Activation of phospholipase A2 by temporin B: Formation of antimicrobial peptide-enzyme amyloid-type cofibrils |
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Authors: | Christian Code J. Antoinette Killian |
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Affiliation: | a Helsinki Biophysics and Biomembrane Group, Medical Biochemistry, Institute of Biomedicine, P.O. Box 63 (Haartmaninkatu 8), FIN-00014, University of Helsinki, Finland b Chemical Biology and Organic Chemistry Research Group, Bijvoet Center for Biomolecular Research/Institute of Biomembranes, Department of Chemistry, Faculty of Science, Utrecht University, The Netherlands |
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Abstract: | Phospholipases A2 have been shown to be activated in a concentration dependent manner by a number of antimicrobial peptides, including melittin, magainin 2, indolicidin, and temporins B and L. Here we used fluorescently labelled bee venom PLA2 (PLA2D) and the saturated phospholipid substrate 1,2-dipalmitoyl-glycero-sn-3-phosphocholine (L-DPPC), exhibiting a lag-burst behaviour upon the initiation of the hydrolytic reaction by PLA2. Increasing concentrations of Cys-temporin B and its fluorescent Texas red derivative (TRC-temB) caused progressive shortening of the lag period. TRC-temB/PLA2D interaction was observed by Förster resonance energy transfer (FRET), with maximum efficiency coinciding with the burst in hydrolysis. Subsequently, supramolecular structures became visible by microscopy, revealing amyloid-like fibrils composed of both the activating peptide and PLA2. Reaction products, palmitic acid and 1-palmitoyl-2-lyso-glycero-sn-3-phosphocholine (lysoPC, both at > 8 mol%) were required for FRET when using the non-hydrolysable substrate enantiomer 2,3-dipalmitoyl-glycero-sn-1-phosphocholine (D-DPPC). A novel mechanism of PLA2 activation by co-fibril formation and associated conformational changes is suggested. |
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Keywords: | apoC-II, apolipoprotein C-II CD, circular dichroism C-temB, cysteine-temporin B L-DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine D-DPPC, 2,3-dipalmitoyl-sn-glycero-1-phosphocholine DHPC, 1,2-dihexadecyl-sn-glycero-3-phosphocholine DIC, differential interference contrast FFA, free fatty acid FRET, Fö rster resonance energy transfer γ, lag time LPL, lipoprotein lipase LUV, large unilamellar vesicles lysoPC, 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine PA, palmitic acid PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis PLA2, phospholipase A2 PLA2D, phospholipase A2 labeled with Alexa 488 (donor) r, anisotropy SDS, sodium dodecyl sulfate sn, stereochemical notation τ, fluorescence lifetime temB, temporin B Tm, main phase transition temperature ThT, Thioflavin T TRC-temB, cysteine-temporin B labelled with Texas Red |
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