Biochemical and functional characterization of a membrane-associated pore-forming protein from the pathogenic ameboflagellate Naegleria fowleri.
A membrane-bound cytolytic pore-forming protein (N-PFP) produced by the pathogenic ameboflagellate Naegleria fowleri was characterized. N-PFP was solubilized from ameba membranes by detergent and enriched 300-fold by gel filtration chromatography. When analyzed by gel electrophoresis, N-PFP migrates with a molecular mass of 66 kDa and 50-54 kDa, under reducing and non-reducing conditions, respectively. In addition to lysing erythrocytes, N-PFP is cytotoxic to several tumor cell lines tested. Its hemolytic activity is not dependent on the presence of divalent cations. N-PFP rapidly depolarizes the membrane potential of microelectrode-impaled chicken embryo myocytes, suggesting that functional channel formation may represent the mode of membrane damage. In planar bilayers, N-PFP forms ion channels with heterogeneous unit conductances ranging between 150 and 400 picosiemens in 0.1 M NaCl and that are relatively resistant to closing by high voltages. Upon heat treatment (75 degrees C, 30 min), N-PFP forms channels with unit conductances that are on average larger than those formed by untreated N-PFP. N-PFP channels are slightly more permeable to cations than to anions. Using a liposome swelling-shrinkage assay, the functional diameter of N-PFP channels is estimated to range between 3.6 and 5.2 nm. N-PFP is immunologically distinct from the PFP/perforin produced by lymphocytes, the terminal components of complement and a PFP from the ameba Entamoeba histolytica, all of which produce pores on target membranes. This protein may have a direct lytic role during target cell killing mediated by N. fowleri.
Young
J D
JD
Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, New York 10021.
Lowrey
D M
DM
D000818:Animals; D002413:Cations, Divalent; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects; D004553:Electric Conductivity; D006461:Hemolysis; D007473:Ion Channels / Q000502:physiology*; D007700:Kinetics; D008051:Lipid Bilayers; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000494:pharmacology / Q000502:physiology; D009253:Naegleria / Q000502:physiology*; D015800:Protozoan Proteins
D002413:Cations, Divalent; D007473:Ion Channels; D008051:Lipid Bilayers; D008565:Membrane Proteins; D015800:Protozoan Proteins; C041670:amoebapore proteins, protozoan