Abstract
The cytotoxin from Pseudomonas aeruginosa is a pore-forming toxin that binds specifically to water channel-related molecules of the erythrocyte membrane. Here, we have defined a domain, Asp197-Gly-Asp-Tyr-His-Tyr202 of the cytotoxin, to be essential for receptor binding. Cytotoxin point mutants from the recombinant gene carrying substitutions in the domain were characterized in terms of inhibiting the binding of radioiodinated natural cytotoxin to rat erythrocyte and producing cytotoxic effects in human granulocytes. A synthetic peptide representing residues 191–211 of the cytotoxin acted as a competitive inhibitor at a concentration of 10−5 M. In contrast, two other cytotoxin-specific peptides were inactive. Structure prediction of the binding sequence shows a loop structure with similarities to the sequence around His332 in Aeromonas aerolysin essential to receptor binding.
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Amann E, Ochs B, Abel KJ (1988) Tightly regulated taq promoter vectors useful for the expression of fused and unfused proteins in Escherichia coli. Gene 69:301–315
Bode W, Chen Z, Bartels K., Kutzbach C, Schmidt-Kastner G, Bartunik H (1983) Refined 2 Å X-ray crystal structure of porcine pancreatic kallikrein A, a specific trypsin-like serine proteinase. J Mol Biol 164:237–282
Chen GC. Yang JT (1977) Two-point calibration of circular dichrometer with d-10-camphorsulfonic acid. Anal Lett 10:1195–1207
Chen Z, Bode W (1983) Refined 2.5 Å X-ray crystal structure of the complex formed by porcine kallikrein A and the bovine pancreatic trypsin inhibitor. J Mol Biol 164:283–311
Debus RJ, Barry BA, Sithole I, Babcock GT, McIntosh L (1988) Direct mutagenesis indicate that the donor to p+ 680 in photosystem II is tyrosine-161 of the D1 polypeptide. Biochemistry 27:9071–9074
Farver O, Licht A, Pecht I (1987) Electron transfer pathways in stellacyanin: a possible homology with plastocyanin. Biochemistry 26:7317–7321
Gladstone GP, van Heyningen WE (1957) Staphylococcal leucocidines. Br J Exp Pathol 38:123–127
Green MJ, Buckley JT (1990) Site-directed mutagenesis of the hole-forming toxin aerolysin: studies on the roles of histidines in receptor binding and oligomerization of the monomer. Biochemistry 29:2177–2180
Greenwood FC, Hunter WM, Glover JS (1963) The preparation of 131I-labelled human growth hormone of high specific radioactivity. Biochem J 89:114–123
Hayashi T, Kamino Y, Hishinuma F, Usami Y, Titani K, Terawaki Y (1989) Pseudomonas aeruginosa cytotoxin: the nucleotide sequence of the gene and the mechanism of the activation of the protoxin. Mol Microbial 3:861–868
Hopp TP, Woods KR (1981) Prediction of protein antigenic determinants from amino acid sequences. Proc Natl Acad Sci USA 78:3824–3828
Jonsson M, Lind J, Eriksen E, Merenyi G (1994) Redox and acidity properties of 4-substituted aniline radical cations in water. J Am Chem Soc 116:1423–1427
Kieber-Emmons T (1993) Strategies in protein-based rational drug design. In: Williams WV, Weiner DB (eds) Biologically active peptides: design, synthesis and utilization. Technomic, Lancaster, Basel
Landt O, Grunert HP, Hahn U (1990) A general method for rapid site-directed mutagenesis using the polymerase chain reaction. Gene 96:125–128
Leszcynski J, Rose GD (1986) Loops in globular proteins: a novel category of secondary structure. Science 234:849–855
Lutgenberg B, Meijers J, Peters R, van der Hoeck P, van Alphen L (1975) Electrophoretic resolution of the ‘major outer membrane protein’ of Escherichia coli K12 into four bands. FEBS Lett 58:254–258
Lutz F (1979) Purification of a cytotoxic protein from Pseudomonas aeruginosa. Toxicon 17:467–475
Lutz F (1986) Interaction of Pseudomonas aeruginosa cytotoxin with plasma membranes from Ehrlich ascites tumor cells. Naunyn-Schmiedeberg's Arch Pharmacol 332:103–110
Lutz F Seeger W, Schischke B, Weiner R, Scharmann W (1983) Effects of a cytotoxic protein from Pseudomonas aeruginosa on phagocytic and pinocytic cells: in vitro and in vivo studies. Toxicon [Suppl] 3:257–260
Lutz F, Mohr M, Grimmig M. Leidolf R, Linder D (1993) Pseudomonas aeruginosa cytotoxin-binding protein in rabbit erythrocyte membranes. An oligomer of 28 kDa with similarity to transmembrane channel proteins. Eur J Biochem 217:1123–1128.
Markwell MAK, Haas SM, Bieber LL, Tolbert NE (1978) A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 87:206–210
Mishra AK, Chandrasekar R, Faraggi M, Klapper MH (1994) Long-range electron transfer in peptides. Tyrosine reduction of the indolyl radical: reaction mechanism, modulation of reaction rate, and physiological considerations. J Am Chem Soc 116:1414–1422
Orlik-Eisel G, Lutz F, Henschen A, Struckmeier M, Kräuter J, Niemann H (1990) The cytotoxin of Pseudomonas aeruginosa: cytotoxicity requires protcolytic activation. Arch Microbiol 153:561–568
Pearson WR (1990) Rapid and sensitive sequence comparison with FASTP and FASTA. Methods Enzymol 183:63–95
Preston GM, Agre P (1991) Isolation of the cDNA for erythrocyte integral membrane protein of 28 kDa: Member of an ancient channel family. Proc Natl Acad Sci USA 88:11110–11114
Rose GD, Roy S (1980) Hydrophobic basis of packing in globular proteins. Proc Natl Acad Sci USA 77:4643–4647
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbour Lab, Cold Spring Harbour, NY
Sanger F, Nicklen S, Coulseon AR (1977) DNA-sequencing with chain termination inhibition. Proc Natl Acad Sci USA 74:5463–5467
Springs B, Haake P (1977) Equilibrium constants for association of guanidinium and ammonium ions with oxyanions. Bioorg Chem 6:181–190
Steck TL, Weinstein RS, Straus JH, Wallach DFH (1970) Inside-out red cell membrane vesicles: preparation and purification. Science 168:255–257
Tanford C (1954) The association of acetate with ammonium and guanidinium ions. J Am Chem Soc 76:945–946
Taniguchi T, Kimura T (1975) Studies on NO2-Tyr82 and NH2-Tyr82 derivates of adrenodoxin. Effects of chemical modification on electron transferring activity. Biochemistry 14: 5573–5578
Wood SP, Blundell TL, Wollmer A, Lazarus NR, Neville RWJ (1975) The relation of conformation and association of insulin to receptor binding; X-ray and circular-dichroism studies on bovine and hystricomorph insulins. Eur J Biochem 55: 531–542
Xiong G, Lutz F (1992) Site-directed mutagenesis of the Pseudomonas aeruginosa cytotoxin for probing toxic activity. Eur J Biochem 204:789–792
Xiong G, Struckmeier M, Alberti U, Lutz F (1991) Relationship of primary structure to functional properties of the cytotoxin protein from Pseudomonas aeruginosa. Naunyn-Schmiedeberg's Arch Pharmacol 343:538–541
Xiong G, Struckmeier M, Lutz F (1994) Pore-forming Pseudomonas aeruginosa cytotoxin. Toxicology 87:69–83
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Struckmeier, M., Xiong, G. & Lutz, F. Pseudomonas aeruginosa cytotoxin: the Asp197-Gly-Asp-Tyr-His-Tyr-His-Tyr202 containing loop is critical for plasma membrane binding. Naunyn-Schmiedeberg's Arch Pharmacol 351, 315–319 (1995). https://doi.org/10.1007/BF00233253
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DOI: https://doi.org/10.1007/BF00233253