Translocation of cell-penetrating peptides involving calcium-dependent interactions between anionic glycosaminoglycans and phosphocholine bilayer

Cell-penetrating peptides can internalize ubiquitously in many, if not all, cell types. To explore the specific targeting issue of cell-penetrating peptides (CPPs), we studied glycosaminoglycan (GAG)-binding peptides previously identified in Otx2 and En2 homeoproteins (HPs), alone or extended with the penetratin-like third helix (H3) of En2. HPs are indeed known to internalize in specific cells, thanks to their GAG-targeting sequence (Joliot et al. 2022; Cardon et al. 2023). We quantified the capacity of these peptides to enter into various cell lines known to express different levels and types of heparan sulfates (HS) and chondroitin sulfates (CS) GAGs. We also analyzed by calorimetry (DSC, ITC) and fluorescence spectroscopy, the binary and ternary interactions between heparin (HI), (4S, 6S)CS (CS-E), zwitterionic phosphocholine (PC) model membranes and those peptides. Altogether, our results demonstrate the existence of Ca2+-dependent interactions between CS-E or HI and PC lipid bilayers, the major phospholipid found in animal cell plasma membrane. Importantly, we show that CS-E can act as a Ca2+-dependent bridge with PC membranes that can be exploited by a chimeric CS-E-recognition motif-H3 peptide to bind and cross the membrane lipid bilayer and get access directly to the cytosol of cells. Altogether, this study brings further information uncovering the molecular mechanism of the translocation process of CPPs that implies specific GAGs at the cell-surface. It also shed light on the role of GAGs in the paracrine activity and cell specificity of HPs.