Identification and Characterization of a New Family of Cell-penetrating Peptides

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 tomembrane 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. Cell-penetrating peptides (CPPs) are short peptides that overcome the barrier of the cell membrane and enter living cells. CPPs can be conjugated with a cargo (i.e. oligonucleotide, peptide sequence, or polysaccharide) and efficiently deliver it inside cells, and so they are of great importance in the field of drug delivery. The most extensively studied CPPs are the Tat peptide, derived from theHIV-1 transactivator of transcription protein (1), and penetratin, derived from the third helix of the Drosophila Antennapedia homeodomain (2). In the last 20 years, many different CPPs have been identified from a range of sources. However, only one cyclic peptide has been reported to pass through cell membranes, i.e. the cyclotide Momordica cochinchinensis trypsin inhibitor II (MCoTI-II) (3–5). Cyclotides are head-to-tail cyclic peptides that contain 30 amino acids, including six conserved cysteine residues that form a cyclic cystine knot (CCK) at the core of their structure (6). The use of the stable CCK motif as a drug scaffold has emerged as an interesting field of research in recent years (7–9). In particular, the exceptional stability of the CCK motif makes it an ideal framework for molecular engineering and drug design applications (10–12). For example, the CCK peptides MCoTI-II and kalata B1 have been successfully engineered to introduce new bioactivities to the molecules (13–16). The versatility of the CCK framework together with the cell-penetrating properties of MCoTI-II makes this mini-protein of special interest for applications in drug design. MCoTI-II has been reported to be internalized into cells by macropinocytosis (3), but the specificmechanism bywhich this occurs has not been determined. Furthermore, possible interactions of MCoTI-II with phospholipids or receptors in cell membranes, and the eventual fate of the molecule within cells, have not been determined. In this study, an analysis of the cellular uptake ofMCoTI-II and the prototypic cyclotide kalata B1 is presented. These two peptides are representative examples of two subfamilies of cyclotides (17) and have very different * This work was supported in part by Australian Research Council Grant DP880105. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1–S3. 1 University of Queensland Research Fellow. 2 Marie Curie International Outgoing Fellow supported by Grant PIOF-GA-2008-220318. 3 Australian Research Council Future Fellow supported by Grant FT100100657 and an honorary National Health and Medical Research Council Senior Research Fellow supported by Grant APP1003470. 4 Queensland Smart State Fellow. 5 National Health and Medical Research Council Fellow. To whom correspondence should be addressed. E-mail: [email protected]. 6 The abbreviations used are: CPP, cell-penetrating peptide; CCK, cyclic cystine knot; CCPP, cyclic cell-penetrating peptide; EIPA, ethylisopropylamiloride; HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; MCoTI-II, M. cochinchinensis trypsin inhibitor II; MCo, M. cochinchinensis trypsin inhibitor II; PA, phosphatidic acid; PI, phosphoinositide; POPC, palmitoyloleoylphosphatidylcholine; POPE, palmitoyloleoylphosphatidylethanolamine; POPG, palmitoyloleoylphosphatidylglycerol; SFTI-1, sunflower trypsin inhibitor 1; SPR, surface plasmon resonance; WGA, wheat germ agglutinin; Fmoc, N-(9-fluorenyl)methoxycarbonyl; RU, response unit; Boc, t-butoxycarbonyl. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 42, pp. 36932–36943, October 21, 2011 © 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.