Binding of Oligoarginine to Membrane Lipids and Heparan Sulfate:  Structural and Thermodynamic Characterization of a Cell-Penetrating Peptide

Abstract

Cell-penetrating peptides (CPPs) comprise a group of arginine-rich oligopeptides that are able to deliver exogenous cargo into cells. A first step in the internalization of CPPs is their binding to the cell surface, a reaction likely to involve membrane phospholipids and/or heparan sulfate proteoglycans (HSPGs). The present work characterizes the interaction of R(9), one of the most efficient CPPs, with either heparan sulfate (HS) or lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG). Isothermal titration calorimetry shows that R(9) binds to HS with high affinity. Assuming that HS has n independent and equivalent binding sites for R(9), we find an association constant of 3.1 x 10(6) M(-1) at 28 degrees C. At this temperature, the reaction enthalpy is DeltaH(degrees)pep = - 5.5 kcal/mol and approximately 7 R(9) molecules bind per HS chain, which is equivalent to approximately 0.95 cationic/anionic charge ratio. Delta decreases in magnitude upon an increase in temperature, and the reaction becomes entropy-driven at higher temperatures (>or=37 degrees C). The positive heat-capacity change entailed by this reaction (DeltaC(degrees)P = +167 cal mol(-1) K(-1)) indicates the loss of polar residues on R(9)-HS binding, suggesting that hydrophobic forces play no major role on binding. Calorimetric analysis of the interaction of R(9) with POPC/POPG (75:25) vesicles reveals an association constant of 8.2 x 10(4) M(-1) at 28 degrees C. Using a surface partition equilibrium model to correct for electrostatic effects, we find an intrinsic partition constant of approximately 900 M(-1), a value that is also confirmed by electrophoretic mobility measurements. This corresponds to an electrostatic contribution of approximately 33% to the total free energy of binding. Deuterium nuclear magnetic resonance (NMR) shows no change in the headgroup conformation of POPC and POPG, suggesting that binding takes place at some distance from the plane of the polar groups. (31)P NMR indicates that the lipid bilayer remains intact upon R(9) binding. The fact that R(9) binds with greater affinity to HS than to anionic lipid vesicles makes the former molecule a more likely target in binding this CPP to the cell surface.

Poly-l-lysines and poly-l-arginines induce leakage of negatively charged phospholipid vesicles and translocate through the lipid bilayer upon electrostatic binding to the membrane

“…Cationic polypeptides also serve as models for cell toxic and antimicrobial peptides [9,10]. A particularly important function of arginine-rich peptides is their assistance of the internalisation of the HI-Virus into cells via Tat protein interactions [11][12][13][14].…”

Smart multifunctional nanoparticles in nanomedicine

“…Arginine rich CPPs such as transactivator of transcription (TAT), penetratin and polyarginine are often used for intracellular delivery of the cargo conjugates [57]. It has been hypothesized that the positively charged CPPs provide a strong electrostatic interaction with anionic species presented at the extracellular surface of cell membranes, including lipid head groups, proteins such as nucleolin, and proteoglycans like heparin sulfate [58,59].…”

Cell-penetrating conjugates of pentaglutamylated methotrexate as potential anticancer drugs against resistant tumor cells

“…Although both octaarginine and penetratin are positively charged with several arginine residues in the sequence, based on the characteristics of their internalisation, the two peptides belong to different classes of cell-penetrating peptides [41]; penetratin is secondary amphipathic [42], while octaarginine is non-amphipathic cell-penetrating peptide [43]. For studying whether the difference between the two CPP's has any effect on in vitro cytostatic activity, MTX-conjugates of both peptides were prepared.…”