Abstract
Cell-penetrating peptides can translocate across the plasma membrane of living cells and thus are potentially useful agents in drug delivery applications. Disulfide-rich cyclic peptides also have promise in drug design because of their exceptional stability, but to date only one cyclic peptide has been reported to penetrate cells, the Momordica cochinchinensis trypsin inhibitor II (MCoTI-II). MCoTI-II belongs to the cyclotide family of plant-derived cyclic peptides that are characterized by a cyclic cystine knot motif. Previous studies in fixed cells showed that MCoTI-II could penetrate cells but kalata B1, a prototypic cyclotide from a separate subfamily of cyclotides, was bound to the plasma membrane and did not translocate into cells. Here, we show by live cell imaging that both MCoTI-II and kalata B1 can enter cells. Kalata B1 has the same cyclic cystine knot structural motif as MCoTI-II but differs significantly in sequence, and the mechanism by which these two peptides enter cells also differs. MCoTI-II appears to enter via macropinocytosis, presumably mediated by interaction of positively charged residues with phosphoinositides in the cell membrane, whereas kalata B1 interacts directly with the membrane by targeting phosphatidylethanolamine phospholipids, probably leading to membrane bending and vesicle formation. We also show that another plant-derived cyclic peptide, SFTI-1, can penetrate cells. SFTI-1 includes just 14 amino acids and, with the exception of its cyclic backbone, is structurally very different from the cyclotides, which are twice the size. Intriguingly, SFTI-1 does not interact with any of the phospholipids tested, and its mechanism of penetration appears to be distinct from MCoTI-II and kalata B1. The ability of diverse disulfide-rich cyclic peptides to penetrate cells enhances their potential in drug design, and we propose a new classification for them, i.e. cyclic cell-penetrating peptides.
Highlights
Cell-penetrating peptides (CPPs)6 are short peptides that overcome the barrier of the cell membrane and enter living cells
We propose that the cyclic peptides examined in this study constitute a new family of CPPs that we refer to as cyclic cell-penetrating peptides (CCPPs)
This study has demonstrated that the disulfide-rich head-totail cyclic peptides MCo, Kalata B1 (kB1), and sunflower trypsin inhibitor 1 (SFTI-1) enter cells, and we propose that they can be regarded as a new class of cell-penetrating peptides that we define here as CCPPs
Summary
Peptide Extraction and Purification—MCoTI-II was extracted from the seeds of M. cochinchinensis as described previously [21]. Biotin Labeling of Peptides—The three Lys residues of MCoTI-II and the Lys of kT20K were conjugated with a 5-fold molar excess of NHS-biotin (Quantum Scientific) in 0.1 M sodium bicarbonate, pH 8, for 2 h at room temperature Both labeled peptides were purified by RP-HPLC using an analytical column, and the mass was confirmed by ES-MS. All solutions were prepared with 10 mM HEPES buffer, pH 7.4, containing 150 mM NaCl. Peptide Interactions with Phosphatidylinositols and Phosphatidic Acid Using Nitrocellulose Membranes—The interaction of MCoTI-II, kalata B1, and SFTI-1 with various lipids was performed with strips of nitrocellulose membranes commercialized as PIP StripsTM (Invitrogen) as described previously [29]. The following day, the cell culture medium was removed, and the cells were incubated for 1 h at 37 °C in fresh medium containing the Alexa-488-conjugated peptide at the specified concentration.
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