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

Abstract

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.

Fig. 1. (a) Sequence alignment of Otx2 and En2 extended homeodomains. (b) Sequence of the peptides used in this study. All peptides have a carboxamide moiety at the C-terminus and contain a biotin-Gly₄ tag at the N-terminus except the chimeric peptides GAG^En-H3 and GAG^Otx-H3 that contain a biotin-Gly₅ tag.

Fig. 2. Quantity of (a) H3 and GAG-binding sequences and (b) chimeric peptides internalized in one million cells after 1 hour incubation with peptides (7 μM) at 37 °C, determined by MALDI-TOF MS. The amounts were normalized relative to H3 internalization in K1 cells.

Fig. 3. MALDI-TOF MS quantification (pmol) of internalized GAG^En-linker-H3 and GAG^Otx-linker-H3 analogues (the linker being either Pro, Gly, Apa, PEG₂ or Gly₄) after incubation at 37 °C with 10 μM peptides with one million CHO-K1 cells.

Fig. 4. MALDI-TOF MS quantification of internalized (pmol) chimeric peptides incubated (7 μM) 1 h at 37 °C with one million SKOV-3 cells submitted to GAG hydrolysis (HepI-III, ChABC) or desulfation (NaClO₃).

Fig. 5. DSC analysis of T_m and enthalpy changes upon addition of heparin (HI) to 1 mg mL⁻¹ DMPC LUVs in (a) HEPES buffer and (b) HEPES buffer plus Ca²⁺, Mg²⁺. Curves correspond to DMPC mixed with different amounts of HI. The pre-transition of DMPC is circled by the dashed rectangle and zoomed in the left top; corresponding main phase transition temperature (c) and enthalpy (d) of DMPC LUVs according to HI/DMPC ratio. The thermograms are from one experiment repeated once. Similar results with DPPC and DSPC in SI. Below thermograms (a) and (b) is represented the hypothesis for the impact of Ca²⁺ on PC organization: in the absence of Ca²⁺, the positively charged choline interacts with the negative phosphate moiety of the polar head, which would create steric hindrance or charge repulsion between the polar head groups of adjacent phospholipids; in the presence of Ca²⁺, the divalent ion bridging phosphate moieties would allow the choline headgroup to reorientate outside the bilayer and induce lateral compression between adjacent alkyl chains.

Fig. 6. DSC analysis of enthalpy changes upon addition of GAG^En-H3 (a)–(c) or GAG^Otx-H3 (d)–(f) to 1 mg mL⁻¹ HI- (grey) or CSE-decorated (pink) DMPC LUVs in HEPES buffer plus Ca²⁺, Mg²⁺. (c) and (f) Curves correspond to the relative ΔH that is the ratio between the enthalpy recorded with GAG-decorated DMPC LUVs and the one with pure DMPC LUVs (blue), according to different ratios of peptide/DMPC LUVs.

Fig. 7. MALDI-TOF MS quantification internalized GAG^En-H3 or GAG^Otx-H3 in pgsA-745 (a) and (b) or CaOV-3 (c) and (d) cells, incubated (7 μM) in the absence or presence of various concentrations of HI (a) and (c) or CS-E (b) and (d). In panels (a) and (b), the quantity of internalized peptides is given in pmoles; in panel (c) and (d), the internalization of the peptide in the presence of HI or CS-E is given relatively (normalized), to the quantity measured in the absence of the exogenously added GAGs.

Fig. 8. Schematic representation of the proposed role of GAGs in the internalization mechanism of the chimeric peptides studied herein: GAG^Otx and GAG^En, H3 and chimeric GAG^Otx-H3 and GAG^En-H3. CS are located at the vicinity of the lipid bilayer while HS are more distal. The chimeric CPPs bind to HS- and CS- proteoglycans (CS-E bind PC lipids in the presence of Ca²⁺, leading to an overall anionic complex). Therefore, there are no longer repulsion forces between the cationic choline headgroup and the cationic peptide, could then insert deeper into the lipid bilayer to cross it.