Biochemical and functional characterization of a membrane-associated pore-forming protein from the pathogenic ameboflagellate Naegleria fowleri

J. D.E. Young, D. M. Lowrey

Research output: Contribution to journalJournal Article peer-review

37 Scopus citations

Abstract

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°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.

Original languageEnglish
Pages (from-to)1077-1083
Number of pages7
JournalJournal of Biological Chemistry
Volume264
Issue number2
StatePublished - 1989
Externally publishedYes

Keywords

  • Animal
  • Cations, Divalent
  • Cell Membrane
  • Cell Survival
  • Electric Conductivity
  • Hemolysis
  • Ion Channels
  • Kinetics
  • Lipid Bilayers
  • Membrane Potentials
  • Membrane Proteins
  • Naegleria
  • Support, Non-U.S. Gov't
  • Support, U.S. Gov't, P.H.S.

Fingerprint

Dive into the research topics of 'Biochemical and functional characterization of a membrane-associated pore-forming protein from the pathogenic ameboflagellate Naegleria fowleri'. Together they form a unique fingerprint.

Cite this