J. Thomas Buckley

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.

Aerolysin--the ins and outs of a model channel-forming toxin

Aerolysin is one of a large group of bacterial proteins that can kill target cells by forming discrete channels in their plasma membranes. The toxin has many properties in common with the porins of the Gram‐negative bacterial outer membrane, including an extensive amount of β‐structure, a high proportion of hydrophilic amino acid side‐chains and no hydrophobic stretches in the primary structure. It also oligomerizes to produce an insertion‐competent state. Aerolysin is secreted as a dimer by members of the Aeromonas family. It binds to a high‐affinity receptor on the target cell that has recently been shown to be a glycosylphosphatidylinositol‐anchored glycoprotein. Binding is followed by heptamerization to form a structure that we propose contains a β‐barrel which can insert into the membrane and produce a channel.

Molecular cloning and expression in Escherichia coli of the structural gene for the hemolytic toxin aerolysin from Aeromonas hydrophila

SummaryThe structural gene for the hemolytic toxin aerolysin has been cloned into the plasmid vectors pBR322 and pEMBL8+. The gene was localized on the hybrid plasmids by analysis of plasmids generated by transposon mutagenesis. The sequence of the first 683 bases of an insert in pEMBL8+ was determined and shown to encode the amino terminus of the protein as well as a typical signal sequence of 23 amino acids. Aerolysin is produced by E. coli cells containing the cloned aerolysin gene and it is processed normally by removal of the signal sequence, however it is not released from the cell. The protein appears to be translocated across the inner membrane of E. coli as its signal sequence is removed and the processed protein can be released by osmotic shock.

Vibrio spp. secrete proaerolysin as a folded dimer without the need for disulphide bond formation

Proaerolysin is an extracellular dimeric protein that is secreted across the inner and outer membranes of Aeromonas spp. in separate steps. To investigate the role of protein folding in the second step, one or more cysteine residues were introduced and the mutant proaerolysins were expressed in Aeromonas hydrophila and Aeromonas salmonicida, as well as Vibrio cholerae. Replacing Met‐41 with Cys resulted in expression of a protein that could form a dimer in which the monomers were linked together by a disulphide bridge. A double mutant was also made, in which Gly‐202 and Ile‐445 were replaced with cysteine in order to allow the formation of an intrachain disulphide bridge when the molecule was correctly folded. The M41C covalent dimer and G202C/I445C proaerolysin with the new intrachain bridge were both easily detected inside the bacteria, and they later appeared in the culture supernatants. Small amounts of incorrectly folded proaerolysin were also observed in the cells, but they were not secreted. We conclude that proaerolysin folds and dimerizes before being released from the cell, and that correct folding is a requirement for secretion to occur. The proton ionophore CCCP reduced release of the folded proteins. Unoxidized protein was secreted by cells grown in β‐mercaptoethanol and by a dsbA mutant of V. cholerae, indicating that disulphide bond formation may not be essential for release.

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.

The erythrocyte receptor for the channel-forming toxin aerolysin is a novel glycosylphosphatidylinositol-anchored protein

The plasma membrane of rat erythrocytes contains a 47‐kDa glycoprotein that binds the channel‐forming toxin aerolysin with high affinity and accounts for the sensitivity of these cells to the toxin. The receptor was purified so that its N‐terminal sequence could be determined after Western blotting. The sequence did not match any sequences in the databases, indicating that the receptor is a novel erythrocyte surface protein. However, it exhibited considerable homology to the N‐termini of a group of membrane proteins that are thought to be involved in ADP‐ribosyl transfer reactions. A common property of these proteins is that they are attached to plasma membranes by C‐terminal glycosylphosphatidylinositol (GPI) anchors. The aerolysin receptor was shown to be anchored in the same way by treating rat erythrocytes with phosphatidylinositol‐specific phospholipase C. This caused the selective release of the receptor and a reduction in the rodent cells’ sensitivity to aerolysin. Human and bovine erythrocytes were shown to contain an aerolysin‐binding protein with similar properties to the rat erythrocyte receptor. Proteins with GPI anchors are thought to have unusually high lateral mobility, and this may be an advantage for a toxin, such as aerolysin, which must oligomerize after binding to become insertion competent.

Aerolysin and pertussis toxin share a common receptor‐binding domain

We have discovered that the bacterial toxins aerolysin and pertussis toxin share a common domain. This is surprising because the two toxins affect cells in very different ways. The common domain, which we call the APT domain, consists of two three‐stranded antiparallel β‐sheets that come together and wrap around a central pair of helices. The APT domain shares a common fold with the C‐type lectins and Link modules, and there appears to be a divergent relationship among the three families. One surface region of the APT domain is highly conserved, raising the possibility that the domains have a common function in both proteins. Mutation of one of the conserved surface residues in aerolysin, Tyr61, results in reduced receptor binding and activity, thus providing evidence that the APT domain may be involved in interaction with the toxins receptor. Structural and biochemical evidence suggests that the APT domain contains a carbohydrate‐binding site that can direct the toxins to their target cells.

Properties of human erythrocyte phosphatidylinositol kinase and inhibition by adenosine, ADP and related compounds

1. An improved assay for the enzyme phosphatidylinositol kinase is described. The kinase activity is increased more than 10-fold by the addition of mercaptoethanol and exogenous phosphatidylinositol in the presence of Triton X-100. The enzyme is solubilized by non-ionic detergents. 2. Phosphatidylinositol kinase is inhibited by physiological concentrations of ADP and by similar levels of adenosine. Cyclic AMP, AMP and theophylline inhibit at higher concentrations. Caffeine is much less effective than theophylline as an inhibitor. Guanine nucleotides do not appreciably inhibit the kinase. All the inhibitors tested seemed to compete with ATP. Theophylline also reduced the rate of polyphosphoinositide synthesis in intact cells. 3. Possible roles of polyphosphoinositides in energy charge maintenance and inversion and resealing are discussed.

Molecular cloning of a phospholipid-cholesterol acyltransferase from Aeromonas hydrophila. Sequence homologies with lecithin-cholesterol acyltransferase and other lipases

We have determined the nucleotide sequence of a gene encoding Aeromonas hydrophila phospholipid-cholesterol acyltransferase, an enzyme which shares many properties with mammalian lecithin:cholesterol acyltransferase. The derived amino acid sequence of the protein contains two regions which are homologous to the proposed active sites and binding sites of the plasma acyltransferase and to similar sequences in other interfacially acting lipolytic enzymes. The amino terminus is preceded by a typical 18 amino acid signal sequence. The protein, which is released into the culture supernatant by Aeromonas hydrophila, is confined to the periplasm of Escherichia coli.

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 glycosylphosphatidylinositol‐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.