Search PubMed⌕ Search

Biomedical subjects

R J Collier

Publications and source records attributed to R J Collier.

At least 55 records · Page 3Linked to original sources
PubMed1999

Translocation of the catalytic domain of diphtheria toxin across planar phospholipid bilayers by its own T domain.

The T domain of diphtheria toxin is known to participate in the pH-dependent translocation of the catalytic C domain of the toxin across the endosomal membrane, but how it does so, and whether cellular proteins are also required for this process, remain unknown. Here, we report results showing that the T domain alone is capable of translocating the entire C domain across model, planar phospholipid bilayers in the absence of other proteins. The T domain therefore contains the entire molecular machinery for mediating transfer of the catalytic domain of diphtheria toxin across membranes.

Amino Acid Sequence↗
PubMed1999

Interaction of diphtheria toxin T domain with molten globule-like proteins and its implications for translocation.

The transmembrane (T) domain of diphtheria toxin has a critical role in the low pH-induced translocation of the catalytic domain (A chain) of the toxin across membranes. Here it is shown that at low pH, addition of proteins in a partly unfolded, molten globule-like conformation converted the T domain from a shallow membrane-inserted form to its transmembrane form. Fluorescence energy transfer demonstrated that molten globule-like proteins bound to the T domain. Thus, the T domain recognizes proteins that are partly unfolded and may function in translocation of the A chain as a transmembrane chaperone.

Antibodies↗
PubMed1999

Membrane translocation of charged residues at the tips of hydrophobic helices in the T domain of diphtheria toxin.

The low pH triggered membrane insertion of the T domain of diphtheria toxin is a critical step in the translocation of the C domain of the toxin across membranes in vivo. We previously established that the T domain can interact with membranes in two distinct conformations, one in which the TH8/TH9 helical hairpin lies close to the bilayer surface and a second in which it inserts more deeply and appears to be transmembraneous. The loss of charge on residues E349 and D352 due to protonation at low pH has been proposed to be a critical step in transmembrane insertion, because they are within a loop connecting TH8 and TH9, and must cross the membrane upon transmembrane insertion. In this report, the role of these residues was examined by measuring the effect of the double substitution E349K/D352K on the conformation of the TH8/TH9 hairpin through a fluorescent group attached to TH9. At pH 4.5, there was shallower insertion of TH8/TH9 of the E349K/D352K mutant relative to T domain with wild-type residues at 349 and 352. In addition, smaller and/or fewer pores were obtained with the E349K/D352K mutant relative to the wild-type. On the other hand, high T domain concentrations, or further decreasing pH, allowed transmembrane insertion of both the wild-type and the 349K/352K mutant as well as induction of larger and/or more numerous pores. Furthermore, the transmembrane insertion process was rapid for both the mutant and wild-type. This shows that the mutant has the capacity to form a transmembrane structure similar to that of the wild-type T domain and, thus, that introduction of charged groups in membrane-penetrating regions of a protein does not introduce an insurmountable barrier to transmembrane movement. The linkage between the ability of the T domain to form the transmembrane conformation and pores suggests that the effects of these mutations in inhibiting pore formation are likely to partly result from the inability to insert properly. Additionally, the observation that decreasing pH allows the 349K/352K mutant to insert deeply indicates that there are residues other than E349 and D352 whose protonation promotes transmembrane insertion.

Biological Transport↗
PubMed1999

Effects of mutations in proline 345 on insertion of diphtheria toxin into model membranes.

Translocation of the catalytic domain of diphtheria toxin (DT) across the endosomal membrane to the cytoplasm of mammalian cells requires the low-pH-dependent insertion of a hydrophobic helical hairpin (TH8-TH9) that is buried within the T domain of the native protein. Mutations of Pro345, which terminates helix TH8, have been reported to block toxicity for Vero cells. We found that mutant toxins in which Pro345 had been replaced by Cys, Glu, or Gly were profoundly defective at low pH in forming channels in planar phospholipid bilayers and in permeabilizing phospholipid vesicles to entrapped fluorophores. Experiments with isolated T domain containing a polarity-sensitive fluorophore attached to Cys at position 332 suggest that the P345E mutation blocks membrane insertion. None of the Pro345 mutations shifted the pH-dependence of binding in solution of the hydrophobic fluorophore, 2-p-toluidinyl-naphthalene 7-sulfonate. The results indicate that proline at position 345 is required for the T domain to insert into phospholipid bilayers or to adopt a functional conformation within the bilayer.

Animals↗
PubMed1999

Anthrax toxin entry into polarized epithelial cells.

We examined the entry of anthrax edema toxin (EdTx) into polarized human T84 epithelial cells using cyclic AMP-regulated Cl- secretion as an index of toxin entry. EdTx is a binary A/B toxin which self assembles at the cell surface from anthrax edema factor and protective antigen (PA). PA binds to cell surface receptors and delivers EF, an adenylate cyclase, to the cytosol. EdTx elicited a strong Cl- secretory response when it was applied to the basolateral surface of T84 cells but no response when it was applied to the apical surface. PA alone had no effect when it was applied to either surface. T84 cells exposed basolaterally bound at least 30-fold-more PA than did T84 cells exposed apically, indicating that the PA receptor is largely or completely restricted to the basolateral membrane of these cells. The PA receptor did not fractionate with detergent-insoluble caveola-like membranes as cholera toxin receptors do. These findings have implications regarding the nature of the PA receptor and confirm the view that EdTx and CT coopt fundamentally different subcellular systems to enter the cell and cause disease.

Animals↗
PubMed1999

Cytotoxic T-lymphocyte epitopes fused to anthrax toxin induce protective antiviral immunity.

We have investigated the use of the protective antigen (PA) and lethal factor (LF) components of anthrax toxin as a system for in vivo delivery of cytotoxic T-lymphocyte (CTL) epitopes. During intoxication, PA directs the translocation of LF into the cytoplasm of mammalian cells. Here we demonstrate that antiviral immunity can be induced in BALB/c mice immunized with PA plus a fusion protein containing the N-terminal 255 amino acids of LF (LFn) and an epitope from the nucleoprotein (NP) of lymphocytic choriomeningitis virus. We also demonstrate that BALB/c mice immunized with a single LFn fusion protein containing NP and listeriolysin O protein epitopes in tandem mount a CTL response against both pathogens. Furthermore, we show that NP-specific CTL are primed in both BALB/c and C57BL/6 mice when the mice are immunized with a single fusion containing two epitopes, one presented by Ld and one presented by Db. The data presented here demonstrate the versatility of the anthrax toxin delivery system and indicate that this system may be used as a general approach to vaccinate outbred populations against a variety of pathogens.

Animals↗
PubMed1999

Production responses to bovine somatotropin in northeast dairy herds.

The commercial response to bovine somatotropin was examined in northeast dairy herds from 1990 to 1998 (4-yr preapproval and 4-yr postapproval). With DHI records and Monsanto customer files, a control group (never purchased Posilac) and a bovine somatotropin (bST) group (used on at least 50% of cows) were identified. A total of 340 herds were involved and, over the 8-yr period, there were over 80,000 cows, 200,000 lactations, and 2 million test days. Herd management comparisons demonstrated the response to bST was relatively constant each year of the postapproval period. Assuming 100% of cows were supplemented, response to bST over a 305-d lactation equaled 894 kg of milk, 27 kg of milk fat, and 31 kg of milk protein. Comparisons of lactation curves were used to identify where the bST response occurred in the lactation cycle. Analysis demonstrated the responses in milk, milk fat, and protein yield were minimal in the early phase of lactation, and then gradually increased until reaching a plateau over the last half of the lactation cycle. Persistency of lactation was also improved by bST, indicating the opportunity exists to extend lactation with combined use of bST and altered reproductive management. Average age and days in milk did not differ between control and bST herds. Thus, stayability and herd-life of animals were not altered by bST treatment. Somatic cell count (SCC) linear scores were minimally affected in herds utilizing bST and the pattern of SCC over the lactation cycle was unaffected. Overall bST improved lactation yield and persistency consistently over the 4-yr postapproval period with no effects on cow stayability and herd-life.

Animals↗
PubMed1998

Membrane topography of the T domain of diphtheria toxin probed with single tryptophan mutants.

The membrane insertion and translocation of diphtheria toxin, which is induced in vivo by low pH, is thought to be directed by the hydrophobic alpha-helices of its transmembrane (T) domain. In this study the structure of membrane-associated T domain was examined. Site-directed mutants of the T domain with single Trp residues were prepared at the two naturally occurring positions, 206 (near the N-terminal end of helix TH1) and 281 (within helix TH5), as well as at three residues in helix TH9, in which the substitutions F355W (near the N-terminal end of TH9), I364W (close to the center of TH9), and Y375W (near the C-terminal end of TH9) were made. All these mutants were found to undergo the low-pH-induced conformational change observed with wild-type T domain and insert into model membranes at low pH. The location of Trp residues relative to the lipid bilayer was characterized in model membrane vesicles by fluorescence emission and by quenching with nitroxide-labeled phospholipids. In TH9, residue 375 was shallowly inserted, residue 364 deeply inserted, and residue 355 located at an intermediate depth. Residues 206 and 281 exhibited moderately deep insertion. It was also found, in agreement with our previous study using bimane-labeled protein (Wang et al. (1997) J. Biol. Chem. 272, 25091-25098), that TH9 switches from a relatively shallowly inserted state to a more deeply inserted state when the concentration of the T domain in the membrane is increased or the thickness of the membrane bilayer is decreased. In particular, the depth of residue 355 was found to increase under the conditions giving deeper insertion. In contrast, residue 375 remained shallowly located in both states, as predicted from its location on the polar C-terminus of TH9. It is concluded that TH1 and TH5 insert into the lipid bilayer in both T domain conformations. In addition, Trp depths suggest that even in the shallowly inserted state there is a significant degree of insertion of TH9. These results suggest regions of the T domain in addition to the hydrophobic TH8/TH9 hairpin insert into membranes. Models for the structure of the membrane-inserted T domain are discussed.

Dimyristoylphosphatidylcholine↗
PubMed1998

Characterization of membrane translocation by anthrax protective antigen.

Solving the crystallographic structure of the ring-shaped heptamer formed by protective antigen (PA), the B moiety of anthrax toxin, has focused attention on understanding how this oligomer mediates membrane translocation of the toxin's A moieties. We have developed an assay for translocation in which radiolabeled ligands are bound to proteolytically activated PA (PA63) at the surface of CHO or L6 cells, and translocation across the plasma membrane is induced by lowering the pH. The cells are then treated with Pronase E to degrade residual surface-bound material, and protected ligands are quantified after fractionation by SDS-PAGE. Translocation was most efficient (35%-50%) with LFN, the N-terminal PA binding domain of the anthrax lethal factor (LF). Intact LF, edema factor (EF), or fusion proteins containing LFN fused to certain heterologous proteins [the diphtheria toxin A chain (DTA) or dihydrofolate reductase (DHFR)] were less efficiently translocated (15%-20%); and LFN fusions to several other proteins were not translocated at all. LFN with different N-terminal residues was found to be degraded according to the N-end rule by the proteasome, and translocation of LFN fused to a mutant form of DHFR with a low affinity for methotrexate (MTX) protected cells from the effects of MTX. Both results are consistent with a cytosolic location of protected proteins. Evidence that a protein must unfold to be translocated was obtained in experiments showing that (i) translocation of LFNDTA was blocked by introduction of an artificial disulfide into the DTA moiety, and (ii) translocation of LFNDHFR and LFNDTA was blocked by their ligands (MTX and adenine, respectively). These results demonstrate that the acid-induced translocation by anthrax toxin closely resembles that of diphtheria toxin, despite the fact that these two toxins are unrelated and form pores by different mechanisms.

Amino Acid Sequence↗
PubMed1998

Identifying transmembrane states and defining the membrane insertion boundaries of hydrophobic helices in membrane-inserted diphtheria toxin T domain.

The membrane topography of proteins that convert between soluble and membrane-inserted states has proven a challenging problem. In particular, it has been difficult to define both whether a transmembrane orientation is achieved and what are the boundaries of membrane-inserted segments. In this report the fluorescence of bimane-labeled Cys residues and the binding of anti-BODIPY antibodies to BODIPY-labeled Cys residues are combined to define these features for helices TH8 and TH9 of the T domain of diphtheria toxin. Using a series of labeled residues the topography of these helices was examined in both conformations of membrane-inserted T domain identified previously (Wang, Y., Malenbaum, S. E., Kachel, K., Zhan, H., Collier, R. J., and London, E. (1997) J. Biol. Chem. 272, 25091-25098). In the shallowly inserted conformation these helices are found to be aligned close to the cis surface of the bilayer all along their sequences. In contrast, in the more deeply inserted conformation most TH8 and TH9 residues examined located in a non-polar environment, with the boundaries of the membrane-inserted sequences close to residues 324 and 372-374 on the cis (insertion) side of the bilayer. It was also found that residues 348 and 349, which are in the loop connecting TH8 and TH9, reached the opposite trans side of the bilayer, but did not protrude fully into the aqueous environment. These boundaries suggest the membrane-inserted segments of TH8 and TH9 form transmembrane helices about 25 residues in length, and suggest that they are connected by a tight turn. It is concluded that this combination of fluorescent techniques can be combined to obtain transmembrane helix topography.

Boron Compounds↗
PubMed1998

Identification of residues lining the anthrax protective antigen channel.

In its activated 63 kDa form, the protective antigen (PA) component of anthrax toxin forms a heptameric prepore, which converts to a pore (channel) in endosomal membranes at low pH and mediates translocation of the toxin's enzymic moieties to the cytosol. It has been proposed that the prepore-to-pore conversion involves a conformational rearrangement of a disordered amphipathic loop (D2L2; residues 302-325), in which loops from the 7 protomers combine to form a transmembrane 14-stranded beta barrel. To test this model, we generated Cys substitutions in 24 consecutive residues of the D2L2 loop, formed channels in artificial bilayers with each mutant, and examined changes in channel conductance after adding the thiol-reactive, bilayer-impermeant reagent methanethiosulfonate ethyltrimethylammonium (MTS-ET) to the trans compartment. The rationale for these experiments is that reaction of MTS-ET with a Cys residue adds a positively charged group and therefore would likely reduce channel conductance if the residue were in the ion-conducting pathway. We found alternating reduction and absence of reduction of conductance in consecutive residues over two stretches (residues 302-311 and 316-325). This pattern is consistent with alternating polar and apolar residues of the two stretches projecting into the pore lumen and into the bilayer, respectively. Residues connecting these two stretches (residues 312-315) were responsive to MTS-ET, consistent with their being in a turn region. Single channels formed by selected mutants (H304C and N306C) showed multiple conductance step changes in response to MTS-ET, consistent with an oligomeric pore. We also found that the binding site for the channel-blocking tetraalkylammonium ions is located cis relative to the inserted D2L2 loops. These findings constitute strong evidence in favor of the model of conversion of the prepore to a 14-stranded beta barrel pore and solidify the foundation for studies to understand the mechanism of translocation by anthrax toxin.

Amino Acid Sequence↗
PubMed1998

The diphtheria toxin channel-forming T domain translocates its own NH2-terminal region across planar bilayers.

The T domain of diphtheria toxin, which extends from residue 202 to 378, causes the translocation of the catalytic A fragment (residues 1-201) across endosomal membranes and also forms ion-conducting channels in planar phospholipid bilayers. The carboxy terminal 57-amino acid segment (322-378) in the T domain is all that is required to form these channels, but its ability to do so is greatly augmented by the portion of the T domain upstream from this. In this work, we show that in association with channel formation by the T domain, its NH2 terminus, as well as some or all of the adjacent hydrophilic 63 amino acid segment, cross the lipid bilayer. The phenomenon that enabled us to demonstrate that the NH2-terminal region of the T domain was translocated across the membrane was the rapid closure of channels at cis negative voltages when the T domain contained a histidine tag at its NH2 terminus. The inhibition of this effect by trans nickel, and by trans streptavidin when the histidine tag sequence was biotinylated, clearly established that the histidine tag was present on the trans side of the membrane. Furthermore, the inhibition of rapid channel closure by trans trypsin, combined with mutagenesis to localize the trypsin site, indicated that some portion of the 63 amino acid NH2-terminal segment of the T domain was also translocated to the trans side of the membrane. If the NH2 terminus was forced to remain on the cis side, by streptavidin binding to the biotinylated histidine tag sequence, channel formation was severely disrupted. Thus, normal channel formation by the T domain requires that its NH2 terminus be translocated across the membrane from the cis to the trans side, even though the NH2 terminus is >100 residues removed from the channel-forming part of the molecule.

Amino Acid Sequence↗
PubMed1998

Anthrax toxin as a molecular tool for stimulation of cytotoxic T lymphocytes: disulfide-linked epitopes, multiple injections, and role of CD4(+) cells.

We have previously demonstrated that anthrax toxin-derived proteins, protective antigen (PA) and the amino-terminal portion of lethal factor (LFn), can be used in combination to deliver heterologous molecules to the cytosol of mammalian cells. In this study we examined the ability of an LFn-peptide disulfide-linked heterodimer to prime cytotoxic T lymphocytes (CTL) in the presence of PA. A mutant of LFn that contains a carboxy-terminal reactive cysteine was generated. This form of LFn could be oxidized with a synthetic cysteine containing peptide to form a heterodimer of the protein and peptide. Mice injected with the heterodimer plus PA mounted a peptide-specific CTL response, indicating that this molecule functioned similarly to the genetically fused forms used previously. We also report the results of an analysis of two aspects of this system important for the development of experimental vaccines. First, CD4 knockout mice were unable to generate a CTL response when treated with PA plus an LFn-epitope fusion protein, suggesting that CD4(+) helper responses are essential for stimulating specific CTL with the PA-LFn system. Second, we now show that primary injection with this system does not generate any detectable antibody response to the vaccine components and that prior immunization has no effect on priming a CTL response to an unrelated epitope upon subsequent injection.

Animals↗
PubMed1998

Anthrax toxin-mediated delivery in vivo and in vitro of a cytotoxic T-lymphocyte epitope from ovalbumin.

We reported earlier that a nontoxic form of anthrax toxin was capable of delivering a cytotoxic T-lymphocyte (CTL) epitope in vivo, such that a specific CTL response was primed against the epitope. The epitope, of bacterial origin, was fused to an N-terminal fragment (LFn) from the lethal-factor component of the toxin, and the fusion protein was injected, together with the protective antigen (PA) component, into BALB/c mice. Here we report that PA plus LFn is capable of delivering a different epitope--OVA(257-264) from ovalbumin. Delivery was accomplished in a different mouse haplotype, H-2Kb and occurred in vitro as well as in vivo. An OVA(257-264)-specific CTL clone, GA-4, recognized EL-4 cells treated in vitro with PA plus as little as 30 fmol of the LFn-OVA(257-264) fusion protein. PA mutants attenuated in toxin self-assembly or translocation were inactive, implying that the role of PA in epitope delivery is the same as that in toxin action. Also, we showed that OVA(257-264)-specific CTL could be induced to proliferate by incubation with splenocytes treated with PA plus LFn-OVA(257-264). These findings imply that PA-LFn may serve as a general delivery vehicle for CTL epitopes in vivo and as a safe, efficient tool for the ex vivo expansion of patient-derived CTL for use in adoptive immunotherapy.

Animals↗
PubMed1997

pH-dependent perforation of macrophage phagosomes by listeriolysin O from Listeria monocytogenes.

The pore-forming toxin listeriolysin O (LLO) is a major virulence factor implicated in escape of Listeria monocytogenes from phagocytic vacuoles. Here we describe the pH-dependence of vacuolar perforation by LLO, using the membrane-impermeant fluorophore 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) to monitor the pH and integrity of vacuoles in mouse bone marrow-derived macrophages. Perforation was observed when acidic vacuoles containing wild-type L. monocytogenes displayed sudden increases in pH and release of HPTS into the cytosol. These changes were not seen with LLO-deficient mutants. Perforation occurred at acidic vacuolar pH (4.9-6.7) and was reduced in frequency or prevented completely when macrophages were treated with the lysosomotropic agents ammonium chloride or bafilomycin A1. We conclude that acidic pH facilitates LLO activity in vivo.

Ammonium Chloride↗
PubMed1997

Identification of shallow and deep membrane-penetrating forms of diphtheria toxin T domain that are regulated by protein concentration and bilayer width.

The alpha-helix-rich, hydrophobic transmembrane (T) domain of diphtheria toxin is believed to play a central role in membrane insertion by the toxin and in the translocation of its catalytic domain across membranes. In this report, T domain structure was studied using site-directed single-Cys mutants. The residues chosen, 322 (near the amino-terminal end of helix TH8), 333 (within helix TH8), and 356 (within helix TH9) were substituted with Cys and labeled with the fluorescent probe bimane. (Residues 333 and 356 should be located within the bilayer in the transmembrane state, and residue 322 should not penetrate the bilayer.) After insertion of T domain into model membrane vesicles, the location of bimane label relative to the lipid bilayer was characterized by its fluorescence emission and by its quenching with nitroxide-labeled phospholipids. It was found that when the T domain is added to dioleoylphosphatidylcholine-containing vesicles, all three residues reside close to the outer surface. However, at high T domain concentration or in thinner dimyristoleoylphosphatidylcholine-containing vesicles, a large fraction of residues 333 and 356 penetrate deeply into the membrane. In contrast, residue 322 remains exposed to aqueous solution under these conditions. These conclusions were confirmed by a novel antibody binding method. Antibodies that quench the fluorescence of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3-indacene++ + (BODIPY) groups were used to evaluate the exposure of BODIPY-labeled 322, 333, and 356. Maximum exposure of residues 333 and 356 to externally added antibody was only observed under conditions in which bimane fluorescence showed that these residues do not penetrate the bilayer. In contrast, residue 322 remained exposed under all conditions. We propose that the deeply penetrating T domain conformation represents a transmembrane or near-transmembrane state. The regulation of the transmembrane/nontransmembrane equilibrium should be a key to understanding diphtheria toxin membrane insertion and translocation. Our results suggest that toxin-toxin interactions may play an important role in regulating this behavior.

Boron Compounds↗
PubMed1997

Crystal structure of the anthrax toxin protective antigen.

Protective antigen (PA) is the central component of the three-part protein toxin secreted by Bacillus anthracis, the organism responsible for anthrax. After proteolytic activation on the host cell surface, PA forms a membrane-inserting heptamer that translocates the toxic enzymes, oedema factor and lethal factor, into the cytosol. PA, which has a relative molecular mass of 83,000 (M(r) 83K), can also translocate heterologous proteins, and is being evaluated for use as a general protein delivery system. Here we report the crystal structure of monomeric PA at 2.1 A resolution and the water-soluble heptamer at 4.5 A resolution. The monomer is organized mainly into antiparallel beta-sheets and has four domains: an amino-terminal domain (domain 1) containing two calcium ions and the cleavage site for activating proteases; a heptamerization domain (domain 2) containing a large flexible loop implicated in membrane insertion; a small domain of unknown function (domain 3); and a carboxy-terminal receptor-binding domain (domain 4). Removal of a 20K amino-terminal fragment from domain 1 allows the assembly of the heptamer, a ring-shaped structure with a negatively charged lumen, and exposes a large hydrophobic surface for binding the toxic enzymes. We propose a model of pH-dependent membrane insertion involving the formation of a porin-like, membrane-spanning beta-barrel.

Amino Acid Sequence↗