. The major alkali-labile oligosaccharide component of both cell types was identified as galactosyl-(beta 1 leads to 3)-N-acetylgalactosaminitol. T-Lymphocytes were characterized by a high proportion of this oligosaccharide and a lower proportion of alkali-stable fraction A glycopeptides, whereas the opposite was observed for B-lymphocytes. The relative proportions of the concanavalin A-binding fractions B and C were similar in both cell types. The differences observed may correlate with the different surface properties of B- and T-lymphocytes.     

Photoaffinity labeling of the beta-adrenergic receptor

A new photoactive beta-adrenergic antagonist, p-azidobenzylcarazolol (pABC) has been synthesized by combining a carbazole moiety with a p-azido-benzyl substituent. The compound has been labeled with tritium to a specific activity of 26 Ci/mmol. In frog erythrocyte membranes, [3H]p-azido-benzylcarazolol binds to the beta-adrenergic receptor with the expected beta 2 specificity and with high affinity (KD congruent to 100 +/- 10 pM). Unlabeled p-azido-benzylcarazolol can irreversibly inactivate the [3H]dihydroalprenolol-binding activity of frog erythrocyte membranes in a photodependent manner which can be prevented by beta-adrenergic agents. Incubation of frog erythrocyte membranes or digitonin-solubilized preparations of these membranes or digitonin-solubilized preparations of these membranes which had been enriched in beta-adrenergic receptors by a Sepharose-alprenolol chromatography step led to covalent incorporation of radioactivity into a Mr = 58,000 peptide. Specific incorporation of [3H]pABC into the Mr = 58,000 peptide could be prevented by both beta-adrenergic agonists and antagonists. This peptide has previously been purified and shown to contain the beta-adrenergic receptor-binding site (Shorr, R. G. L., Lefkowitz, R. J., and Caron, M. G. (1981) J. Biol. Chem. 256, 5820-5826). Thus, photoaffinity labeling of the beta-adrenergic receptor protein directly identifies the same hormone-binding subunit as has been isolated by conventional purification techniques.     

Identification of three oligosaccharide binding sites in ricin.

The galactoside-binding sites of ricin B chain can be blocked by affinity-directed chemical modification using a reactive ligand derived from asialoglycopeptides containing triantennary N-linked oligosaccharides. The terminal galactosyl residue of one branch of the triantennary oligosaccharide is modified to contain a reactive dichlorotriazine moiety. Two separate galactoside-binding sites have been clearly established in the ricin B chain by X-ray crystallography [Rutenber, E., and Robertus, J. D. (1991) Proteins 10, 260-269], and it is necessary to covalently attach two such reactive ligands to the B chain to block its binding to galactoside affinity matrixes. A method was developed using thiol-specific labeling of the ligand combined with subsequent immunoaffinity chromatography which allowed the isolation of ricin B chain peptides covalently linked to the ligand from proteolytic digests of purified blocked ricin. The sites of covalent attachment of the two ligands in blocked ricin were inferred from sequence analysis to be Lys 62 in domain 1 of the B chain and Tyr 148 in domain 2. A minor species of blocked ricin contains a third covalently attached ligand. From the analysis of peptides derived from blocked ricin enriched in this species, it is inferred that Tyr 67 in domain 1 is the specific site on the ricin B chain where a third reactive ligand becomes covalently linked to the protein. These results are interpreted as providing support for the notion that the ricin B chain has three oligosaccharide binding sites.     

The inhibitory guanine nucleotide-binding protein (Ni) purified from bovine brain is a high affinity GTPase

Using modifications of the methods of Bokoch et al. (Bokoch, G.M., Katada, T., Northup, J. K., Ui, M., and Gilman, A. G. (1984) J. Biol. Chem. 259, 3560-3567) and Codina et al. (Codina, J., Hildebrandt, J. D., Sekura, R. D., Birnbaumer, M., Bryan, J., Manclark, C. R., Iyengar, R., and Birnbaumer, L. (1984) J. Biol. Chem. 259, 5871-5886), we have purified a pertussis toxin substrate with the expected characteristics of the inhibitory guanine nucleotide-binding protein (Ni) essentially to homogeneity. The purified protein consists of 3 subunits of Mr 40,000, 35,000, and less than 10,000. The Mr 40,000 band is found, upon close examination, to consist of a poorly resolved doublet. Starting with the membranes from 1,320 g of bovine forebrain we purified the protein some 100-fold with approximately 20% yield to obtain 13 mg of a greater than 95% pure protein. Chromatography on octyl-Sepharose provided efficient separation of Ni from Ns (the stimulatory guanine nucleotide-binding protein). Analytical ultracentrifugation indicates an Mr of 82,000 and a sedimentation coefficient S20,w of 5.1. The protein is able to restore opiate-mediated inhibition of adenylate cyclase to membranes prepared from NG 108-15 cells which had been treated with pertussis toxin. Bovine brain Ni has the enzymatic properties of a low Km GTPase with a turnover number of 0.3 and affinities for nucleotides in the order GppNHp greater than or equal to GTP greater than or equal to GDP much greater than ATP, CTP, UTP, and GMP. Na+ specifically stimulates the GTPase and low concentrations of Mg2+ (less than 50 microM) are inhibitory. Some Mg2+ is apparently necessary because EDTA, but not EGTA, abolishes the GTPase activity.     

Single-step purification of pertussis toxin and its subunits by heat-treated fetuin-sepharose affinity chromatography

A general procedure for purifying biologically active pertussis toxin from Bordetella pertussis fermentation broth using affinity chromatography on heat-treated fetuin-Sepharose CL-4B is described. Diethanolamine is used as eluent in this single-step purification to prepare endotoxin-free pertussis toxin in good yield (70%) and high purity (greater than 95%). This one-step affinity chromatography procedure can be easily applied for large-scale preparation of pertussis toxin S1 subunit and its B-component. The affinity-purified S1 subunit is devoid of any of the histamine-sensitizing activity normally associated with pertussis toxin. The chromatographically purified pertussis toxin and its subunits retained their immunogenicity and could induce high levels of anti-toxin neutralizing antibodies.     

The amine transporter from bovine chromaffin granules. Partial purification

We have partially purified the amine transporter from bovine adrenal chromaffin granules in a single step utilizing affinity chromatography. A 5-hydroxytryptamine moiety has been coupled to a Sepharose 4B matrix in a position ortho to the hydroxyl group. When membranes solubilized with sodium cholate are chromatographed on the above matrix a 45,000 Mr polypeptide is highly enriched. The enrichment is dependent on the presence of the proper ligand on the matrix and is inhibited if the column is previously equilibrated with a soluble ligand. Enrichment of the above polypeptide is accompanied by an increase in the specific activity of the transporter as measured by its labeling by 4-azido-3-nitrophenylazo(5-hydroxytryptamine). The ability of reserpine, a competitive inhibitor of binding and transport, to inhibit labeling of the purified transporter correlates well with its known kinetic constants in the native membranes. The polypeptide purified is identical to the one previously identified as the putative transporter based on specific labeling by a photoaffinity label (Gabizon, R., Yetinzon, T., and Schuldiner, S. (1982) J. Biol. Chem. 257, 15145-15150). The results clearly support the contention that the 45,000 Mr peptide is the amine transporter or one of its subunits.     

Type analysis of the oligosaccharide chains on microheterogeneous components of bovine pancreatic DNAase by the lectin-nitrocellulose sheet method.

The oligosaccharide chains of microheterogeneous bovine pancreatic DNAases were characterized by the lectin-nitrocellulose sheet method. The active fractions of the DNAases from column chromatography showed four major and several minor spots on a two-dimensional polyacrylamide gel. They were transferred on to nitrocellulose sheets and treated with glycosidases (neuraminidase, endo-beta-N-acetyl glucosaminidase H or F, or peptide N-glycosidase F) and treated with peroxidase-coupled lectins (concanavalin A, Ricinus communis agglutinin or wheat-germ agglutinin). From the results, the most probable oligosaccharide types were proposed to be as follows: the four major spots contained components which had high-mannose type or hybrid-type oligosaccharides, such as those susceptible to endo-beta-N-acetylglucosaminidase H. In addition, spot 1 contained a complex-type biantennary oligosaccharide without sialic acid and spot 3 contained a tri- or tetra-antennary complex-type oligosaccharide with sialic acid. The component corresponding to spot 2 had a hybrid-type oligosaccharide chain with a 'bisecting' acetylglucosamine, linked 1-4 to the beta-mannose residue of the trimannosyl core, and the component corresponding to spot 4 had a high-mannose-type oligosaccharide chain.     

Preparation and some properties of the haemoglobin-binding protein of bovine blood

Bovine haptoglobin was prepared from sera which showed peroxidase activity in the complex with haemoglobin. The procedure consisted of precipitation with ammonium sulphate at 0.5 saturation, affinity chromatography on Sepharose 4B coupled to bovine apoglobin, and gel filtration on Sepharose 4B. The preparation was heterogeneous, migrating in 7.5% polyacrylamide gel electrophoresis at pH 8.3 as four haemoglobin-binding bands. On immunoelectrophoresis with bovine antiserum the preparation formed a single precipitin arc with the mobility of alpha 2-globulin; the preparation was found to be a glycoprotein, the sugar moiety of which amounted to 16%.     

Studies on the chemical nature of the lipidic J blood-group substance of cattle

Total lipids extracted from J-positive cattle serum, erythrocytes or spleen exhibit J blood-group activity. The J subsance is concentrated in a lipid fraction obtained by column chromatography. Following mild alkaline hydrolysis or reduction with complex hydrides (LiAlH4, LiBH4), the J activity remains detectable in this lipid fraction even though all acyl ester groups have been destroyed as revealed by ester group determination. This disagrees with the suggestion that fatty acyl esters are essential for J activity. This was confirmed by experiments with a water-soluble J-active product prepared by ozone treatment of glycosphingolipids from bovine spleen. The results of these experiments are in favour of a glycosphingolipid containing anunusually lang oligosaccharide chain. Furthermore, it appears that the terminal moiety of the J determinant is not necessarily an N-acetyl galactosamine unit as suggested previously.     

Association of two pertussis toxin-sensitive G-proteins with the D2-dopamine receptor from bovine striatum.

The solubilized D2-dopamine receptor from bovine striatum exhibits high and low affinity states for dopaminergic agonists. Guanine nucleotides and pertussis toxin convert the solubilized receptor from a high affinity state to a low one. A D2-receptor preparation partially purified by affinity chromatography on a haloperidol adsorbent, exhibited agonist-stimulated GTPase activity. [32P]ADP-ribosylation by pertussis toxin of this receptor preparation resulted in the specific labeling of two protein bands corresponding to mol. wts of 39 and 41 kd, in SDS-PAGE. Association of these G-proteins with the receptor was specifically inhibited by Gpp(NH)p. Immunoblot analysis of these G-proteins indicated that the 41- and 39-kd protein bands are analogous to brain Gi and Go respectively. These experiments demonstrate that two distinct pertussis toxin-sensitive G-proteins are functionally associated with bovine striatum D2-dopamine receptor.     

Novel oligosaccharide constituents of the cellulase complex of Bacteroides cellulosolvens.

The multiple cellulase-containing protein complex, isolated from the cellulolytic bacterium Bacteroides cellulosolvens, contains oligosaccharides which are O-linked mainly to a 230-kDa subunit. The oligosaccharide chains were liberated by alkaline-borohydride treatment and fractionated as oligosaccharide alditols via gel-permeation chromatography and HPLC. The fractions were investigated by one- and two-dimensional (correlation, homonuclear Hartmann-Hahn, rotating-frame nuclear Overhauser enhancement) 500-MHz 1H-NMR spectroscopy in combination with monosaccharide and methylation analyses and with fast-atom-bombardment mass spectrometry. The following carbohydrate structures could be established: [formula: see text] The results indicate an interesting similarity between the oligosaccharide moieties of the cellulase complex of B. cellulosolvens and of Clostridium thermocellum [Gerwig, G. J., Kamerling, J. P., Vliegenthart, J. F. G., Morag (Morgenstern), E., Lamed, R. & Bayer, E. A. (1991) Eur. J. Biochem. 196, 115-122], having 3, 5 and 6 as common elements. The furanose form of a terminal alpha-D-galactose residue demonstrated an inhibitory effect on the interaction of Griffonia simplicifolia I isolectin B4 with the cellulosome-like entity of B. cellulosolvens.     

Effects of antibodies to the filamentous haemagglutinin and to the pertussis toxin of Bordetella pertussis on adherence and toxic effects to 3T3 cells

Abstract Adherence of B. pertussis to NIH3T3 mouse fibroblasts was efficiently inhibited by a mouse immune serum reacting specifically with the filamentous haemagglutinin (FHA), whereas a mouse immune serum reacting specifically with the pertussis toxin (Ptx) produced partial inhibition only significant after 3 h infection. Protection against cytopathic effects on infected 3T3 cells with anti-FHA antibodies was at least as effective (83.3%± 7.5) as with anti-Ptx antibodies (75%± 4). This suggests that adherence of B. pertussis to eukaryotic receptors is a primary mechanism determining both bacterial proliferation and toxic effects in susceptible cells, and that prevention of B. pertussis attachment to cell receptors might be sufficient to protect against both infectious and toxic processes in whooping cough.     

Studies on the chemical nature of the lipidic J blood-group substance of cattle

Total lipids extracted from J-positive cattle serum, erythrocytes or spleen exhibit J blood-group activity. The J subsance is concentrated in a lipid fraction obtained by column chromatography. Following mild alkaline hydrolysis or reduction with complex hydrides (LiAlH₄, LiBH₄), the J activity remains detectable in this lipid fraction even though all acyl ester groups have been destroyed as revealed by ester group determination. This disagrees with the suggestion that fatty acyl esters are essential for J activity. This was confirmed by experiments with a water-soluble J-active product prepared by ozone treatment of glycosphingolipids from bovine spleen. The results of these experiments are in favour of a glycosphingolipid containing an unusually lang oligosaccharide chain. Furthermore, it appears that the terminal moiety of the J determinant is not necessarily an N-acetyl galactos-amine unit as suggested previously.