Site-directed mutagenesis of the hole-forming toxin aerolysin: studies on the roles of histidines in receptor binding and oligomerization of the monomer

The six histidines of the channel-forming protein aerolysin have been replaced one at a time with asparagine by site-directed mutagenesis, and each of the modified proteins has been purified. Three proteins had the same hemolytic activity as native toxin, but the others, those changed at His107, His132, or His332, were less able to disrupt both human and rat erythrocytes. The largest reduction in activity, more than 100-fold, was observed with the His132 mutant protein. Studies with radioiodinated samples showed that it had approximately the same affinity as native aerolysin for the rat erythrocyte receptor. However, once bound to either rat or human erythrocytes, it was much less able to carry out the next essential step in hole formation, aggregation to form a stable oligomer. Aggregation was also reduced by replacing His107, but the contrast with native aerolysin and the effect on hemolytic activity were less pronounced. The protein modified at His332 behaved in a different way from those substituted at positions 107 and 132. Its affinity for the rat erythrocyte receptor was considerably lower than the affinity of the wild-type protein, but when bound it appeared to aggregate normally. The results suggest that His132 and perhaps His107 are involved in the aggregation of aerolysin whereas His332 may be at or near the receptor binding site.     

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.     

Partial purification of the rat erythrocyte receptor for the channel-forming toxin aerolysin and reconstitution into planar lipid bilayers

The cytolytic toxin aerolysin binds to a receptor on the surface of eukaryotic cells. Murine erythrocytes are among the most sensitive to the toxin. Here we describe the detergent solubilization and partial purification of the receptor from rat erythrocytes. We show that it can be successfully incorporated into planar lipid bilayers, greatly decreasing the concentration of aerolysin required to form channels. Exploiting the ability of the receptor to bind aerolysin after SDS electrophoresis and blotting, we obtain evidence that it is a 47 kDa glycoprotein that is sensitive to proteases and N-glycosidase. It may correspond to CHIP28, the water channel of the human erythrocyte.     

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.     

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.     

Use of Clostridium septicum alpha toxins for isolation of various glycosylphosphatidylinositol-deficient cells

In eukaryotic cells, various proteins are anchored to the plasma membrane through glycosylphosphatidylinositol (GPI). To study the biosynthetic pathways and modifications of GPI, various mutant cells have been isolated from the cells of Chinese hamster ovaries (CHO) supplemented with several exogenous genes involved in GPI biosynthesis using aerolysin, a toxin secreted from gram-negative bacterium Aeromonas hydrophila. Alpha toxin from Gram-positive bacterium Clostridium septicum is homologous to large lobes (LL) of aerolysin, binds GPI-anchored proteins and possesses a cell-destroying mechanism similar to aerolysin. Here, to determine whether alpha toxins can be used as an isolation tool of GPI-mutants, like aerolysin, CHO cells stably transfected with several exogenous genes involved in GPI biosynthesis were chemically mutagenized and cultured in a medium containing alpha toxins. We isolated six mutants highly resistant to alpha toxins and deficient in GPI biosynthesis. By genetic complementation, we determined that one mutant cell was defective of the second subunit of dolichol phosphate mannose synthase (DPM2) and other five cells were of a putative catalytic subunit of inositol acyltransferase (PIG-W). Therefore, C. septicum alpha toxins are a useful screening probe for the isolation of various GPI-mutant cells.     

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.     

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.     

Generation and characterization of Clostridium septicum alpha toxin mutants and their use in diagnosing paroxysmal nocturnal hemoglobinuria

Glycosylphosphatidylinositol (GPI) anchors various proteins to the membrane of eukaryotic cells. Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell disorder that is primarily due to the lack of GPI-anchored proteins on the surface of blood cells. To detect the GPI-deficient cells in PNH patients, we modified alpha toxin, a pore-forming toxin of the Gram-positive bacterium Clostridium septicum. We first showed that aerolysin, a homologous toxin from Aeromonas hydrophila, bound to both of Chinese hamster ovary cells deficient of N-glycan maturation as well as GPI biosynthesis at a significant level. However, alpha toxin bound to the mutant cells of N-glycosylation, but not to GPI-deficient cells. It suggested that alpha toxin could be used as a specific probe to differentiate only GPI-deficient cells. As a diagnostic probe, alpha toxin must be the least cytotoxic while maintaining its affinity for GPI. Thus, we constructed several mutants. Of these, the mutants carrying the Y155G or S189C/S238C substitutions bound to GPI as well as the wild-type toxin. These mutants also efficiently underwent proteolytic activation and aggregated into oligomers on the cell surface, which are events that precede the formation of a pore in the host cell membrane, leading to cell death. Nevertheless, these mutants almost completely failed to kill host cells. It was revealed that the substitutions affect the events that follow oligomerization. The S189C/S238C mutant toxin differentiated GPI-deficient granulocyte and PMN, but not red blood cells, of a PNH patient from GPI-positive cells at least as sensitively as the commercial monoclonal antibodies that recognize the CD59 or CD55 GPI proteins on blood cells. Thus, this modified bacterial toxin can be employed instead of costly monoclonal antibodies to diagnose PNH patients.     

Requirement of N-glycan on GPI-anchored proteins for efficient binding of aerolysin but not Clostridium septicum alpha-toxin

Aerolysin of the Gram-negative bacterium Aeromonas hydrophila consists of small (SL) and large (LL) lobes. The alpha-toxin of Gram-positive Clostridium septicum has a single lobe homologous to LL. These toxins bind to glycosylphosphatidylinositol (GPI)-anchored proteins and generate pores in the cell's plasma membrane. We isolated CHO cells resistant to aerolysin, with the aim of obtaining GPI biosynthesis mutants. One mutant unexpectedly expressed GPI-anchored proteins, but nevertheless bound aerolysin poorly and was 10-fold less sensitive than wild-type cells. A cDNA of N-acetylglucosamine transferase I (GnTI) restored the binding of aerolysin to this mutant. Therefore, N-glycan is involved in the binding. Removal of mannoses by alpha-mannosidase II was important for the binding of aerolysin. In contrast, the alpha-toxin killed GnTI-deficient and wild-type CHO cells equally, indicating that its binding to GPI-anchored proteins is independent of N-glycan. Because SL bound to wild-type but not to GnTI-deficient cells, and because a hybrid toxin consisting of SL and the alpha-toxin killed wild-type cells 10-fold more efficiently than GnTI- deficient cells, SL with its binding site for N-glycan contributes to the high binding affinity of aerolysin.     

Genome-Wide Screening of Genes Required for Glycosylphosphatidylinositol Biosynthesis

Glycosylphosphatidylinositol (GPI) is synthesized and transferred to proteins in the endoplasmic reticulum (ER). GPI-anchored proteins are then transported from the ER to the plasma membrane through the Golgi apparatus. To date, at least 17 steps have been identified to be required for the GPI biosynthetic pathway. Here, we aimed to establish a comprehensive screening method to identify genes involved in GPI biosynthesis using mammalian haploid screens. Human haploid cells were mutagenized by the integration of gene trap vectors into the genome. Mutagenized cells were then treated with a bacterial pore-forming toxin, aerolysin, which binds to GPI-anchored proteins for targeting to the cell membrane. Cells that showed low surface expression of CD59, a GPI-anchored protein, were further enriched for. Gene trap insertion sites in the non-selected population and in the enriched population were determined by deep sequencing. This screening enriched 23 gene regions among the 26 known GPI biosynthetic genes, which when mutated are expected to decrease the surface expression of GPI-anchored proteins. Our results indicate that the forward genetic approach using haploid cells is a useful and powerful technique to identify factors involved in phenotypes of interest.     

Diagnosis of paroxysmal nocturnal hemoglobinuria by fluorescent clostridium septicum alpha toxin

Paroxysmal nocturnal hemoglobinuria (PNH), a hematopoietic stem cell disorder, is caused by the loss of glycosylphosphatidylinositol (GPI)-anchored proteins on the cell membrane. PNH can be simply diagnosed by flow cytometry using monoclonal antibodies against GPI-anchored proteins or fluorescent-tagged aerolysin, a bacterial toxin that binds GPI anchored proteins. Clostridium septicum alpha toxin is homologous to aerolysin and specifically binds GPI-anchored proteins. Previously, we found that an alpha toxin m45 mutant with two amino acid changes, S189C/S238C, lost cytotoxicity but still possessed binding activity for GPI-anchored proteins. To use this mutant toxin as a diagnostic probe in flow cytometry, we constructed the EGFP-AT(m45) expression vector, comprising a S189C/S238C alpha toxin mutant with EGFP and His tags at the N and C termini, respectively. The recombinant EGFP-AT(m45) was easily purified using single-step affinity chromatography against His tag from Escherichia coli. EGFP-AT(m45) bound to CHO and HeLa cells in a similar manner to monoclonal antibodies against GPI-anchored proteins or aerolysin. In whole blood from a PNH patient, GPI-deficient granulocytes could be differentiated by EGFP-AT(m45) using the same procedure as that employed with commercially available monoclonal antibodies. Therefore, nontoxic EGFP-conjugated C. septicum alpha toxin could be used clinically for PNH diagnosis.     

Site-Specific Chemoenzymatic Labeling of Aerolysin Enables the Identification of New Aerolysin Receptors

Aerolysin is a secreted bacterial toxin that perforates the plasma membrane of a target cell with lethal consequences. Previously explored native and epitope-tagged forms of the toxin do not allow site-specific modification of the mature toxin with a probe of choice. We explore sortase-mediated transpeptidation reactions (sortagging) to install fluorophores and biotin at three distinct sites in aerolysin, without impairing binding of the toxin to the cell membrane and with minimal impact on toxicity. Using a version of aerolysin labeled with different fluorophores at two distinct sites we followed the fate of the C-terminal peptide independently from the N-terminal part of the toxin, and show its loss in the course of intoxication. Making use of the biotinylated version of aerolysin, we identify mesothelin, urokinase plasminogen activator surface receptor (uPAR, CD87), glypican-1, and CD59 glycoprotein as aerolysin receptors, all predicted or known to be modified with a glycosylphosphatidylinositol anchor. The sortase-mediated reactions reported here can be readily extended to other pore forming proteins.     

Listeria monocytogenes phosphatidylinositol (PI)-specific phospholipase C has low activity on glycosyl-PI-anchored proteins.

The ability of the phosphatidylinositol-specific phospholipase C (PI-PLC) from Listeria monocytogenes to hydrolyze glycosyl phosphatidylinositol (GPI)-anchored membrane proteins was compared with the ability of the PI-PLC from Bacillus thuringiensis to hydrolyze such proteins. The L. monocytogenes enzyme produced no detectable release of acetylcholinesterase from bovine, sheep, and human erythrocytes. The cleavage of the GPI anchors of alkaline phosphatase from rat and rabbit kidney slices was less than 10% of the cleavage seen with the PI-PLC from B. thuringiensis. Activity for release of Fc gamma receptor IIIB (CD16) on human granulocytes was also low. Variations in pH and salt concentration had little effect on the release of GPI-anchored proteins. Our data show that L. monocytogenes PI-PLC has low activity on GPI-anchored proteins.     

Efficient glycosylphosphatidylinositol (GPI) modification of membrane proteins requires a C-terminal anchoring signal of marginal hydrophobicity

Many plasma membrane proteins are anchored to the membrane via a C-terminal glycosylphosphatidylinositol (GPI) moiety. The GPI anchor is attached to the protein in the endoplasmic reticulum by transamidation, a reaction in which a C-terminal GPI-attachment signal is cleaved off concomitantly with addition of the GPI moiety. GPI-attachment signals are poorly conserved on the sequence level but are all composed of a polar segment that includes the GPI-attachment site followed by a hydrophobic segment located at the very C terminus of the protein. Here, we show that efficient GPI modification requires that the hydrophobicity of the C-terminal segment is "marginal": less hydrophobic than type II transmembrane anchors and more hydrophobic than the most hydrophobic segments found in secreted proteins. We further show that the GPI-attachment signal can be modified by the transamidase irrespective of whether it is first released into the lumen of the endoplasmic reticulum or is retained in the endoplasmic reticulum membrane.     

Sorting of the human folate receptor in MDCK cells

The human folate receptor (hFR) is a glycosylphosphatidy-linositol (GPI) linked plasma membrane protein that mediates delivery of folates into cells. We studied the sorting of the hFR using transfection of the hFR cDNA into MDCK cells. MDCK cells are polarized epithelial cells that preferentially sort GPI-linked proteins to their apical membrane. Unlike other GPI-tailed proteins, we found that in MDCK cells, hFR is functional on both the apical and basolateral surfaces. We verified that the same hFR cDNA that transfected into CHO cells produces the hFR protein that is GPI-linked. We also measured the hFR expression on the plasma membrane of type III paroxysmal nocturnal hemoglobinuria (PNH) human erythrocytes. PNH is a disease that is characterized by the inability of cells to express membrane proteins requiring a GPI anchor. Despite this defect, and different from other GPI-tailed proteins, we found similar levels of hFR in normal and type III PNH human erythrocytes. The results suggest the hypothesis that there may be multiple mechanisms for targeting hFR to the plasma membrane.     

New mutant Chinese hamster ovary cell representing an unknown gene for attachment of glycosylphosphatidylinositol to proteins

Aerolysin, a secreted bacterial toxin from Aeromonas hydrophila, binds to glycosylphosphatidylinositol (GPI)-anchored protein and kills the cells by forming pores. Both GPI and N-glycan moieties of GPI-anchored proteins are involved in efficient binding of aerolysin. We isolated various Chinese hamster ovary (CHO) mutant cells resistant to aerolysin. Among them, CHOPA41.3 mutant cells showed several-fold decreased expression of GPI-anchored proteins. After transfection of N-acetylglucosamine transferase I (GnT1) cDNA, aerolysin was efficiently bound to the cells, indicating that the resistance against aerolysin in this cells was mainly ascribed to the defect of N-glycan maturation. CHOPA41.3 cells also accumulated GPI intermediates lacking ethanolamine phosphate modification on the first mannose. After stable transfection of PIG-N cDNA encoding GPI-ethanolamine phosphate transferase1, a profile of accumulated GPI intermediates became similar to that of GPI transamidase mutant cells. It indicated, therefore, that CHOPA41.3 cells are defective in GnT1, ethanolamine phosphate modification of the first mannose, and attachment of GPI to proteins. The GPI accumulation in CHOPA41.3 cells carrying PIG-N cDNA was not normalized after transfection with cDNAs of all known components in GPI transamidase complex. Microsomes from CHOPA41.3 cells had normal GPI transamidase activity. Taken together, there is an unknown gene required for efficient attachment of GPI to proteins.     

Assessment of the roles of ordered lipid microdomains in post-endocytic trafficking of glycosyl-phosphatidylinositol-anchored proteins in mammalian fibroblasts

We have used artificial phosphatidylethanolamine-polyethylene glycol (PE-PEG)-anchored proteins, incorporated into living mammalian cells, to evaluate previously proposed roles for ordered lipid 'raft' domains in the post-endocytic trafficking of glycosylphosphatidylinositol (GPI)-anchored proteins in CHO and BHK cells. In CHO cells, endocytosed PE-PEG protein conjugates colocalized strongly with the internalized GPI-anchored folate receptor, concentrating in the endosomal recycling compartment, regardless of the structure of the hydrocarbon chains of the PE-PEG 'anchor'. However, internalized PE-PEG protein conjugates with long-chain saturated anchors recycled to the plasma membrane at a slow rate comparable to that measured for the GPI-anchored folate receptor, whereas conjugates with short-chain or unsaturated anchors recycled at a faster rate similar to that observed for the transferrin receptor. These findings support the proposal (Mayor et al. Cholesterol-dependent retention of GPI-anchored proteins in endosomes. EMBO J 1998;17:4628-4638) that the slow recycling of GPI proteins in CHO cells rests on their affinity for ordered lipid domains. In BHK cells, internalized PE-PEG protein conjugates with either saturated or unsaturated 'anchors' colocalized strongly with simultaneously endocytosed folate receptor and, like the folate receptor, gradually accumulated in late endosomes/lysosomes. These latter findings do not support previous suggestions that the sorting of GPI proteins to late endosomes in BHK cells depends on their association with lipid rafts.     

Transfer of glycosyl-phosphatidylinositol membrane anchors to polypeptide acceptors in a cell-free system

Glycosylinositol phospholipid (GPI) membrane anchors are the sole means of membrane attachment of a large number of cell surface proteins, including the variant surface glycoproteins (VSGs) of the parasitic protozoan, Trypanosoma brucei. Biosynthetic data suggest that GPI-anchored proteins are synthesized with carboxy-terminal extensions that are immediately replaced by GPI, suggesting the existence of preformed GPI species available for transfer to the nascent protein in the ER. Candidate precursor glycolipids having a linear sequence indistinguishable from the conserved core structure found on all GPI anchors, have been characterized in T. brucei. In this paper we describe the transfer of three GPI variants to endogenous VSG in vitro. GPI addition is not reduced by inhibitors of protein synthesis and does not require ATP or GTP, consistent with a transpeptidation mechanism.     

A Pore-forming Toxin Interacts with a GPI-anchored Protein and Causes Vacuolation of the Endoplasmic Reticulum

In this paper, we have investigated the effects of the pore-forming toxin aerolysin, produced by Aeromonas hydrophila, on mammalian cells. Our data indicate that the protoxin binds to an 80-kD glycosyl-phosphatidylinositol (GPI)-anchored protein on BHK cells, and that the bound toxin is associated with specialized plasma membrane domains, described as detergent-insoluble microdomains, or cholesterol-glycolipid “rafts.” We show that the protoxin is then processed to its mature form by host cell proteases. We propose that the preferential association of the toxin with rafts, through binding to GPI-anchored proteins, is likely to increase the local toxin concentration and thereby promote oligomerization, a step that it is a prerequisite for channel formation. We show that channel formation does not lead to disruption of the plasma membrane but to the selective permeabilization to small ions such as potassium, which causes plasma membrane depolarization. Next we studied the consequences of channel formation on the organization and dynamics of intracellular membranes. Strikingly, we found that the toxin causes dramatic vacuolation of the ER, but does not affect other intracellular compartments. Concomitantly we find that the COPI coat is released from biosynthetic membranes and that biosynthetic transport of newly synthesized transmembrane G protein of vesicular stomatitis virus is inhibited. Our data indicate that binding of proaerolysin to GPI-anchored proteins and processing of the toxin lead to oligomerization and channel formation in the plasma membrane, which in turn causes selective disorganization of early biosynthetic membrane dynamics.