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Action mechanism of tachyplesin I and effects of PEGylation
Authors:Yuichi Imura  Yoshiyuki Ogawa  Katsumi Matsuzaki
Institution:a Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
b Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
Abstract:PEGylation of protein and peptide drugs is frequently used to improve in vivo efficacy. We investigated the action mechanism of tachyplesin I, a membrane-acting cyclic antimicrobial peptide from Tachypleus tridentatus and the effects of PEGylation on the mechanism. The PEGylated peptide induced the leakage of calcein from egg yolk l-α-phosphatidylglycerol/egg yolk l-α-phosphatidylcholine large unilamellar vesicles similarly to the parent peptide. Both peptides induced lipid flip-flop coupled to leakage and was translocated into the inner leaflet of the bilayer, indicating that tachyplesin I forms a toroidal pore and that PEGylation did not alter the basic mechanism of membrane permeabilization of the parent peptide. Despite their similar activities against model membranes, the peptides showed very different biological activities. The cytotoxicity of tachyplesin I was greatly reduced by PEGylation, although the antimicrobial activity was significantly weakened. We investigated the enhancement of the permeability of inner membranes induced by the peptides. Our results suggested that outer membranes and peptidoglycan layers play an inhibitory role in the permeation of the PEG moiety. Furthermore, a reduction in DNA binding by PEGylation may also contribute to the weak activity of the PEGylated peptide.
Keywords:AMEM  Alpha modification of Eagle's medium  C6-NBD-PC  1-palmitoyl-2-[6-[(7-nitrobenz-2-oxa-1  l-α-phosphatidylcholine" target="_blank">3-diazol-4-yl) amino]caproyl]-l-α-phosphatidylcholine  CD  circular dichroism  CFU  colony forming unit  CHO  Chinese Hamster Ovary  DNS-PE  N-(5-dimethylaminonaphthalene-1-sulfonyl)-1  2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine  triethylammonium salt  FBS  fetal bovine serum  Fmoc  9-fluorenylmethoxycarbonyl  HPLC  high-performance liquid chromatography  L/P  lipid-to-peptide molar ratio  LUVs  large unilamellar vesicles  MIC  minimal inhibitory concentration  MLVs  multilamellar vesicles  NAPB  10   mM sodium phosphate/100   mM NaCl  pH 7  4 buffer  ONPG  d-galactoside" target="_blank">o-nitrophenyl-β-d-galactoside  PC  l-α-phosphatidylcholine" target="_blank">egg yolk l-α-phosphatidylcholine  PEG  polyethyleneglycol  PG  l-α-phosphatidyl-dl-glycerol enzymatically converted from PC" target="_blank">l-α-phosphatidyl-dl-glycerol enzymatically converted from PC  Pyrene-PC  1-hexadecanoyl-2-(1-pyrenehexanoyl)-sn-glycero-3-phosphocholine  SUVs  small unilamellar vesicles  TSB  tryptic soy broth  WST-1  2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2  4-disulfophenyl)-2H-tetrazolium  monosodium salt
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