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J T Buckley

Publications and source records attributed to J T Buckley.

At least 19 recordsLinked to original sources
PubMed2001

The channel-forming protein proaerolysin remains a dimer at low concentrations in solution.

Proaerolysin, the proform of the channel-forming protein aerolysin, is secreted as a dimer by Aeromonas sp. The protein also exists as a dimer in the crystal, as well as in solution, at least at concentrations in the region of 500 microg/ml. Recently it has been argued that proaerolysin becomes monomeric at concentrations below 100 microg/ml and that only the monomeric form of the protoxin can bind to cell surface receptors (Fivaz, M., Velluz, M.-C., and van der Goot, F. G. (1999) J. Biol. Chem. 274, 37705-37708). Here we show, using non-denaturing polyacrylamide electrophoresis, chemical cross-linking, and analytical ultracentrifugation, that proaerolysin remains dimeric at the lowest concentrations of the protein that we measured (less than 5 microg/ml) and that the dimeric protoxin is quite capable of receptor binding.

Aeromonas↗
PubMed2001

Multilineage glycosylphosphatidylinositol anchor-deficient haematopoiesis in untreated aplastic anaemia.

Aplastic anaemia and paroxysmal nocturnal haemoglobinuria (PNH) are closely related disorders. In PNH, haematopoietic stem cells that harbour PIGA mutations give rise to blood elements that are unable to synthesize glycosylphosphatidylinositol (GPI) anchors. Because the GPI anchor is the receptor for the channel-forming protein aerolysin, PNH cells do not bind the toxin and are unaffected by concentrations that lyse normal cells. Exploiting these biological differences, we have developed two novel aerolysin-based assays to detect small populations of PNH cells. CD59 populations as small as 0.004% of total red cells could be detected when cells were pretreated with aerolysin to enrich the PNH population. All PNH patients displayed CD59-deficient erythrocytes, but no myelodysplastic syndrome (MDS) patient or control had detectable PNH cells before or after enrichment in aerolysin. Only one aplastic anaemia patient had detectable PNH red cells before exposure to aerolysin. However, 14 (61%) had detectable PNH cells after enrichment in aerolysin. The inactive fluorescent proaerolysin variant (FLAER) that binds the GPI anchors of a number of proteins on normal cells was used to detect a global GPI anchor deficit on granulocytes. Flow cytometry with FLAER showed that 12 out of 18 (67%) aplastic anaemia patients had FLAER-negative granulocytes, but none of the MDS patients or normal control subjects had GPI anchor-deficient cells. These studies demonstrate that aerolysin-based assays can reveal previously undetectable multilineage PNH cells in patients with untreated aplastic anaemia. Thus, clonality appears to be an early feature of aplastic anaemia.

Adult↗
PubMed2001

Type II secretion by Aeromonas salmonicida: evidence for two periplasmic pools of proaerolysin.

Aeromonas salmonicida containing the cloned gene for proaerolysin secretes the protein via the type II secretory pathway. Here we show that altering a region near the beginning of aerA led to a dramatic increase in the amount of proaerolysin that was produced and that a large amount of the protein was cell associated. All of the cell-associated protein had crossed the cytoplasmic membrane, because the signal sequence had been removed, and all of it was accessible to processing by trypsin during osmotic shock. Enlargement of the periplasm was observed by electron microscopy in overproducing cells, likely caused by the osmotic effect of the very large concentrations of accumulated proaerolysin. Immunogold electron microscopy localized nearly all of the proaerolysin in the enlarged periplasm; however, only half of the protoxin was released from the cells by osmotic shocking. Cross-linking studies showed that this fraction contained normal dimeric proaerolysin but that proaerolysin in the fraction that was not shockable had not dimerized, although it appeared to be correctly folded. Both periplasmic fractions were secreted by the cells; however, the nonshockable fraction was secreted much more slowly than the shockable fraction. We estimated a rate for maximal secretion of proaerolysin from the bacteria that was much lower than the rates that have been estimated for inner membrane transit, which suggests that transit across the outer membrane is rate limiting and may account for the periplasmic accumulation of the protein. Finally, we show that overproduction of proaerolysin inhibited the release of the protease that is secreted by A. salmonicida.

Aeromonas↗
PubMed2000

Lipids favoring inverted phase enhance the ability of aerolysin to permeabilize liposome bilayers.

Channel formation by the bacterial toxin aerolysin follows oligomerization of the protein to produce heptamers that are capable of inserting into lipid bilayers. How insertion occurs is not understood, not only for aerolysin but also for other proteins that can penetrate membranes. We have studied aerolysin channel formation by measuring dye leakage from large unilamellar egg phosphatidylcholine vesicles containing varying amounts of other lipids. The rate of leakage was enhanced in a dose-dependent manner by the presence of phosphatidylethanolamine, diacylglycerol, cholesterol, or hexadecane, all of which are known to favor a lamellar-to-inverted hexagonal (L-H) phase transition. Phosphatidylethanolamine molecular species with low L-H transition temperatures had the largest effects on aerolysin activity. In contrast, the presence in the egg phosphatidylcholine liposomes of lipids that are known to stabilize the lamellar phase, such as sphingomyelin and saturated phosphatidylcholines, reduced the rate of channel formation, as did the presence of lysophosphatidylcholine, which favors positive membrane curvature. When two different lipids that favor hexagonal phase were present with egg PC in the liposomes, their stimulatory effects were additive. Phosphatidylethanolamine and lysophosphatidylcholine canceled each other's effect on channel formation.

Animals↗
PubMed2000

Channel formation by the glycosylphosphatidylinositol-anchored protein binding toxin aerolysin is not promoted by lipid rafts.

Glycosylphosphatidylinositol-anchored proteins may be concentrated in membrane microdomains (lipid rafts) that are also enriched in cholesterol and sphingolipids. The glycosyl anchor of these proteins is a specific, high affinity receptor for the channel-forming protein aerolysin. We wished to determine if the presence of rafts promotes the activity of aerolysin. Treatment of T lymphocytes with methyl-beta-cyclodextrin, which destroys lipid rafts by sequestering cholesterol, had no measurable effect on the sensitivity of the cells to aerolysin; nor did similar treatment of erythrocytes decrease the rate at which they were lysed by the toxin. We also studied the rate of aerolysin-induced channel formation in liposomes containing glycosylphosphatidylinositol-anchored placental alkaline phosphatase, which we show is a receptor for aerolysin. In liposomes containing sphingolipids as well as glycerophospholipids and cholesterol, most of the enzyme was Triton X-100-insoluble, indicating that it was localized in rafts, whereas in liposomes prepared without sphingolipids, all of the enzyme was soluble. Aerolysin was no more active against liposomes containing rafts than against those that did not. We conclude that lipid rafts do not promote channel formation by aerolysin.

Alkaline Phosphatase↗
PubMed2000

Improved detection and characterization of paroxysmal nocturnal hemoglobinuria using fluorescent aerolysin.

Paroxysmal nocturnal hemoglobinuria (PNH) is caused by a somatic mutation in the gene PIGA, which encodes an enzyme essential for the synthesis of glycosylphosphatidylinositol (GPI) anchors. The PIGA mutation results in absence or marked deficiency of more than a dozen proteins on PNH blood cells. Current flow cytometric assays for PNH rely on the use of labeled antibodies to detect deficiencies of specific GPI anchor proteins, such as CD59. However, because no single GPI anchor protein is always expressed in all cell lineages, no one monoclonal antibody can be used with confidence to diagnose PNH. We describe a new diagnostic test for PNH, based on the ability of a fluorescently labeled inactive variant of the protein aerolysin (FLAER) to bind selectively to GPI anchors. We compared GPI anchor protein expression in 8 patients with PNH using FLAER and anti-CD59. In all cases, FLAER detected similar or higher proportions of PNH monocytes and granulocytes compared with anti-CD59. Because of the increased sensitivity of detection, FLAER could detect small abnormal granulocyte populations in patients to a level of about 0.5%; samples from healthy control subjects contained substantially fewer FLAER-negative cells. FLAER gives a more accurate assessment of the GPI anchor deficit in PNH.

Anemia, Aplastic↗
PubMed1999

Clostridium septicum alpha toxin uses glycosylphosphatidylinositol-anchored protein receptors.

The alpha toxin produced by Clostridium septicum is a channel-forming protein that is an important contributor to the virulence of the organism. Chinese hamster ovary (CHO) cells are sensitive to low concentrations of the toxin, indicating that they contain toxin receptors. Using retroviral mutagenesis, a mutant CHO line (BAG15) was generated that is resistant to alpha toxin. FACS analysis showed that the mutant cells have lost the ability to bind the toxin, indicating that they lack an alpha toxin receptor. The mutant cells are also resistant to aerolysin, a channel-forming protein secreted by Aeromonas spp., which is structurally and functionally related to alpha toxin and which is known to bind to glycosylphosphatidylinositol (GPI)-anchored proteins, such as Thy-1. We obtained evidence that the BAG15 cells lack N-acetylglucosaminyl-phosphatidylinositol deacetylase-L, needed for the second step in GPI anchor biosynthesis. Several lymphocyte cell lines lacking GPI-anchored proteins were also shown to be less sensitive to alpha toxin. On the other hand, the sensitivity of CHO cells to alpha toxin was increased when the cells were transfected with the GPI-anchored folate receptor. We conclude that alpha toxin, like aerolysin, binds to GPI-anchored protein receptors. Evidence is also presented that the two toxins bind to different subsets of GPI-anchored proteins.

Animals↗
PubMed1999

Analysis of receptor binding by the channel-forming toxin aerolysin using surface plasmon resonance.

Aerolysin is a channel-forming bacterial toxin that binds to glycosylphosphatidylinositol (GPI) anchors on host cell-surface structures. The nature of the receptors and the location of the receptor-binding sites on the toxin molecule were investigated using surface plasmon resonance. Aerolysin bound to the GPI-anchored proteins Thy-1, variant surface glycoprotein, and contactin with similar rate constants and affinities. Enzymatic removal of N-linked sugars from Thy-1 did not affect toxin binding, indicating that these sugars are not involved in the high affinity interaction with aerolysin. Aerolysin is a bilobal protein, and both lobes were shown to be required for optimal binding. The large lobe by itself bound Thy-1 with an affinity that was at least 10-fold weaker than that of the whole toxin, whereas the small lobe bound the GPI-anchored protein at least 1000-fold more weakly than the intact toxin. Mutation analyses provided further evidence that both lobes were involved in GPI anchor binding, with certain single amino acid substitutions in either domain leading to reductions in affinity of as much as 100-fold. A variant with single amino acid substitutions in both lobes of the protein was completely unable to bind the receptor. The membrane protein glycophorin, which is heavily glycosylated but not GPI-anchored, bound weakly to immobilized proaerolysin, suggesting that interactions with cell-surface carbohydrate structures other than GPI anchors may partially mediate toxin binding to host cells.

Bacterial Toxins↗
PubMed1999

Resistance of paroxysmal nocturnal hemoglobinuria cells to the glycosylphosphatidylinositol-binding toxin aerolysin.

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal stem cell disorder caused by a somatic mutation of the PIGA gene. The product of this gene is required for the biosynthesis of glycosylphosphatidylinositol (GPI) anchors; therefore, the phenotypic hallmark of PNH cells is an absence or marked deficiency of all GPI-anchored proteins. Aerolysin is a toxin secreted by the bacterial pathogen Aeromonas hydrophila and is capable of killing target cells by forming channels in their membranes after binding to GPI-anchored receptors. We found that PNH blood cells (erythrocytes, lymphocytes, and granulocytes), but not blood cells from normals or other hematologic disorders, are resistant to the cytotoxic effects of aerolysin. The percentage of lysis of PNH cells after aerolysin exposure paralleled the percentage of CD59(+) cells in the samples measured by flow cytometry. The kinetics of red blood cell lysis correlated with the type of PNH erythrocytes. PNH type III cells were completely resistant to aerolysin, whereas PNH type II cells displayed intermediate sensitivity. Importantly, the use of aerolysin allowed us to detect PNH populations that could not be detected by standard flow cytometry. Resistance of PNH cells to aerolysin allows for a simple, inexpensive assay for PNH that is sensitive and specific. Aerolysin should also be useful in studying PNH biology.

Bacterial Toxins↗
PubMed1999

Expression and properties of an aerolysin--Clostridium septicum alpha toxin hybrid protein.

Aerolysin is a bilobal channel-forming toxin secreted by Aeromonas hydrophila. The alpha toxin produced by Clostridium septicum is homologous to the large lobe of aerolysin. However, it does not contain a region corresponding to the small lobe of the Aeromonas toxin, leading us to ask what the function of the small lobe is. We fused the small lobe of aerolysin to alpha toxin, producing a hybrid protein that should structurally resemble aerolysin. Unlike aerolysin, the hybrid was not secreted when expressed in Aeromonas salmonicida. The purified hybrid was activated by proteolytic processing in the same way as both parent proteins and, after activation, it formed oligomers that corresponded to the aerolysin heptamer. Like aerolysin, the hybrid was far more active than alpha toxin against human erythrocytes and mouse T lymphocytes. Both aerolysin and the hybrid bound to human glycophorin, and both were inhibited by preincubation with this erythrocyte glycoprotein, whereas alpha toxin was unaffected. We conclude that aerolysin contains two receptor binding sites, one for glycosyl-phosphatidylinositol-anchored proteins that is located in the large lobe and is also found in alpha toxin, and a second site, located in the small lobe, that binds a surface carbohydrate determinant.

Aeromonas↗
PubMed1999

The channel-forming toxin aerolysin neutralizes human immunodeficiency virus type 1.

Aerolysin is a channel-forming toxin secreted by Aeromonas spp. that binds to glycosyl phosphatidylinositol (GPI)-anchored proteins, such as Thy-1, on sensitive target cells. Receptor binding is followed first by oligomerization of the toxin and then by insertion of the oligomers into the membrane to form stable channels that disrupt the permeability barrier. Human immunodeficiency virus type 1 (HIV-1) produced from T cells is known to incorporate Thy-1 and other GPI-anchored proteins into its membrane. Here, we show that aerolysin is capable of neutralizing HIV-1 in a dose-dependent manner and that neutralization depends upon the presence of these proteins in the viral envelope. Pretreatment with phosphatidylinositol-specific phospholipase C to remove GPI-anchored proteins greatly reduced HIV-1 sensitivity to the toxin, and virus originating from a mutant cell line that lacks GPI-anchored proteins was not neutralized. Aerolysin variants with single amino acid changes that prevent oligomerization or insertion of the toxin were unable to inactivate the virus, implying that channel formation is necessary for neutralization to occur. These findings represent the first evidence that a pathogenic human virus can be neutralized by a bacterial toxin.

Anti-HIV Agents↗
PubMed1999

Channels formed by subnanomolar concentrations of the toxin aerolysin trigger apoptosis of T lymphomas.

Aerolysin is a channel-forming toxin that binds to glycosylphosphatidylinositol (GPI)-anchored proteins, such as Thy-1, on target cells. Here, we show that subnanomolar concentrations of aerolysin trigger apoptosis of T lymphomas. Using inactive aerolysin variants, we determined that apoptosis was not directly triggered by binding to GPI-anchored receptors, nor was it caused by receptor clustering induced by toxin oligomerization. Apoptosis was caused by the production of a small number of channels in the cell membrane. Channel formation resulted in a rapid increase in intracellular calcium, which may have been the signal for apoptosis. Overexpression of the antiapoptotic protein bcl-2 blocked aerolysin-induced apoptosis, although this effect was overcome at higher toxin concentrations.

Animals↗
PubMed1998

The pore-forming toxin proaerolysin is activated by furin.

Aerolysin is secreted as an inactive dimeric precursor by the bacterium Aeromonas hydrophila. Proteolytic cleavage within a mobile loop near the C terminus of the protoxin is required for oligomerization and channel formation. This loop contains the sequence KVRRAR432, which should be recognized by mammalian proprotein convertases such as furin, PACE4, and PC5/6A. Here we show that these three proteases cleave proaerolysin after Arg-432 in vitro, yielding active toxin. We also investigated the potential role of these enzymes in the in vivo activation of the protoxin. We found that Chinese hamster ovary cells were able to convert the protoxin to aerolysin in the absence of exogenous proteases and that activation did not require internalization of the toxin. The furin inhibitor alpha1-antitrypsin Portland reduced the rate of proaerolysin activation in vivo, and proaerolysin processing was even further reduced in furin-deficient FD11 Chinese hamster ovary cells. The cells were also less sensitive to proaerolysin than wild type cells; however, transient transfection of FD11 cells with the cDNA encoding furin conferred normal sensitivity to the protoxin. Together these findings argue that furin catalyzes the cell-surface activation of proaerolysin in vivo.

Animals↗
PubMed1998

Glycosylphosphatidylinositol anchors of membrane glycoproteins are binding determinants for the channel-forming toxin aerolysin.

Cells that are sensitive to the channel-forming toxin aerolysin contain surface glycoproteins that bind the toxin with high affinity. Here we show that a common feature of aerolysin receptors is the presence of a glycosylphosphatidylinositol anchor, and we present evidence that the anchor itself is an essential part of the toxin binding determinant. The glycosylphosphatidylinositol (GPI)-anchored T-lymphocyte protein Thy-1 is an example of a protein that acts as an aerolysin receptor. This protein retained its ability to bind aerolysin when it was expressed in Chinese hamster ovary cells, but could not bind the toxin when expressed in Escherichia coli, where the GPI anchor is absent. An unrelated GPI-anchored protein, the variant surface glycoprotein of trypanosomes, was shown to bind aerolysin with similar affinity to Thy-1, and this binding ability was significantly reduced when the anchor was removed chemically. Cathepsin D, a protein with no affinity for aerolysin, was converted to an aerolysin binding form when it was expressed as a GPI-anchored hybrid in COS cells. Not all GPI-anchored proteins bind aerolysin. In some cases this may be due to differences in the structure of the anchor itself. Thus the GPI-anchored proteins procyclin of Trypanosoma congolense and gp63 of Leishmania major did not bind aerolysin, but when gp63 was expressed with a mammalian GPI anchor in Chinese hamster ovary cells, it bound the toxin.

Animals↗
PubMed1998

Movement of a loop in domain 3 of aerolysin is required for channel formation.

Aerolysin is a channel-forming toxin that must oligomerize in order to become insertion-competent. Modeling based on the crystal structure of the proaerolysin dimer and electron microscopic images of the oligomer indicated that a loop in domain 3 must move away from the beta-sheet that forms the main body of the protein before oligomerization can proceed. In order to determine if movement actually occurs, strategically located amino acids in the loop and in the sheet were replaced with cysteines by site-directed mutagenesis. A double mutant was produced in which the new cysteines, at position 253 on the loop and position 300 in the sheet, were close enough together to allow formation of a disulfide bridge. The double mutant was unable to oligomerize, and it was completely inactive, showing not only that the bridge had formed but also that movement of the loop was essential for formation of the oligomer. The existence of the bridge was confirmed by X-ray crystallography. The reduced form of the protein and the single mutants T253C and A300C were as active as wild type, indicating that the amino acid replacements themselves had no functional consequences. Labeling studies using an environment-sensitive fluorescent sulfhydryl-reactive probe confirmed that the structure of the protein changes in the loop region as a consequence of proteolytic activation of proaerolysin, a step which also must precede oligomerization.

Aeromonas↗
PubMed1998

Aerolysin--a paradigm for membrane insertion of beta-sheet protein toxins?

The determination of the crystal structure of the bacterial protein proaerolysin provided the first view of a pore-forming toxin constructed mainly from beta-sheet. The structure that was obtained and subsequent crystallographic and biochemical studies have together allowed us to explain how the toxin is transformed from a water-soluble dimer to a heptameric transmembrane pore. Recent discoveries of structural similarities between aerolysin and other toxins suggest that the structure/function studies we have made may prove useful in understanding the actions of a number of pore-forming proteins.

Amino Acid Sequence↗
PubMed1998

Secretion and properties of the large and small lobes of the channel-forming toxin aerolysin.

Aerolysin is a dimeric protein secreted by Aeromonas spp. that binds to glycosylphosphatidylinositol-anchored receptors on target cells and becomes insertion competent by oligomerizing. The protein comprises two lobes joined by a short arm. The large lobe is thought to be responsible for channel formation, whereas the small lobe is believed to stabilize the dimer, and it may also contain the receptor binding site. We cloned and expressed the DNA for both lobes of the toxin separately and together in A. salmonicida. The large lobe produced alone was secreted, although more poorly than native protein. The small lobe with the arm produced by itself was not secreted. When the large lobe without the arm was co-produced with the small lobe with the arm, both were secreted, and they co-purified as a stoichiometric complex. Analytical ultracentrifugation showed that they form a heterotetramer corresponding to the native dimer. The purified product was nearly as active as aerolysin, but lost activity and became trypsin sensitive above 25 degreesC. The large lobe with the arm was also purified. It was shown to be monomeric, confirming that the small lobe is responsible for dimer stabilization. The large lobe had very low channel-forming activity, although it was correctly processed by trypsin, and it could form stable oligomers. Surprisingly, the large lobe was found to bind to several glycosylphosphatidylinositol-anchored proteins, indicating that it contains at least part of the receptor-binding domain.

Aeromonas↗