Biomedical subjects

J T Buckley

Publications and source records attributed to J T Buckley.

At least 55 records · Page 3Linked to original sources

PubMed1994

Structure of the Aeromonas toxin proaerolysin in its water-soluble and membrane-channel states.

Aerolysin is chiefly responsible for the pathogenicity of Aeromonas hydrophila, a bacterium associated with diarrhoeal diseases and deep wound infections. Like many other microbial toxins, the protein changes in a multistep process from a completely water-soluble form to produce a transmembrane channel that destroys sensitive cells by breaking their permeability barriers. Here we describe the structure of proaerolysin determined by X-ray crystallography at 2.8 A resolution. The protoxin (M(r) 52,000) adopts a novel protein fold. Images of an aerolysin oligomer derived from electron microscopy have assisted in constructing a model of the membrane channel and have led to the proposal of a scheme to account for insertion of the protein into lipid bilayers to form ion channels.

Aeromonas↗

PubMed1994

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.

Animals↗

PubMed1993

Physical and chemical characterization of the oligomerization state of the Aeromonas hydrophila lipase/acyltransferase.

Aeromonas glycerophospholipid:cholesterol acyl transferase undergoes a conformational transition upon activation by treatment with trypsin. Chemical cross-linking and sedimentation velocity analysis showed that the lipase dimerizes due to removal of a region near its C-terminus. The lipase monomer has a sedimentation coefficient s20.w = 2.83 S, whereas the dimer has s20.w = 3.65 +/- 0.22 S. Hydrodynamic analysis using these sedimentation values and the masses determined by mass spectrometry indicated that the monomers are aligned side-by-side in the dimer. An important change occurs in the apparent partial specific volume of the molecule upon activation.

Acyltransferases↗

PubMed1993

Dimerization stabilizes the pore-forming toxin aerolysin in solution.

Aerolysin is a channel-forming protein secreted as a protoxin by Aeromonas hydrophila. Analytical centrifugation measurements showed that proaerolysin is a dimer in solution, and this was confirmed by chemical cross-linking with dimethyl suberimidate. Dissociation of proaerolysin with low concentrations of SDS resulted in the loss of tertiary structure, assessed by near ultraviolet circular dichroism. This was accompanied by an increase in the protein's ability to bind the hydrophobic dye 1-anilino-8-naphthalene sulfonate, as well as by increased sensitivity to proteolytic degradation. However, the monomer was not fully unfolded by the detergent, as the tryptophans remained in a hydrophobic environment, and the secondary structure measured by far ultraviolet circular dichroism did not seem to be affected. Aerolysin, the active form of the protein, was also shown to be a dimer, and its stability was found to be no different from the stability of the protoxin dimer. Substituting tryptophan 371 or tryptophan 373 with leucine greatly reduced the stability of dimeric proaerolysin. These substitutions are known to increase the protein's ability to oligomerize, supporting the conclusion that dimer dissociation is necessary for oligomerization to occur.

Amino Acid Sequence↗

PubMed1993

Oligomerization of the channel-forming toxin aerolysin precedes insertion into lipid bilayers.

Oligomerization is a necessary step in channel formation by the bacterial toxin aerolysin. We have identified a region of aerolysin containing two tryptophans which influence the ability of the protein to oligomerize. Changing the tryptophan at position 371 or 373 to leucine resulted in mutant proteins that oligomerized at much lower concentrations than the wild-type toxin. Near-ultraviolet circular dichroism measurements showed that the tertiary structures of the L-371 and L-373 mutant toxins may be slightly different from the structure of wild type. Other single amino acid replacements in the same region of the protein as the two tryptophans appeared to have little or no effect on any properties of the protein. None of the changes we made had any measured effect on secretion of the protein by the bacteria. The L-373 and L-371 proteins induced chloride release from liposomes at lower concentrations than native toxin. Wild-type aerolysin solutions were completely unable to cause release when oligomeric toxin was absent or when it was removed by centrifugation. Aerolysin changed at H-132, which cannot form oligomers, was also inactive against liposomes. We conclude that aerolysin channels are produced by direct insertion of oligomers formed in solution, or assembled on the surface of the cell after binding to the receptor, and not by lateral diffusion of the monomer after it enters the lipid bilayer.

Amino Acid Sequence↗

PubMed1993

Aeromonas spp. can secrete Escherichia coli alkaline phosphatase into the culture supernatant, and its release requires a functional general secretion pathway.

Aerolysin is a channel-forming protein secreted by Aeromonas hydrophila. To determine if regions of aerolysin could direct the secretion of another protein, portions of aerA were fused to phoA, the Escherichia coli alkaline phosphatase gene and cloned into E. coli, Aeromonas salmonicida, and A. hydrophila. We were surprised to find that secretion of the enzyme by both Aeromonas spp. was independent of the aerolysin segments fused to it. The smallest fusion product contained only the signal sequence and two amino acids of aerolysin. The largest had more than 90% of the aerolysin molecule. The fusion proteins were found in the periplasms of E. coli and A. salmonicida grown in LB medium containing glucose, as well as in the shocked cells. Aerolysin itself was secreted by A. salmonicida under these conditions. In contrast, when A. salmonicida containing any of the fused genes was grown in LB medium without glucose, most of the alkaline phosphatase activity was extracellular, whereas beta-lactamase remained in its normal periplasmic location. Similar results were obtained with A. hydrophila. The change in location of the enzyme in A. salmonicida appeared to be related to the pH of the growth medium. A. salmonicida and A. hydrophila also secreted native E. coli alkaline phosphatase, but A. hydrophila strains with mutations in the general secretion pathway were unable to release the enzyme. We conclude that the Aeromonas secretion system can recognize the E. coli enzyme as an extracellular protein and direct it outside the cell.

Aeromonas↗

PubMed1992

Spectroscopic study of the activation and oligomerization of the channel-forming toxin aerolysin: identification of the site of proteolytic activation.

The channel-forming protein aerolysin is secreted as a protoxin which can be activated by proteolytic removal of a C-terminal peptide. The activation and subsequent oligomerization of aerolysin were studied using a variety of spectroscopic techniques. Mass spectrometric determination of the molecular weights of proaerolysin and aerolysin permitted identification of the sites at which the protoxin is processed by trypsin and chymotrypsin. The results of far- and near-UV circular dichroism measurements indicated that processing with trypsin does not lead to major changes in secondary or tertiary structure of the protein. An increase in tryptophan fluorescence intensity and a small red shift in the maximum emission wavelength of tryptophans could be observed, suggesting that there is a change in the environment of some of the tryptophans. There was also a dramatic increase in the binding of the hydrophobic fluorescent probe 1-anilino-8-naphthalenesulfonate during activation, leading us to conclude that a hydrophobic region in the protein is exposed by trypsin treatment. Using measurements of light scattering, various parameters influencing oligomerisation of trypsin-activated aerolysin were determined. Oligomerization rates were found to increase with the concentration of aerolysin, whereas they decreased with increasing ionic strength.

Aeromonas hydrophila↗

PubMed1992

Crossing three membranes. Channel formation by aerolysin.

Aerolysin is a channel-forming toxin responsible for the pathogenicity of Aeromonas hydrophila. It crosses the inner and outer membranes of the bacteria in separate steps and is released as a 52-kDa inactive protoxin which is activated by proteolytic removal of approximately 40 amino acids from the C terminus. The toxin binds to the erythrocyte transmembrane protein glycophorin and oligomerizes before inserting into the membrane, producing a voltage gated, anion selective channel about 1 nm in diameter. Remarkably, proaerolysin appears to be dimeric, whereas the oligomer is a heptamer. Using chemical modification and site-directed mutagenesis, we have identified some of the regions of the molecule which appear to be involved in secretion and in channel formation.

Bacterial Toxins↗

PubMed1992

Stereochemical and positional specificity of the lipase/acyltransferase produced by Aeromonas hydrophila.

Aeromonas species secrete a glycerophospholipid-cholesterol acyltransferase (GCAT) which shares many properties with mammalian plasma lecithin-cholesterol acetyltransferase (LCAT). We have studied the stereochemical and positional specificity of GCAT against a variety of lipid substrates using NMR spectroscopy as well as other assay methods. The results show that both the primary and secondary acyl ester bonds of L-phosphatidylcholine can be hydrolyzed but only the sn-2 fatty acid can be transferred to cholesterol. The enzyme has an absolute requirement for the L configuration at the sn-2 position of phosphatidylcholine. The secondary ester bond of D-phosphatidylcholine cannot be hydrolyzed, and this lipid is not a substrate for acyl transfer. In contrast to the phospholipases, but similar to LCAT, the enzyme does not interact stereochemically with the phosphorus of phosphatidylcholine. In fact, the phosphorus is not required for enzyme activity, as GCAT will also hydrolyze monolayers of diglyceride, although at much lower rates.

Acyltransferases↗

PubMed1992

The aerolysin membrane channel is formed by heptamerization of the monomer.

The cytolytic toxin aerolysin has been found to form heptameric oligomers by SDS-PAGE electrophoresis, STEM mass measurements of single oligomers and image analysis of two-dimensional membrane crystals. Two types of crystal, flat sheets and long regular tubes, have been obtained by reconstitution of purified protein and Escherichia coli phospholipids. A noise-filtered image of the best crystalline sheets reveals a structure with 7-fold symmetry containing a central strongly stain-excluding ring that encircles a dark stain-filled channel 17 A in diameter. The ring is surrounded by seven arms each made up of two unequal sized domains. By combining projected views and side-views, a simplified model of the aerolysin channel complex has been constructed. The relevance of this structure to the mode of action of aerolysin is discussed.

Bacterial Toxins↗

PubMed1992

Purification of Aeromonas hydrophila major outer-membrane proteins: N-terminal sequence analysis and channel-forming properties.

Four outer-membrane proteins of Aeromonas hydrophila were purified and their N-terminal sequences and channel-forming properties were determined. Three could be matched with proteins from other species. One was a maltoporin, as its level increased when cells were grown in maltose-containing media, and the channel it formed was blocked by maltose. Another was like OmpF and OmpC of Escherichia coli, except that its channel fluctuated much more rapidly. The third protein, which was produced in low-phosphate medium, exhibited several properties of the general anion porin PhoE. The fourth showed no similarity to any known proteins. It had a unique N-terminus and it formed small sharply-defined cation-selective channels. Two other proteins which corresponded to OmpW of Vibrio cholerae and E. coli OmpA were partly characterized.

Aeromonas hydrophila↗

PubMed1992

The channel-forming toxin aerolysin.

Aeromonas sp. secrete a precursor of the cytolytic protein aerolysin into the culture medium, where it is activated by proteolytic removal of a C-terminal fragment. Activation can be achieved by a variety of mammalian proteases as well as by proteases released by the bacteria itself. Activated toxin binds with high affinity to the transmembrane protein glycophorin on the surface of eucaryotic cells. Binding is followed by oligomerization and the formation of transmembrane channels, leading to cell death. Using chemical modification and site-directed mutagenesis, we have identified regions of the molecule which are important in transfer across the outer membrane of the bacteria, and in proteolytic activation, binding, and oligomerization. A preliminary electron density map of proaerolysin crystals indicates that the protein is organized into three domains. Analysis of two-dimensional crystals of aerolysin suggests that the oligomeric form of the protein is heptameric.

Amino Acid Sequence↗

PubMed1991

Site-directed mutagenesis of a single tryptophan near the middle of the channel-forming toxin aerolysin inhibits its transfer across the outer membrane of Aeromonas salmonicida.

The channel-forming protein aerolysin must cross both the inner and outer bacterial membranes during its secretion from Aeromonas hydrophila or from Aeromonas salmonicida containing the cloned structural gene. We examined the fate of three mutant proteins in which Trp-227, near the middle of the amino acid chain, was replaced with glycine, leucine, or phenylalanine by site-directed mutagenesis. All three proteins crossed the inner membrane and entered the periplasm in the same way as wild-type, and in each case the signal sequence was removed correctly. Little or none of the proaerolysin substituted with glycine or leucine was released into the culture supernatant. Instead, significant amounts became associated with the outer membrane. The Phe-227 protoxin was secreted by the bacteria but at a reduced rate. The leucine and phenylalanine mutant proteins were purified and compared with native proaerolysin. They were processed correctly to the mature forms by treatment with trypsin, and like native aerolysin, both were resistant to further proteolysis. In each case, processing was followed by the formation of oligomers similar to those produced by native toxin. The hemolytic activity of the processed Phe-227 mutant was one-quarter that of wild-type toxin whereas Leu-227 aerolysin had less than one-hundredth the wild-type activity. These results are further evidence that aerolysin is secreted in at least two steps. As well, they show that the last step, crossing the outer membrane, can be blocked by an apparently small change in the structure of the protein.

Aeromonas↗

PubMed1991

Action of a microbial lipase/acyltransferase on phospholipid monolayers.

Vibrio species release a lipase which shares many properties with mammalian lecithin-cholesterol acyltransferase. We have studied the action of the enzyme on phospholipid monolayers. At similar surface pressures, reaction velocities were higher with monolayers of dilauroylphosphatidylcholine than with the corresponding phosphatidylglycerol or phosphatidylethanolamine. The dependence of reaction velocity on molecular density was very similar for phosphatidylcholine and phosphatidylethanolamine monolayers. Lag times were shortest with phosphatidylglycerol at low molecular densities, but maximum velocity was reached at considerably lower densities than with the other two lipids. We have found [Hilton, S., McCubbin, N. D., Kay, C., & Buckley, J.T. (1990) Biochemistry 29, 9072-9078] that nicking of the enzyme with trypsin or other proteases results in an increase in its activity against lipids in membranes. Here we show that trypsin treatment results in a large change in the surface activity of the lipase, allowing it to penetrate monolayers at pressures higher than 40 mN.m-1.

Acyltransferases↗

PubMed1991

Secretion and mechanism of action of the hole-forming toxin aerolysin.

Aeromonas hydrophila exports aerolysin as a protoxin which is activated by proteolysis after release. Aerolysin binds to the eucaryotic cell receptor glycophorin and oligomerizes, forming holes in the membrane. Important regions of the molecule have been identified by site-directed mutagenesis, and channel formation has been studied in planar lipid bilayers.

Aeromonas↗

PubMed1991

Studies on the reaction mechanism of a microbial lipase/acyltransferase using chemical modification and site-directed mutagenesis.

Aeromonas hydrophila releases a protein which is a member of the lipase superfamily, similar in reaction mechanism to the important mammalian plasma enzyme lecithin-cholesterol acyltransferase. We have used chemical modification and site-directed mutagenesis of the protein to identify amino acids which may be involved in catalysis. The enzyme was unaffected by phenylmethylsulfonyl fluoride, but it was almost completely inhibited by another serine-reactive compound, diethyl p-nitrophenyl phosphate. A serine selectively modified by this reagent was identified by sequencing the amino-terminal region of the protein. It was located at position 16, in the short consensus sequence shared by the enzyme with other lipases. When this serine was changed to asparagine the product was an enzymatically inert protein which nevertheless retained the surface activity of the wild-type enzyme, suggesting its ability to bind to substrate was not changed. Diethylpyrocarbonate treatment drastically reduced the rate of acyl transfer by the native enzyme, but this did not appear to be due to modification of an essential histidine, since inhibition was not reversed by addition of hydroxylamine. We have shown that only two of the histidines in the enzyme can be involved in catalysis (Hilton, S., McCubbin, W. D., Kay, C.M., and Buckley, J. T. (1990) Biochemistry, 29, 9072-9078). Replacing both of these with asparagines had little or no effect on enzyme activity. These results indicate that, in apparent contrast to other lipases, histidine does not participate in the reaction catalyzed by the microbial enzyme. Since catalysis was not inhibited by sulfhydryl reagents, we conclude that a free cysteine is also not required for activity. This may distinguish the microbial enzyme from the mammalian acyltransferase.

Acyltransferases↗

PubMed1991

Site-directed mutagenesis at histidines of aerolysin from Aeromonas hydrophila: a lipid planar bilayer study.

The role of histidine residues in the formation of channels by the cytolytic toxin aerolysin has been studied in planar lipid bilayers by substituting each of the six histidines in the native protein with asparagine. His341 or His186 mutants had the same channel-forming ability as native toxin, whereas the His332 and His121 mutants were less active. Mutations at His132 and His107, which interfere with the oligomerization of the toxin, drastically reduce pore formation. These findings support the conclusion that oligomerization of the toxin must precede channel formation, and that at least two of the six histidine residues are essential for this to occur. The aerolysin channel is a water-filled pore with an approximate diameter of 9.3 +/- 0.4 A.

Aeromonas hydrophila↗

PubMed1990

Purification and spectral study of a microbial fatty acyltransferase: activation by limited proteolysis.

A fatty acyltransferase with a reaction mechanism similar to that of mammalian lecithin: cholesterol acyltransferase has been purified from culture supernatants of a mutant Aeromonas salmonicida containing the cloned Aeromonas hydrophila structural gene. Typically, more than 35 mg of protein were isolated from 2 L of culture supernatant. The amino-terminal sequence, amino acid composition, and molecular weight of the purified protein corresponded to predictions based on the sequence of the gene, indicating that the signal sequence had been correctly removed during export but that no further processing had occurred. Analysis of the far-UV circular dichroic (CD) spectrum of the enzyme showed that it consists of 31% alpha-helix, 21% beta-sheet, and 16% beta-turn, with 12% of aperiodic form. Treatment of the purified protein with a variety of proteases resulted in nicking near the C-terminus. This led to an increase in enzyme activity against lipids in erythrocyte membranes and increased rate of hydrolysis of p-nitrophenyl butyrate. Activation was accompanied by a change in the CD spectrum and a change in its aggregation state. The trypsin cut site was located between the two cysteines in the enzyme. Evidence is presented that the cysteines are joined by a disulfide bond and therefore cannot participate in acyl transfer. This may distinguish the microbial enzyme from lecithin:cholesterol acyltransferase. This is the second extracellular A. hydrophila protein that we have shown can be activated by proteolysis after it is released.

Acyltransferases↗