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J. Biol. Chem., Vol. 278, Issue 2, 1310-1315, January 10, 2003
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From the Graduate School of Biostudies, Kyoto University, Sakyo-ku,
Kyoto 606-8501, Japan
Cationic antimicrobial peptides play
important roles in innate immunity. Compared with extensive studies on
peptide-bacteria interactions, little is known about peptide-human cell
interactions. Using human cervical carcinoma HeLa and fibroblastic TM12
cells, we investigated the cellular uptake of fluorescent analogues of the two representative antimicrobial peptides magainin 2 and buforin 2 in comparison with the representative Arg-rich cell-penetrating Tat-(47-57) peptide (YGRKKRRQRRR). The dose, time, temperature, and
energy dependence of translocation suggested that the three peptides
cross cell membranes through different mechanisms. The magainin peptide
was internalized within a time scale of tens of minutes. The
cooperative concentration dependence of uptake suggested that the
peptide forms a pore as an intermediate similar to the observations in
model membranes. Furthermore, the translocation was coupled with
cytotoxicity, which was larger for tumor HeLa cells. In contrast, the
buforin peptide translocated within 10 min by a
temperature-independent, less concentration-dependent passive mechanism without showing any significant cytotoxicity at the
highest concentration investigated (100 µM). The
uptake of the Tat peptide was proportional to the peptide
concentration, and the concentration dependence was lost upon ATP
depletion. The peptide exhibited a moderate cytotoxicity at higher
concentrations. The time course did not show saturation even after 120 min. The buforin peptide, covalently attached to the 28-kDa
green fluorescent protein, also entered cells, suggesting a potency of
the peptide as a vector for macromolecular delivery into cells.
However, the mechanism appeared to be different from that of the parent peptide.
Translocation of Analogues of the Antimicrobial
Peptides Magainin and Buforin across Human Cell Membranes*
*
This work was supported in part by the Mochida Memorial
Foundation for Medical and Pharmaceutical Research, NOVARTIS Foundation (Japan) for the Promotion of Science, the Inamori Foundation, the
Shimizu Foundation for the Promotion of Immunology Research, the Japan
Securities Scholarship Foundation, and Grant-in-aids for Scientific
Research (14572091, 13024244, and 14017048) from the Ministry of
Education, Culture, Sports, Science and Technology of Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Graduate School of
Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan. Tel.:
81-75-753-4574; Fax: 81-75-761-2698; E-mail:
katsumim@pharm.kyoto-u.ac.jp.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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