title: Human perforin permeabilizing activity, but not binding to lipid membranes, is affected by pH.
abstract: The various steps that perforin (PFN), a critical mediator of innate immune response, undertakes to form a transmembrane pore remains poorly understood. We have used surface plasmon resonance (SPR) to dissect mechanism of pore formation. The membrane association of PFN was calcium dependent irrespective of pH. However, PFN does not permeabilize large or giant unilamellar vesicles (GUV) at pH 5.5 even though the monomers bind to the membranes in the presence of calcium. It was possible to activate adsorbed PFN and to induce membrane permeabilization by simply raising pH to a physiological level (pH 7.4). These results were independently confirmed on GUV and Jurkat cells. The conformational state of PFN at either pH was further assessed with monoclonal antibodies Pf-80 and Pf-344. Pf-344 maps to a linear epitope within region 373-388 of epidermal growth factor (EGF)-like domain while the Pf-80 appears to recognize a conformational epitope. Pf-344 interacts with the EGF-like domain after PFN monomers undergo pore formation, the site recognized by Pf-80 is only accessible at acidic but not neutral pH. Thus, the Pf-80 mAb likely interacts with a region of the monomer that participates in oligomerization prior to insertion of the monomer into the lipid bilayer and thus may have therapeutic utility against PFN-mediated immunopathology.
authors.affiliation: Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia.
authors.forename: Tilen
authors.initials: T
authors.lastname: Praper
authors.forename: Mojca Podlesnik
authors.initials: MP
authors.lastname: Besenicar
authors.forename: Helena
authors.initials: H
authors.lastname: Istinic
authors.forename: Zdravko
authors.initials: Z
authors.lastname: Podlesek
authors.forename: Sunil S
authors.initials: SS
authors.lastname: Metkar
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Froelich
authors.forename: Gregor
authors.initials: G
authors.lastname: Anderluh
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions; D002118:Calcium / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects*; D000939:Epitopes / Q000276:immunology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D019169:Jurkat Cells; D008051:Lipid Bilayers; D008081:Liposomes; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins / Q000737:chemistry* / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D012965:Sodium Chloride / Q000494:pharmacology; D020349:Surface Plasmon Resonance
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D008051:Lipid Bilayers; D008081:Liposomes; C510468:PRF1 protein, human; D052899:Pore Forming Cytotoxic Proteins; D054353:Perforin; D012965:Sodium Chloride; D002118:Calcium