Relationships between Membrane Binding, Affinity and Cell Internalization Efficacy of a Cell-Penetrating Peptide: Penetratin as a Case Study

Penetratin is a positively charged cell-penetrating peptide (CPP) that has the ability to bind negatively charged membrane components, such as glycosaminoglycans and anionic lipids. Whether this primary interaction of penetratin with these cell surface components implies that the peptide will be further internalized is not clear.Using mass spectrometry, the amount of internalized and membrane bound penetratin remaining after washings, were quantified in three different cell lines: wild type (WT), glycosaminoglycans- (GAG(neg)) and sialic acid-deficient (SA(neg)) cells. Additionally, the affinity and kinetics of the interaction of penetratin to membrane models composed of pure lipids and membrane fragments from the referred cell lines was investigated, as well as the thermodynamics of such interactions using plasmon resonance and calorimetry.Penetratin internalized with the same efficacy in the three cell lines at 1 µM, but was better internalized at 10 µM in SA(neg)>WT>GAG(neg). The heat released by the interaction of penetratin with these cells followed the ranking order of internalization efficiency. Penetratin had an affinity of 10 nM for WT cells and µM for SA(neg) and GAG(neg) cells and model membrane of phospholipids. The remaining membrane-bound penetratin after cells washings was similar in WT and GAG(neg) cells, which suggested that these binding sites relied on membrane phospholipids. The interaction of penetratin with carbohydrates was more superficial and reversible while it was stronger with phospholipids, likely because the peptide can intercalate between the fatty acid chains.These results show that accumulation and high-affinity binding of penetratin at the cell-surface do not reflect the internalization efficacy of the peptide. Altogether, these data further support translocation (membrane phospholipids interaction) as being the internalization pathway used by penetratin at low micromolecular concentration, while endocytosis is activated at higher concentration and requires accumulation of the peptide on GAG and GAG clustering

In-capillary (electrophoretic) digestion-reduction-separation: A smart tool for middle-up analysis of mAb

International audienceComprehensive characterization of physicochemical properties of monoclonal antibodies (mAbs) is a critical process to ensure their quality, efficacy, and safety. For this purpose, mAb analysis at different levels (bottom-up, middle-up) is a common approach that includes rather complex multistep sample preparation (reduction, digestion). To ensure high analysis performance, the development of fully integrated methodologies is highly valuable. Capillary zone electrophoresis is a particularly well-adapted technique for the multistep implementation of analytical strategies from sample preparation to detection. This feature was employed to develop novel integrated methodologies for the analysis of mAb at the middle-up level. Multiple in-line reactions (simultaneous reduction and digestion) were performed for the first time in the separation capillary. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was used as an effective reducing agent under a broad pH range and IdeS (Immunoglobulin degrading enzyme from Streptococcus) as a highly specific enzyme for mAb digestion. Transverse diffusion of laminar flow profile (TDLFP) was applied for reactants mixing. Both in-line sample preparation and separation parameters were optimized under non-denaturing and denaturing conditions. The developed in-line methodologies provided good reproducibility and higher peak efficiencies comparing with off-line assays. They were successfully applied to different mAbs

Amount (pmoles) of A) internalized and B) high-affinity membrane bound penetratin in WT, GAG^neg and SA^neg cells.

<p>Corresponding intracellular penetratin concentrations (µM) are also indicated (A) taken the volume of one cell as being 1 pL.</p

Formation of a lipid bilayer containing WT membrane fragments and penetratin interaction with this membrane monitored by PWR.

<p>Panels A) and B) correspond to the PWR spectra obtained for the buffer (1) and the lipid bilayer (2) and Panels C) and D) for spectra obtained after addition of about 0.1 µM of penetratin (+) to the bilayer (solid line) obtained for p- and s-polarized light, respectively. The resonance position shifts obtained for p- (•) and s- (▪) polarizations for the incremental addition of penetratin are represented in Panel E, together with the hyperbolic binding (affinity constants are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024096#pone-0024096-t002" target="_blank">table 2</a>). Panel F corresponds to the kinetic measurements obtained for the p-pol light upon addition of penetratin (0.05 µM) to the lipid bilayer (rate constants are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024096#pone-0024096-t003" target="_blank">Table 3</a>).</p

(A) Isothermal calorimetric titration of 50 nmol penetratin to a suspension of 1.5 million CHO cells at 37°C.

<p>(B) Released heat measured after the addition of 50 nmol (white, final concentration 32 µM) and 7.75 nmol (black, final concentration 5 µM) penetratin to WT, GAG^neg and SA^neg cells.</p

Direction, magnitude and graphical analysis of the spectral changes observed by PWR upon penetratin interaction with the synthetic lipid model membrane.

<p>Direction, magnitude and graphical analysis of the spectral changes observed by PWR upon penetratin interaction with the synthetic lipid model membrane.</p

Kinetic analysis of the interaction of penetratin with lipid membranes; data obtained by PWR spectroscopy.

<p>Note: experiment was repeated 3 times.</p