A kinetic model of intermediate formation during assembly of cholera toxin B-subunit pentamers

Cholera toxin is the most important virulence factor produced by Vibrio cholerae. The pentameric B-subunit of the toxin can bind to GM1-ganglioside receptors, leading to toxin entry into mammalian cells. Here, the in vitro disassembly and reassembly of CtxB(5) (the B subunit pentamer of cholera toxin) is investigated. When CtxB(5) was acidified at pH 1.0 and then neutralized, the B-subunits disassembled and could no longer migrate as SDS-stable pentamers on polyacrylamide gels or be captured by GM1. However, continued incubation at neutral pH resulted in the B-subunits regaining the capacity to be detected by GM1 enzyme-linked immunosorbent assay (t(12) approximately 8 min) and to migrate as SDS-stable pentamers (t(12) approximately 15 min). Time-dependent changes in Trp fluorescence intensity during B-subunit reassembly occurred with a half-time of approximately 8 min, similar to that detected by GM1 enzyme-linked immunosorbent assay, suggesting that both methods monitor earlier events than B-pentamer formation alone. Based on the Trp fluorescence intensity measurements, a kinetic model of the pathway of CtxB(5) reassembly was generated that depended on trans to cis isomerization of Pro-93 to give an interface capable of subunit-subunit interaction. The model suggests formation of intermediates in the reaction, and these were successfully detected by glutaraldehyde cross-linking.     

Gas phase characterization of the noncovalent quaternary structure of cholera toxin and the cholera toxin B subunit pentamer

Cholera toxin (CTx) is an AB5 cytotonic protein that has medical relevance in cholera and as a novel mucosal adjuvant. Here, we report an analysis of the noncovalent homopentameric complex of CTx B chain (CTx B5) using electrospray ionization triple quadrupole mass spectrometry and tandem mass spectrometry and the analysis of the noncovalent hexameric holotoxin usingelectrospray ionization time-of-flight mass spectrometry over a range of pH values that correlate with those encountered by this toxin after cellular uptake. We show that noncovalent interactions within the toxin assemblies were maintained under both acidic and neutral conditions in the gas phase. However, unlike the related Escherichia coli Shiga-like toxin B5 pentamer (SLTx B), the CTx B5 pentamer was stable at low pH, indicating that additional interactions must be present within the latter. Structural comparison of the CTx B monomer interface reveals an additional alpha-helix that is absent in the SLTx B monomer. In silico energy calculations support interactions between this helix and the adjacent monomer. These data provide insight into the apparent stabilization of CTx B relative to SLTx B.     

Hamster female protein, a pentameric oligomer capable of reassociation and hybrid formation

Syrian hamster female protein (SFP), a serum oligomer composed of five identical subunits, was reassociated in vitro from monomer subunits. The reconstituted pentamer was genuine by morphologic, antigenic, and structural criteria. Another female protein (FP), a homologue from Armenian hamsters (AFP), also reassociated into a pentamer after dissociation with 5 M guanidine hydrochloride. These two FP's hybridized when a mixture of them was dissociated and then reassociated. Differences between the parent FP's were used to show that the recombinant pentamer contained monomer subunits from both SFP and AFP. Reassociation of both FP's was enhanced by increasing FP concentration and also by adding Ca2+ during reassembly. The two FP's differed in their reassociation profile in that SFP was especially efficient in reassembly, whereas AFP was more dependent upon Ca2+. Female protein is a homologue of C-reactive protein and amyloid P component, and all of these proteins (pentraxins) share a similar structure. The in vitro dissociation-reassociation of female protein described herein may reflect an in vivo dissociation-reassociation which is functionally important and a common metabolic feature within this family of proteins.     

霍乱毒素B亚单位基因（CtxB）的克隆及其表达

从霍乱弧菌中抽提基因组ＤＮＡ,用ＰＣＥＲ方法获取霍乱毒素Ｂ亚单位基因（ＣｔｘＢ）。序列分析结果表明,ＣｔｘＢ基因编码１２４个氨基酸,其中编码６２位Ｔｈｒ的密码子与文献报道有差异。将ＣｔｘＢ基因插入质粒ｐＧＥＸ－４Ｔ－２,构建ｐＧＥＸ－ＣＴＸＢ表达质粒,转化大肠相菌ＢＬ２１（ＤＥ３０,筛选表达菌株ＣＴＸＢ／ＢＬ２１。工程株经ＩＰＴＧ诱导表达,可产生大量的表达蛋白,经ＳＤＳ－ＰＡＧＥ分析,融合蛋白分子     

Identification of the Shiga toxin A-subunit residues required for holotoxin assembly.

Recent X-ray crystallographic analyses have demonstrated that the receptor-binding (B) subunits of Shiga toxin (STX) are arranged as a doughnut-shaped pentamer. The C terminus of the enzymatic (A) subunit presumably penetrates the nonpolar pore of the STX B pentamer, and the holotoxin is stabilized by noncovalent interactions between the polypeptides. We identified a stretch of nine nonpolar amino acids near the C terminus of StxA which were required for subunit association by using site-directed mutagenesis to introduce progressive C-terminal deletions in the polypeptide and assessing holotoxin formation by a receptor analog enzyme-linked immunosorbent assay, immunoprecipitation, and a cytotoxicity assay. Tryptophan and aspartic acid residues which form the N-terminal boundary, as well as two arginine residues which form the C-terminal boundary of the nine-amino-acid sequence, were implicated as the stabilizers of subunit association. Our model proposes that residues 279 to 287 of the 293-amino-acid STX A subunit penetrate the pore while the tryptophan, aspartic acid, and 2 arginine residues interact with other charged or aromatic amino acids outside the pore on the planar surfaces of the STX B pentamer.     

Sequence determination and comparison of the exfoliative toxin A and toxin B genes from Staphylococcus aureus.

The DNA encoding the exfoliative toxin A gene (eta) of Staphylococcus aureus was cloned into bacteriophage lambda gt11 and subsequently into plasmid pLI50 on a 1,391-base-pair DNA fragment of the chromosome. Exfoliative toxin A is expressed in the Escherichia coli genetic background, is similar in length to the toxin purified from culture medium, and is biologically active in an animal assay. The nucleotide sequence of the DNA fragment containing the gene was determined. The protein deduced from the nucleotide sequence is a polypeptide of 280 amino acids. The mature protein is 242 amino acids. The DNA sequence of the exfoliative toxin B gene was also determined. Corrections indicate that the amino acid sequence of exfoliative toxin B is in accord with chemical sequence data.     

Structural inferences for Cholera toxin mutations in Vibrio cholerae

Cholera is a global disease that has persisted for millennia. The cholera toxin (CT) from Vibrio cholerae is responsible for the clinical symptoms of cholera. This toxin is a hetero-hexamer (AB(5)) complex consisting of a subunit A (CTA) with a pentamer (B(5)) of subunit B (CTB). The importance of the AB(5) complex for pathogenesis is established for the wild type O1 serogroup using known structural and functional data. However, its role is not yet documented in other known serogroups harboring sequence level residue mutations. The sequences for the toxin from different serogroups are available in GenBank (release 177). Sequence analysis reveals mutations at several sequence positions in the toxin across serogroups. Therefore, it is of interest to locate the position of these mutations in the AB(5) structure to infer complex assembly for its functional role in different serogroups. We show that mutations in the CTA are at the solvent exposed regions of the AB(5) complex, whereas those in the CTB are at the CTB/CTB interface of the homo-pentamer complex. Thus, the role of mutations at the CTB/CTB interface for B(5) complex assembly is implied. It is observed that these mutations are often non-synonymous (e.g. polar to non-polar or vice versa). The formation of the AB(5) complex involves inter-subunit residue-residue interactions at the protein-protein interfaces. Hence, these mutations, at the structurally relevant positions, are of importance for the understanding of pathogenesis by several serogroups. This is also of significance in the improvement of recombinant CT protein complex analogs for vaccine design and their use against multiple serogroups.     

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)     

Identification, cloning and sequence analysis of the Bacillus sphaericus 1593 41.9 kD larvicidal toxin gene

A number of strains of the widespread aerobic soil bacterium, Bacillus sphaericus, possess crystalline inclusions of a toxin lethal to a variety of insect (larvae) which are vectors of major tropical diseases. Partial amino acid sequence data from one strain, B. sphaericus 2362 have permitted us to design oligonucleotide probes for identifying the toxin gene in the closely related B. sphaericus 1593. The gene was found to be contained within an EcoRI-HindIII fragment and was cloned in its entirety in the bacterial plasmid pUC12. The DNA sequence was determined together with the upstream and downstream controlling elements, and a sequence of 370 amino acids was deduced for the toxin protein. This is the first reported sequence of a B. sphaericus toxin gene and will facilitate further work in characterizing the genes from other strains of different virulence and host range. The data do not support the suggestion that the toxin is derived by proteolysis of a protoxin precursor.     

Evidence that a globular conformation is not compatible with FhaC-mediated secretion of the Bordetella pertussis filamentous haemagglutinin

The 220 kDa Bordetella pertussis filamentous haemagglutinin (FHA) is the major extracellular protein of this organism. It is exported using a signal peptide-dependent pathway, and its secretion depends on one specific outer membrane accessory protein, FhaC. In this work, we have investigated the influence of conformation on the FhaC-mediated secretion of FHA using an 80 kDa N-terminal FHA derivative, Fha44. In contrast to many signal peptide-dependent secretory proteins, no soluble periplasmic intermediate of Fha44 could be isolated. In addition, cell-associated Fha44 synthesized in the absence of FhaC did not remain competent for extracellular secretion upon delayed expression of FhaC, indicating that the translocation steps across the cytoplasmic and the outer membrane might be coupled. A chimeric protein, in which the globular B subunit of the cholera toxin, CtxB, was fused at the C-terminus of Fha44, was not secreted in B. pertussis or in Escherichia coli expressing FhaC. The hybrid protein was only secreted when both disulphide bond-forming cysteines of CtxB were replaced by serines or when it was produced in DsbA^?E. coli. The Fha44 portion of the secretion-incompetent hybrid protein was partly exposed on the cell surface. These results argue that the Fha44–CtxB hybrid protein transited through the periplasmic space, where disulphide bond formation is specifically catalysed, and that secretion across the outer membrane was initiated. The folded CtxB portion prevented extracellular release of the hybrid, in contrast to the more flexible CtxB domain devoid of cysteines. We propose a secretion model whereby Fha44 transits through the periplasmic space on its way to the cell surface and initiates its translocation through the outer membrane before being released from the cytoplasmic membrane. Coupling of Fha44 translocation across both membranes would delay the acquisition of its folded structure until the protein emerges from the outer membrane. Such a model would be consistent with the extensive intracellular proteolysis of FHA derivatives in B. pertussis.     

The disassembly and reassembly of mutants of Escherichia coli heat-labile enterotoxin: replacement of proline 93 does not abolish the reassembly-competent and reassembly-incompetent states

The carrier moiety of heat-labile enterotoxin of Escherichia coli (EtxB) is formed by the noncovalent association of identical monomeric subunits, which assemble, in vivo and in vitro, into exceptionally stable pentameric complexes. In vitro, acid disassembly followed by neutralization results in reassembly yields of between 20% and 60% depending on the identity of the salts present during the acid denaturation process. Loss of reassembly competence has been attributed to isomerization of the native cis-proline residue at position 93. To characterize this phenomenon further, two mutants of EtxB at proline 93 (P93G and P93A) were generated and purified. The proline variants reveal only minor differences in their biophysical and biochemical properties relative to wild-type protein, but major changes were observed in the kinetics of pentamer disassembly and reassembly. Additionally, a loss of assembly competence was observed following longer term acid treatment, which was even more marked than that of the wild-type protein. We present evidence that the loss of assembly competence of these mutants is best explained by a cis/trans peptidyl isomerization of the unfolded mutant subunits in acid conditions; this limited reassembly competence and the biophysical properties of the native P93 mutant pentamers imply the retention of the native cis conformation in the nonproline peptide bond between residues 92 and 93 in the mutated proteins.     

The refolding and reassembly of Escherichia coli heat-labile enterotoxin B-subunit: analysis of reassembly-competent and reassembly-incompetent unfolded states

The B-subunit pentamer of Escherichia coli heat-labile enterotoxin (EtxB) is an exceptionally stable protein maintaining its quaternary structure over the pH value range 2.0-11.0. Up to 80% yields of reassembled pentamer can be obtained in vitro from material disassembled for very short incubation periods in KCl-HCl, pH 1.0. However, when the incubation period in acid is extended, the reassembly yield decreases to no more than 20% (Ruddock et al. (1996) J. Biol. Chem. 271 19118-19123). Here we demonstrate that the ion species present in the disassembly conditions strongly influence the reassembly competence of EtxB showing that 60% reassembly yields can be achieved, even after prolonged incubations, by the use of a phosphate buffer for acid disassembly. Using this system, we have fully characterized the disassembly and reassembly behavior of EtxB by electrophoretic, immunochemical, and spectroscopic techniques and compared it with that previously observed. Depending on the denaturation system used, the acid-denatured monomer is either in a predominantly reassembly-competent state (H(3)PO(4) system) or in a predominantly reassembly-incompetent conformation (KCl-HCl system). Interconversion between these two conformations in the denatured state is possible by the addition of salts to the denatured protein. The results are consistent with the previous hypothesis that the conversion between reassembly-competent and -incompetent states corresponds to a cis/trans isomerization of a peptide bond, presumably that to Pro93.     

Integrin-mediated adhesion regulates membrane order

The properties of cholesterol-dependent domains (lipid rafts) in cell membranes have been controversial. Because integrin-mediated cell adhesion and caveolin both regulate trafficking of raft components, we investigated the effects of adhesion and caveolin on membrane order. The fluorescent probe Laurdan and two-photon microscopy revealed that focal adhesions are highly ordered; in fact, they are more ordered than caveolae or domains that stain with cholera toxin subunit B (CtxB). Membrane order at focal adhesion depends partly on phosphorylation of caveolin1 at Tyr14, which localizes to focal adhesions. Detachment of cells from the substratum triggers a rapid, caveolin-independent decrease in membrane order, followed by a slower, caveolin-dependent decrease that correlates with internalization of CtxB-stained domains. Endocytosed CtxB domains also become more fluid. Thus, membrane order is highly dependent on caveolae and focal adhesions. These results show that lipid raft properties are conferred by assembly of specific protein complexes. The ordered state within focal adhesions may have important consequences for signaling at these sites.     

Molecular cloning of pertussis toxin genes.

We have cloned a 4.5 kb EcoRI/BamHI DNA fragment from Bordetella pertussis which contains at least two genes responsible for expression of pertussis toxin. The S4 subunit of the toxin was isolated by high pressure liquid chromatography and the NH2-terminal amino acid sequence determined. Using a mixed synthetic oligonucleotide probe designed by reverse translation of a portion of the protein sequence, a cloned DNA fragment was identified which contains the coding information for at least the S4 structural subunit of the toxin. Sequence analyses indicate that the mature protein is derived by proteolytic cleavage of a precursor molecule. Southern blot analyses of Tn5-induced B. pertussis toxin-deficient mutants show that the Tn5 DNA is inserted 1.3 kb downstream from the S4 subunit gene. This second gene could code for another subunit required for assembly of the mature toxin or a non-structural transport protein, possibly in the same polycistronic operon. The molecular cloning of pertussis toxin genes provides the basis for development of a safer recombinant "new generation" vaccine for whooping cough.     

Ubiquitin fusion enhances cholera toxin B subunit expression in transgenic plants and the plant-expressed protein binds GM1 receptors more efficiently

Developing plant based systems for the production of therapeutic recombinant proteins requires the development of efficient expression strategies and characterization of proteins made in heterologous cellular environment. In this study, the expression of cholera toxin B subunit (CtxB) was examined in the leaves of transgenic tobacco plants. A synthetic gene encoding CtxB was designed for high level expression in plant cells and cloned as ubiquitin (Ub) fusion in a plant expression vector. Tobacco plants were genetically engineered by nuclear transformation to express the CtxB or Ub-CtxB fusion proteins under the control of CaMV35S duplicated enhancer promoter. Functionally active CtxB accumulated in tobacco leaves at 2.5-fold higher level in the Ub-CtxB plants. In the best expressors, CtxB accumulated at 0.9% of the total soluble leaf protein. In both the constructs, molecular mass of the plant-expressed CtxB was 14.6 kDa in contrast to 11.6 kDa for the authentic CtxB. Schiff's test, retention on concanavalin A column and chemical and enzymatic deglycosylation established that the higher molecular mass was due to glycosylation of the CtxB expressed in plant cells. The glycosylated CtxB made in tobacco leaves had higher affinity of binding to the GM1 receptors.     

Expression and site-specific mutagenesis of phospholamban. Studies of residues involved in phosphorylation and pentamer formation

Full-length cDNAs encoding either dog cardiac or rabbit skeletal muscle phospholamban were expressed transiently in COS-1 cells. The expressed protein displayed the mobility of a pentamer when dissolved in sodium dodecyl sulfate and separated in polyacrylamide gels, and of a monomer when boiled prior to polyacrylamide gel separation. Site-specific mutagenesis was used to analyze the roles of several amino acids in the structure and function of the protein. Ser16 and Thr17 were shown to be phosphorylated uniquely by cAMP- and calmodulin-dependent protein kinases, respectively, confirming earlier observations on the native protein (Simmerman, H. K. B., Collins, J. H., Theibert, J.L., Wegener, A.D., and Jones, L.R. (1986) J. Biol. Chem. 261, 13333-13341). Arg13 and Arg14 were shown to be essential for both types of phosphorylation, and Arg9 was shown to be essential for calmodulin-dependent phosphorylation. In studies of pentamer stability, mutation of Gln22-Gln23 to Ala-Ala or Glu-Glu, of Gln26-Asn27 to Glu-Asp, or of Gln29-Asn30 to Glu-Asp had no effect on thermal stability of the pentamer, suggesting that hydrogen bonding involving these residues in domain IB is not important for pentamer stability. By contrast, mutation of Cys36, Cys41, and Cys46 in transmembrane domain II to Ser, Ala, or Phe diminished the stability of the pentamer when microsomal proteins were dissociated in sodium dodecyl sulfate and separated by polyacrylamide gel electrophoresis. In particular, the Cys41 to Phe mutant existed as a monomer at ambient temperature. These results suggest that the intramembranous cysteine residues are important for pentamer formation even though they are not disulfide-bonded.     

Activation of Shiga toxin type 2d (Stx2d) by elastase involves cleavage of the C-terminal two amino acids of the A2 peptide in the context of the appropriate B pentamer

Shiga toxins (Stx) are potent ribosome-inactivating toxins that are produced by Shigella dysenteriae type 1 or certain strains of Escherichia coli. These toxins are composed of one A subunit that can be nicked and reduced to an enzymatically active A1(approximately 27 kDa) and an A2 peptide (approximately 4 kDa) as well as a pentamer of B subunits (approximately 7 kDa/monomer) that binds the eukaryotic cell. Purified Shiga toxin type 2d is activated 10- to 1000-fold for Vero cell toxicity by preincubation with mouse or human intestinal mucus or purified mouse elastase, whereas Stx2, Stx2c, Stx2e and Stx1 are not activatable. E. coli strains that produce the activatable Stx2d are more virulent in a streptomycin (str)-treated mouse model of infection [lethal dose 50% (LD50) = 101] than are E. coli strains that produce any other type of Stx (LD50 = 1010). To identify the element(s) of Stx2d that are required for mucus-mediated activation, toxin genes were constructed such that the expressed mutant toxins consisted of hybrids of Stx2d and Stx1, Stx2 or Stx2e, contained deletions of up to six amino acids from the C-terminus of the A2 of Stx2d or were altered in one or both of the two amino acids of the A2 of Stx2d that represent the only amino acid differences between the activatable Stx2d and the non-activatable Stx2c. Analysis of these mutant toxins revealed that the A2 portion of Stx2d is required for toxin activation and that activation is abrogated if the Stx1 or Stx2e B subunit is substituted for the Stx2d B polypeptide. Furthermore, mass spectrometry performed on buffer- or elastase-treated Stx2d indicated that the A2 peptide of the activated Stx2d was two amino acids smaller than the A2 peptide from buffer-treated Stx2d. This finding, together with the toxin hybrid results, suggests that activation involves B pentamer-dependent cleavage by elastase of the C-terminal two amino acids from the Stx2d A2 peptide.     

Sequence of a cDNA clone encoding the zinc metalloproteinase hemorrhagic toxin e from Crotalus atrox: evidence for signal, zymogen, and disintegrin-like structures.

The sequence of two overlapping cDNA clones for the zinc metalloproteinase hemorrhagic toxin e (also known as atrolysin e, EC 3.4.24.44) from the venom gland of Crotalus atrox, the Western diamondback rattlesnake, is presented. The assembled cDNA sequence is 1975 nucleotides in length and encodes an open reading frame of 478 amino acids. The mature hemorrhagic toxin e protein as isolated from the crude venom has a molecular weight of approximately 24,000 and thus represents the processed product of this open reading frame. From the deduced amino acid sequence, it can be hypothesized that the enzyme is translated with a signal sequence of 18 amino acids, an amino-terminal propeptide of 169 amino acids, a central hemorrhagic proteinase domain of 202 amino acids, and a carboxy-terminal sequence of 89 amino acids. The propeptide has a short region similar to the region involved in the activation of matrix metalloproteinase zymogens. The proteinase domain is similar to other snake venom metalloproteinases, with over 57% identity to the low molecular weight proteinases HR2a and H2-proteinase from the Habu snake Trimeresurus flavoviridis. The carboxy-terminal region, which is not observed in the mature protein, strongly resembles the protein sequence immediately following the proteinase domain of HR1B (a high molecular weight hemorrhagic proteinase from the venom of T. flavoviridis) and the members of a different family of snake venom polypeptides known for their platelet aggregation inhibitory activity, the disintegrins. The cDNA sequence bears striking similarity to a previously reported sequence for a disintegrin cDNA. This report is evidence that this subfamily of venom metalloproteinases is synthesized in a proenzyme form which must be proteolytically activated.(ABSTRACT TRUNCATED AT 250 WORDS)     

幽门螺杆菌HspA融合蛋白口服疫苗的构建

构建表达幽门螺杆菌的保护性抗原分热休克蛋白A亚单位(HspA）和霍乱毒素B亚单位（CtxB）的重组融合蛋白的生物工程菌株,以此制备幽门螺杆菌的口服疫苗。用PCR方法扩增hspA和ctxB两个目的的基因片段,将它们分别克隆至pSK（＋）质粒上,然后插入含T7启动子ET－22b（＋）的表达载体中,构建嗓基因的表达质量pET－hct,转化E.coliBL21(DE3）,经IPTG诱导表达融合蛋白HCT。经测序,hspA-ctxB(hct)融合基因片段由726bp组成,可以编码242个氨基酸残基的多肽。经SDS－PAGE和免疫印迹分析检测发现,融合基因表达的蛋白质相对分子质量约为30kD。融合蛋白经镍离子柱纯化、复性后,和HspA共同标记同位素^125I,然后给小鼠灌胃,结果观察到HCT组小鼠血清中的^125I的放射量要明显高于HspA组（P＜0．001）,且吸收峰值时间明显提前。融合蛋白中的CtxB可明显促进小鼠对HspA的吸收,HCT融合蛋白可以作为预防和治疗幽门螺杆菌感染的侯选口服疫苗。     

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)