Dependence of the lytic activity of the N-terminal domain of human perforin on membrane lipid composition--implications for T-cell self-preservation.
The kinetics and thermodynamics of pore formation by the 22-residue N-terminal domain of human perforin-(1-22)-peptide have been studied for a variety of model phospholipid membranes. Peptide binding and aggregation, and cell lysis were monitored through the change in the fluorescence of Trp, or vesicle-encapsulated carboxyfluorescein, respectively. Peptide binding was analyzed in terms of a model that incorporates non-ideal interactions and aggregation in a membrane bilayer. The minimum number of peptide monomers required to form an active pore averaged from four to six, according to the lipid composition of the vesicle. This combined kinetic and thermodynamic approach has provided quantitative information that allows a direct comparison of the binding behavior of the perforin-(1-22)-peptide in different lipid vesicles and affords molecular insight on the factors controlling pore formation. Pore formation is most favorable in thinner membranes with low melting temperatures. No significant difference in activity is observed for different zwitterionic headgroups. Rather, the gel state of the lipid chain, which diminishes the incorporation and aggregation of the perforin-(1-22)-peptide shows the strongest influence. This effect is observed in both the thermodynamic (incorporation isotherm) and kinetic (carboxyfluorescein release) studies. Insertion and aggregation are more facile in membranes with less densely packed lipids. The dependence of pore-forming activity on lipid composition provides important clues to understanding the self-protection mechanism employed by cytotoxic T lymphocytes (CTL) against perforin-mediated lysis.
Rochel
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Evans Laboratory of Chemistry, The Ohio State University, Columbus 43210, USA.
Cowan
J A
JA
D003602:Cytotoxicity, Immunologic; D006801:Humans; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D008563:Membrane Lipids / Q000737:chemistry* / Q000378:metabolism*; D008954:Models, Biological; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology / Q000378:metabolism*; D013816:Thermodynamics
D008051:Lipid Bilayers; D008562:Membrane Glycoproteins; D008563:Membrane Lipids; D010446:Peptide Fragments; D052899:Pore Forming Cytotoxic Proteins; D054353:Perforin