Translocation of penetratin-like peptides involving calcium-dependent interactions between glycosaminoglycans and phosphocholine headgroups of the membrane lipid bilayer

Cell-penetrating peptides (CPPs) can internalize ubiquitously in cells. To explore the specific targeting issue of CPPs, we used glycosaminoglycan (GAG)-binding peptides previously identified in Otx2 and En2 homeoproteins (HPs). The Otx2 sequence preferentially recognizes highly sulfated chondroitin (CS) and the En2 one, heparan sulfates (HS) GAGs. The two HPs internalize in specific cells thanks to their GAG-targeting sequence. We studied the capacity of chimeric peptides containing a GAG-targeting and a penetratin-like sequences to enter into various cell lines known to express different levels and types of GAGs. Since GAGs are found at the vicinity the membrane lipid bilayer, we also analyzed the putative binary and ternary interactions between heparin (HI), (4S,6S)-CS (CS-E), zwitterionic phosphocholine (PC) model membranes and those chimeric peptides. Altogether, our results demonstrate the existence of Ca²⁺-dependent interactions between GAGs and PC lipid bilayers, the major phospholipid headgroup found in animal cell plasma membrane. In addition, the interaction of CS-E (but not HI), with PC favors the binding of the chimeric CS-E-recognition motif-penetratin-like peptide and its subsequent crossing of the lipid membrane to access directly to the cytosol of cells. Altogether, this study brings further understanding of translocation mechanism of CPPs, which requires specific GAGs at the cell-surface. It also shed light on the role of GAGs in the cell transfer specificity and paracrine activity of HPs.