A Novel Cell-Penetrating Peptide Derived from Human Eosinophil Cationic Protein

Abstract

Cell-penetrating peptides (CPPs) are short peptides which can carry various types of molecules into cells; however, although most CPPs rapidly penetrate cells in vitro, their in vivo tissue-targeting specificities are low. Herein, we describe cell-binding, internalization, and targeting characteristics of a newly identified 10-residue CPP, denoted ECP32–41, derived from the core heparin-binding motif of human eosinophil cationic protein (ECP). Besides traditional emphasis on positively charged residues, the presence of cysteine and tryptophan residues was demonstrated to be essential for internalization. ECP32–41 entered Beas-2B and wild-type CHO-K1 cells, but not CHO cells lacking of cell-surface glycosaminoglycans (GAGs), indicating that binding of ECP32–41 to cell-surface GAGs was required for internalization. When cells were cultured with GAGs or pre-treated with GAG-digesting enzymes, significant decreases in ECP32–41 internalization were observed, suggesting that cell-surface GAGs, especially heparan sulfate proteoglycans were necessary for ECP32–41 attachment and penetration. Furthermore, treatment with pharmacological agents identified two forms of energy-dependent endocytosis, lipid-raft endocytosis and macropinocytosis, as the major ECP32–41 internalization routes. ECP32–41 was demonstrated to transport various cargoes including fluorescent chemical, fluorescent protein, and peptidomimetic drug into cultured Beas-2B cells in vitro, and targeted broncho-epithelial and intestinal villi tissues in vivo. Hence this CPP has the potential to serve as a novel vehicle for intracellular delivery of biomolecules or medicines, especially for the treatment of pulmonary or gastrointestinal diseases.

New Amphiphilic Amino Acid Derivatives for Efficient DNA Transfection <i>in Vitro</i>

“…All amphiphiles showed concentration-dependent hemolysis at Ph 7.4. However, the hemolytic activity observed is markedly lower than the activity observed for similar molecules [40]. In fact, for a concentration of about 6 µM Xu et al [41] detected between 10% and 60% hemolysis while, at the same concentration, our amphiphiles reached between 7% and 20% hemolysis.…”

“…The design of these amphiphiles is new. The literature describes the importance of the coexistence of tryptophane and cysteine in the sequence of some CPP for cellular internalization [40], but there are no reports on amphiphilic amino acid derivatives which combine tryptophan, cysteine, and ornithine residues in the same structure. We believe that this combination could be the explanation for the fact that the lipoplexes formed from these amphiphiles are much more easily internalized in CHO-K1 cells than in other cell lines, which should be further researched.…”

Endocytotic Routes of Cobra Cardiotoxins Depend on Spatial Distribution of Positively Charged and Hydrophobic Domains to Target Distinct Types of Sulfated Glycoconjugates on Cell Surface

“…Multiple mechanisms have been proposed for the internalization and/or penetration of cationic peptide into living cells, such as temperature/energy dependence (47), the involvement of proteoglycans (48,49), hydrophobicity (50), amphiphilicity (51), and distinct endocytotic pathways (52)(53)(54). The CTX homologues in the cobra venom are cationic polypeptide with different ligand-binding specificities and cellular targets.…”

Cell Penetrating Peptide as a High Safety Anti-Inflammation Ingredient for Cosmetic Applications

“…Here a 10-residue peptide, covering major GAG binding motif of a human ribonuclease, is identified as a CPP AIF (anti-inflammatory CPP). CPP AIF has been shown to possess epithelial cell, GAG and lipid binding properties as well as cell penetrating activity through macropinocytosis [20,21]. Notably, CPP AIF is able to deliver small fluorescent molecules, recombinant proteins, nanoparticles, and peptidomimetic drugs into cells [20].…”