Abstract
Cell penetrating peptides (CPPs) are powerful tools to transport cell impermeable cargo in cytoplasm without damaging cell membrane. Cationic CPPs may interact with glycosaminoglycans (GAGs) prior to cell internalization. Human eosinophil cationic protein (ECP) has multiple in vivo functions including antihelminthic, antiparastic, antiviral, antibacterial, cytotoxic and immunemodulation activities and in vitro functions including heparan sulfate (HS), lipopolysaccharide, lipid, cellular binding, ribonucleolytic, and endocytotic activities. Here, we have discovered and identified a novel cell penetrating peptide derived from a major heparin binding motif of ECP (CPPecp). A series of CPPecp variants were synthesized to examine correlation between peptide sequence/length and internalization activity. CPPecp is capable of specifically binding to cells through interaction with cell surface proteoglycans. Cellular uptake of CPPecp was clearly observed in wild type Chinese hamster ovary (CHO)-K1 cells rather than HS-deficient pgsD-677 cells and GAG-deficient pgsA-745 cells. Additionally, when cells were co-cultured with GAGs or pre-treated with GAG-digesting enzymes, significant decrease in CPPecp internalization was observed, suggesting that cell surface GAGs, especially HS, were necessary for CPPecp attachment and penetration. Furthermore, treatment with pharmacological agents identified two forms of energy-dependent endocytosis, lipid-raft endocytosis and macropinocytosis, as the major internalization routes for CPPecp. CPPecp-tagged protein was preferentially routed to broncho epithelial and intestinal villi tissues in rat. In comparison with intact ECP, non-cytotoxic CPPecp has multiple functions including GAG, HS, lipid, cellular binding and endocytotic activities and acts as a key element associated with characteristic features of ECP. In terms of novelty, our CPPecp is the first heparin binding CPP derived from human ECP, and also the first CPP discovered with in vivo tissue targeting activity. Taken together, CPPecp is a promising new vehicle for intracellular cargo delivery mainly to lung and intestinal tissues and a potential candidate for drug development in translational medicine.