Structure-function relationship of islet-activating protein, pertussis toxin: biological activities of hybrid toxins reconstituted from native and methylated subunits

Islet-activating protein (IAP), pertussis toxin, is a hexameric protein composed of an A protomer and a B oligomer, the residual pentamer having such a subunit assembly that two different dimers, dimer 1 and dimer 2, are connected with each other by means of the smallest C subunit. Incubation of IAP with formaldehyde and pyridine-borane produced the modified toxin in which most of the free amino groups were dimethylated. The methylated and nonmethylated (native) IAP were disintegrated into their respective constituent components, which were then cross combined to reconstitute hybrid toxins with the original hexameric structure. The binding of the B oligomer to the mammalian cell surface via dimer 2 was, but the binding via dimer 1 was not, seriously impaired by methylation of amino groups in the protein. The binding of the B oligomer allowed the A protomer to enter cells and to catalyze ADP-ribosylation of a membrane Mr 41 000 protein. The diverse biological activities of IAP occurring by this mechanism were mimicked by not only methylated IAP but also all hybrid toxins, indicating that the free amino groups in the protein were not essential for the enzyme activity of the A protomer and that the A protomer was able to enter cells if the B oligomer bound to cells "monovalently" via dimer 1. An additional effect of the B oligomer binding, i.e., the direct stimulation, without the transport of the A protomer, of cells leading to mitosis in lymphocytes in vitro or increases in circulating lymphocytes in vivo, was not mimicked by hybrid toxins containing methylated dimer 2.(ABSTRACT TRUNCATED AT 250 WORDS)     

The subunit S1 is important for pertussis toxin secretion

Pertussis toxin is a protein containing five noncovalently linked subunits which are assembled into the monomer A (containing the subunit S1) and the oligomer B (containing subunits S2, S3, S4, and S5 in a 1:1:2:1 ratio). Each of the five subunits is synthesized as a precursor containing a secretory leader peptide and is secreted into the periplasm of Bordetella pertussis where the five subunits are assembled into the oligomeric structure and then released into the culture medium. In the absence of subunit S3 the remaining subunits are not secreted into the medium, thus suggesting that the assembled structure is necessary for the release of the toxin into the supernatant. In this study we describe four B. pertussis mutants which secrete into the medium low amounts of the B oligomer of pertussis toxin. These mutants have single or multiple changes in the gene encoding the S1 subunit and synthesize S1 proteins with altered conformation which are not assembled into the holotoxin and are apparently degraded in the periplasm. These data indicate that while the B oligomer alone has the structural information necessary for the extracellular export of pertussis toxin, the S1 subunit is required for its efficient release into the medium.     

The ins and outs of pertussis toxin

Pertussis toxin, produced and secreted by the whooping cough agent Bordetella pertussis, is one of the most complex soluble bacterial proteins. It is actively secreted through the B. pertussis cell envelope by the Ptl secretion system, a member of the widespread type IV secretion systems. The toxin is composed of five subunits (named S1 to S5 according to their decreasing molecular weights) arranged in an A-B structure. The A protomer is composed of the enzymatically active S1 subunit, which catalyzes ADP-ribosylation of the α subunit of trimeric G proteins, thereby disturbing the metabolic functions of the target cells, leading to a variety of biological activities. The B oligomer is composed of 1S2:1S3:2S4:1S5 and is responsible for binding of the toxin to the target cell receptors and for intracellular trafficking via receptor-mediated endocytosis and retrograde transport. The toxin is one of the most important virulence factors of B. pertussis and is a component of all current vaccines against whooping cough.     

Structure-activity analysis of the activation of pertussis toxin

Bordetella pertussis, the causative agent of whooping cough, releases pertussis toxin in an inactive form. The toxin consists of an A protomer containing one S1 peptide subunit and a B oligomer containing several other peptide subunits. The toxin binds to cells via the B oligomer, and the S1 subunit is activated and expresses ADP-ribosyltransferase and NAD glycohydrolase activities. Treatment of purified toxin with dithiothreitol (DTT) in vitro increases both activities. ATP and the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) synergistically reduce the A0.5 (activation constant) for DTT from greater than 100 mM to 200 microM. We studied the structure-activity relationships of activators of the toxin. In the presence of CHAPS (1%) and DTT (10 mM) the following compounds increased the NAD glycohydrolase activity of the toxin with the following A0.5's in microM and fraction of the ATP effect in parentheses: ATP, 0.2 (1.0); ADP, 6 (0.8); UTP, 15 (0.7); GTP, 35 (0.6); pyrophosphate, 45 (0.7); triphosphate, 60 (0.6); tetraphosphate, greater than or equal to 170 (greater than or equal to 0.4). Thus, the polyphosphate moiety is sufficient to stimulate the toxin, and the adenosine moiety confers upon ATP its extraordinary affinity for the toxin. Phospholipid and detergents could substitute for CHAPS in the activation of the toxin. Glutathione substituted for DTT with an A0.5 of 2 mM, a concentration within the range found in eucaryotic cells. Thus, membrane lipids and cellular concentrations of glutathione and ATP are sufficient to activate pertussis toxin without the need for a eucaryotic enzymatic process.     

The A protomer of islet-activating protein,pertussis toxin,as an active peptide catalyzing ADP-ribosylation of a membrane protein

Islet-activating protein (IAP), pertussis toxin, is an oligomeric protein composed of an A protomer and a B oligomer. IAP and its A protomer were equipotent, on a molar basis, in enhancing GTP-dependent adenylate cyclase activity and in causing ADP-ribosylation of the 41,000 M_r protein when directly added to the cell-free membrane preparation from rat C6 glioma cells. Similar actions of IAP observed upon its addition to intact C6 cells were not mimicked by its A protomer, indicating that the A protomer had to be associated with the B oligomer to become accessible to its site of action on the inner surface of the membrane of intact cells. The A protomer, but not IAP, exhibited NAD-glycohydrolase activity in the reaction mixture lacking cellular components but containing dithiothreitol. Their actions on membranes were not accelerated by dithiothreitol, but markedly suppressed by oxidized glutathione. Thus, C6 cell membranes may possess certain “processing” enzyme(s) responsible for releasing the A protomer from the IAP molecule and for reductive cleavage of an intrachain disulfide bond in the released protomer, thereby producing an active peptide which functions to cause ADP-ribosylation of one of the subunits of guanine nucleotide regulatory protein in the receptor-adenylate cyclase system.     

Expression, activity and cytotoxicity of pertussis toxin S1 subunit in transfected mammalian cells

Pertussis toxin (PT) comprises an active subunit (S1), which ADP-ribosylates the alpha subunit of several mammalian G proteins, and the B oligomer (S2–S5), which binds glycoconjugate receptors on cells. In a previous report, expression of S1 in Cos cells resulted in no observable cytotoxicity, and it was hypothesized that either S1 failed to locate its target proteins or the B oligomer was also necessary for cytotoxicity. To address this, we stably transfected S1 with and without a signal peptide into mammalian cells. Immunofluorescence analysis confirmed the function of the signal peptide. Surprisingly, we found that S1 was active in both transfectants, as determined by clustering of transfected Chinese hamster ovary (CHO) cells and ADP-ribosylation of G proteins. Constructs with a cysteine-to-serine change at residue 201 or a truncated S1 (residues 1–181) were also active when transfected into cells. Constructs with an inactive mutant S1 had no activity, confirming that the observed results were due to the activity of the toxin subunit. We conclude that S1 is active when expressed in mammalian cells without the B oligomer, that secretion into the endoplasmic reticulum does not prevent this activity and that the C-terminal portion of S1 is not required for its activity in cells.     

In vitro induction of antigen specific antibody synthesis and proliferation of T lymphocytes with acellular pertussis vaccines, pertussis toxin and filamentous haemagglutinin in humans

The in vitro response of human B- and T-lymphocytes to the acellular vaccines JNIH-6 (containing pertussis toxoid and filamentous hemagglutinin), and JNIH-7 (containing pertussis toxoid), and to the purified components JNIH-4 (filamentous hemagglutinin) and JNIH-5 (pertussis toxin) was investigated. Pertussis toxoid and filamentous hemagglutinin induced specific Ig synthesis in vitro in lymphocytes obtained from convalescent pertussis patients as target cells. The antigen-dependent Ig production was demonstrated in lymphocyte culture supernatants by ELISA techniques and by a chinese hamster ovary cell toxin neutralization assay. Particularly with JNIH-4, -6 and -7, high antibody titers were obtained. At optimal antigen concentrations a marked lymphocyte blast transformation was found in lymphocyte cultures from whooping cough patients, but not in cultures of lymphocytes obtained from healthy volunteers. At high concentrations native pertussis toxin as well as the B oligomer (S2-5) of the toxin induced a strong proliferation of patient as well as control lymphocytes, indicating non-specific mitogenic activity. At lower concentrations lymphocyte blast transformation was seen in patient cultures only, which indicates an antigen-specific T-cell response. The A protomer (S1), dimer 1 (S2 + 4) and dimer 2 (S3 + 4) induced proliferation of patient lymphocytes, which demonstrates the presence of T-cell epitopes on these peptides. The in vitro B-cell response and the lymphocyte blast transformation assay are both useful tools for estimating the potency of acellular pertussis vaccines in man. Spontaneously acquired and vaccine induced immunity to Bordetella pertussis can be investigated at the level of B- and T-lymphocytes.     

Adenine nucleotides promote dissociation of pertussis toxin subunits

Pertussis toxin is composed of an enzymatically active A subunit and a binding component (B oligomer). Both the holotoxin and the isolated A subunit have previously been shown to exhibit NAD glycohydrolase activity although the A subunit is more active on a molar basis than the holotoxin. We have investigated the mechanism by which ATP stimulates the activity of this toxin. Since dissociation of pertussis toxin subunits would result in increased NAD glycohydrolase activity, the ability of ATP to promote release of the A subunit from the B oligomer was examined. In the presence of the zwitterionic detergent 3-(3-cholamidopropyldimethyl)-1-ammonio)-propanesulfonate, concentrations of ATP as low as 1 microM promoted subunit dissociation. The concentration of ATP required for release of the A subunit was similar to that required for stimulation of NAD glycohydrolase activity. Both ATP and ADP promoted subunit dissociation and stimulated NAD glycohydrolase activity. In contrast, AMP and adenosine did not alter NAD glycohydrolase activity or affect subunit structure. The ability of ATP to decrease the affinity of the A subunit for the B oligomer may play a role in nucleotide stimulation of pertussis toxin activity.     

Binding of ATP by pertussis toxin and isolated toxin subunits.

The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of [3H]ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP greater than ATP greater than GTP greater than CTP greater than TTP for pertussis toxin and ATP greater than GTP greater than TTP greater than CTP for the B oligomer. Phosphate ions inhibited the binding of [3H]ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of [3H]ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site.     

Structure-function studies of cholera toxin and its A and B protomers. Modification of tryptophan residues

The tryptophan residues on cholera toxin and its A and B protomers have been modified by reaction with 2-nitrophenylsulfenyl chloride and 2,4-dinitrophenylsulfenyl chloride. Modification of the tryptophan residues of cholera toxin results in complete loss of toxicity measured in a skin permeability assay. Modification of cholera toxin and its B protomer results in the complete loss of binding activity toward membrane receptors, the ganglioside galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylceramide (GM1), and the oligosaccharide moiety of the ganglioside GM1. Modification of cholera toxin and its A protomer results in a complete loss of the ADP-ribosylation activity exhibited by their native counterparts. Modification of the A protomer results in no apparent change in its physical properties by sedimentation velocity in the ultracentrifuge or by gel filtration chromatography. Modification of the B protomer, either directly or when it remains a component part of the holo toxin structure, results in a change in its sedimentation value and its elution from gel filtration columns. The changes are compatible with a conversion of the B protomer from a pentameric moiety in aqueous solvents to its existence as a monomer unit, i.e. to the individual polypeptide chains comprising the native B pentamer. Thiolysis of the 2,4-dinitrophenylsulfenyl chloride derivative of the B protomer reaggregates the individual-polypeptide chains but does not return its ability to interact with GM1.     

Protease treatment of pertussis toxin identifies the preferential cleavage of the S1 subunit.

Trypsin digestion of pertussis toxin (PT) preferentially cleaved the S1 subunit at Arg-218 without detectable degradation of the B oligomer. The fragment produced, termed the tryptic S1 fragment, appears to remain associated with the B oligomer. Chymotrypsin digestion of PT also preferentially cleaved the S1 subunit without detectable degradation of the B oligomer. The chymotryptic S1 fragment possessed a slightly lower apparent molecular weight than the tryptic S1 fragment and was more accessible to the respective protease. Trypsin- and chymotrypsin-treated PT and PT required the presence of dithiothreitol and ATP for optimal enzymatic activity. Trypsin-treated PT showed approximately a 2-4-fold higher level of expression of ADP-ribosyltransferase and NAD-glycohydrolase activities than PT. Chymotrypsin-treated PT also exhibited approximately a 2-fold greater level of ADP-ribosyltransferase activity than PT. The observed increase in activity of protease-treated PT was due primarily to a shorter time for activation in PT mediated ADP-ribosylation of transducin. In addition, trypsin-digested PT possessed the same cytotoxic potential for Chinese hamster ovary cell clustering as PT. One possible role for the generation of a proteolytic fragment of the S1 subunit of PT would be to produce a catalytic fragment with increased efficiency for ADP-ribosylation of G proteins in vivo.     

Proteolytic cleavage of pertussis toxin S1 subunit is not essential for its activity in mammalian cells

Background  

Pertussis toxin (PT) is an exotoxin virulence factor produced by Bordetella pertussis, the causative agent of whooping cough. PT consists of an active subunit (S1) that ADP-ribosylates the alpha subunit of several mammalian G proteins, and a B oligomer (S2–S5) that binds glycoconjugate receptors on cells. PT appears to enter cells by endocytosis, and retrograde transport through the Golgi apparatus may be important for its cytotoxicity. A previous study demonstrated that proteolytic processing of S1 occurs after PT enters mammalian cells. We sought to determine whether this proteolytic processing of S1 is necessary for PT cytotoxicity.     

Crystal structure of the Vibrio cholerae cytolysin (VCC) pro-toxin and its assembly into a heptameric transmembrane pore

Pathogenic Vibrio cholerae secrete V. cholerae cytolysin (VCC), an 80 kDa pro-toxin that assembles into an oligomeric pore on target cell membranes following proteolytic cleavage and interaction with cell surface receptors. To gain insight into the activation and targeting activities of VCC, we solved the crystal structure of the pro-toxin at 2.3A by X-ray diffraction. The core cytolytic domain of VCC shares a fold similar to the staphylococcal pore-forming toxins, but in VCC an amino-terminal pro-domain and two carboxy-terminal lectin domains decorate the cytolytic domain. The pro-domain masks a protomer surface that likely participates in inter-protomer interactions in the cytolytic oligomer, thereby explaining why proteolytic cleavage and movement of the pro-domain is necessary for toxin activation. A single beta-octyl glucoside molecule outlines a possible receptor binding site on one lectin domain, and removal of this domain leads to a tenfold decrease in lytic activity toward rabbit erythrocytes. VCC activated by proteolytic cleavage assembles into an oligomeric species upon addition of soybean asolectin/cholesterol liposomes and this oligomer was purified in detergent micelles. Analytical ultracentrifugation and crystallographic analysis indicate that the resulting VCC oligomer is a heptamer. Taken together, these studies define the architecture of a pore forming toxin and associated lectin domains, confirm the stoichiometry of the assembled oligomer as heptameric, and suggest a common mechanism of assembly for staphylococcal and Vibrio cytolytic toxins.     

Interaction of pertussis toxin with cells and model membranes.

The interaction of pertussis toxin (PT) with cells and model membranes was investigated by examining PT-induced intoxication of Chinese hamster ovary cells and by studying the binding of PT and its subunits to phospholipid vesicles. Since certain bacterial toxins require an acidic environment for efficient interaction with membranes and subsequent entry into the cell, the requirement for an acidic environment for PT action was examined. PT, unlike bacterial toxins such as diphtheria toxin, did not require an acidic environment for efficient intoxication of Chinese hamster ovary cells. Potential modes by which PT might interact with biological membranes were studied by examining the binding of PT to a model membrane system. PT was found to be capable of interacting with phospholipid vesicles, however, efficient binding of the toxin to the vesicles occurred only in the presence of both ATP and reducing agent. The A subunit portion of the toxin bound preferentially to the vesicles while little binding of the B oligomer portion of PT to the model membranes was observed. Isolated A subunit, in the absence of the B oligomer, also bound to the vesicles with optimal binding occurring in the presence of reducing agent. After cleavage of the A subunit by trypsin, probably at Arg-181, Arg-182, and/or Arg-193, large fragments which lacked the C-terminal portion of the A subunit of PT no longer associated with the lipid vesicles. These results suggest that the A subunit of PT can interact directly with a lipid matrix and, if freed from the constraints imposed by the B oligomer, may be capable of interacting with cellular membranes.     

Preliminary X-ray crystallographic analysis of holotoxin from Bordetella pertussis

Pertussis (whooping cough) is a serious infectious disease caused by the bacterium Bordetella pertussis. One of the major virulence factors is a protein known as pertussis toxin, which is composed of six subunits, with a total molecular weight of 106,000. Enzymatic transfer of ADP-ribose from NAD to a family of GTP-binding proteins is effected by the largest subunit (S1 or the A monomer), while binding of host cells and entry of S1 to the interior is a function of the other subunits (the B oligomer). The holotoxin crystallizes in the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 98.4 A, b = 164.2 A and c = 195.2 A. The crystals are suitable for high-resolution X-ray diffraction analysis.     

Lectin-like binding of pertussis toxin to a 165-kilodalton Chinese hamster ovary cell glycoprotein

Chinese hamster ovary (CHO) cells cluster in the presence of pertussis toxin, a response that is correlated with the ADP-ribosylation of a Mr = 41,000 membrane protein by the toxin. A ricin-resistant line of CHO cells (CHO-15B) which specifically lacks the terminal NeuAc----Gal beta 4GlcNAc oligosaccharide sequence on glycoproteins did not cluster in response to pertussis toxin. These cells do contain the Mr = 41,000 protein substrate for the enzymatic activity of the toxin which suggests that pertussis toxin, like certain plant lectins, does not bind to or is not internalized by the CHO-15B cells. There was no evidence of pertussis toxin binding to gangliosides or neutral glycolipids isolated from CHO cells but the toxin bound to a Mr = 165,000 component in N-octyglucoside extracts of CHO cells that had been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted to nitrocellulose. Plant lectins from Ricinus communis and Erythina cristagalli detected a similar size band in CHO cells and also did not react with CHO-15B cells. Unlike pertussis toxin, these plant lectins recognized two other major bands in CHO cell extracts and reacted best after sialidase treatment of nitrocellulose transfers containing CHO cell extracts. Conversely, sialidase treatment abolished binding a pertussis toxin and wheat germ agglutinin, a plant lectin that reacts with multivalent sialic acid residues on glycoproteins, to the Mr = 165,000 band. Purified B oligomer of pertussis toxin also uniquely detected a Mr = 165,000 component in CHO cell extracts while the A subunit of pertussis toxin was unreactive. These results indicate that pertussis toxin binds to a CHO cell glycoprotein with N-linked oligosaccharides and that sialic acid contributes to the complementary receptor site for the toxin. In addition, they suggest that a glycoprotein may serve as a cell surface receptor for pertussis toxin and that this interaction is mediated by a lectin-like binding site located on the B oligomer.     

Thermal stability and intersubunit interactions of cholera toxin in solution and in association with its cell-surface receptor ganglioside GM1

The thermal stability of cholera toxin free in solution and in association with its cell-surface receptor ganglioside GM1 has been studied by using high-sensitivity differential scanning calorimetry and differential solubility thermal gel analysis. In the absence of ganglioside GM1, cholera toxin undergoes two distinct thermally induced transitions centered at 51 and 74 degrees C, respectively. The low-temperature transition has been assigned to the irreversible thermal denaturation of the active A subunit. The second transition has been assigned to the reversible unfolding of the B subunit pentamer. The isolated B subunit pentamer exhibits a single transition also centered at 74 degrees C, suggesting that the attachment of the A subunit does not contribute to the stability of the pentamer. In the intact toxin, the A subunit dissociates from the B subunit pentamer at a temperature that coincides with the onset of the B subunit thermal unfolding. In aqueous solution, the denatured A subunit precipitates after dissociation from the B subunit pentamer. This phenomenon can be detected calorimetrically by the appearance of an exothermic heat effect. In the presence of ganglioside GM1, the B subunit is greatly stabilized as indicated by an increase of 20 degrees C in the transition temperature. In addition, ganglioside GM1 greatly enhances the cooperative interactions between B subunits. In the absence of ganglioside, each monomer within the B pentamer unfolds in an independent fashion whereas the fully ganglioside-bound pentamer behaves as a single cooperative unit. On the contrary, the thermotropic behavior of the A subunit is only slightly affected by the presence of increasing concentrations of ganglioside GM1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzymic activity of cholera toxin. II. Relationships to proteolytic processing, disulfide bond reduction, and subunit composition.

Cholera toxin containing intact A chain (Mr = 29,000) was isolated, and its enzymic properties were characterized. The "unnicked" form of the toxin, produced by a protease-deficient, hypertoxinogenic mutant of Vibrio cholerae 569B, had greatly reduced activity in catalyzing the NAD+-glycohydrolase and ADP-ribosyltransferase reactions as compared to the naturally nicked form commonly isolated. In the latter, the intact A chain has been cleaved by bacterial proteases to yield disulfide-linked A1 and A2 chains (Mr = 23,000 and 6,000, respectively). Digestion of unnicked toxin with trypsin or elastase yielded a nicked form similar to or identical with the naturally nicked toxin, but chymotryptic digestion did not. Disulfide bond reduction was necessary for expression of enzymic activity by naturally nicked or trypsin-nicked toxin, or the A1A2 protomer. Fractionation of thiol-treated, nicked cholera toxin by ion exchange, molecular exclusion, or affinity chromatography gave results suggesting that the reduced toxin displays enzymic activity while remaining structurally intact.