Discovery of a Cell‐penetrating Peptide via Heparin‐binding from Pereskia bleo

Pereskia bleo or rose cactus belongs to the Cactaceae family and is commonly found in South‐east Asia. Traditionally, it is used for cancer treatment, inflammatory diseases and wound healing1. Wound healing involves the extracellular matrix and a complex network of protein‐protein interactions2. Protein‐protein interactions is a vast underexplored space for drug targeting. Disruption of intracellular protein‐protein interactions is largely limited in the ability of molecules capable of specific entry via receptor‐mediated mechanisms and non‐specific entry by membrane penetrations. Extracellular matrices, particularly glycosaminoglycans have been exploited to increase the efficiency of intracellular uptake through non‐receptor mediated entry mechanisms3. Heparin/heparain sulfate, commonly found in the extracellular matrix, can be involved in the cellular uptake of cell‐penetrating peptides3. Here we report the discovery from Pereskia bleo , bleotide pB1, a novel heparin‐binding peptide with cell‐penetrating capabilities. Bleotide pB1 was isolated and purified by a series of liquid chromatography. Transcriptomic and proteomic analyses showed that bleotide pB1 is a 36‐residue peptide with a cystine‐knot arrangement. Similar to known cystine‐knot peptides, bleotide pB1 has high metabolic stability4. Bleotide pB1 is not cytotoxic and hemolytic at concentrations up to 100 μM. Using heparin affinity chromatography and in silico modeling; our results demonstrated that bleotide pB1 is a heparin‐binding peptide. To examine the cell‐penetrating properties of bleotide pB1, N‐terminal TAMRA‐conjugated bleotide pB1 was synthesized by solid‐phase peptide synthesis and oxidative‐folded to the native state. Using flow cytometry and confocal microscopy, we show that TAMRA‐pB1 can be internalized into living cells. CHO‐K1 (wild type) and pgsA‐745 (glycosaminoglycan‐deficient) cells were used to show that the cellular uptake of TAMRA‐pB1 is dependent on glycosaminoglycan expressions. Taken together, this study reports the discovery of a novel heparin‐binding hyper‐stable peptide with cell penetrating properties which could be useful for drug development. Support or Funding Information This project was supported in part by the National Research Foundation (NRF‐CRP8‐2011‐05) of the Prime Minister's Office of Singapore.