Tryptophan within basic peptide sequences triggers glycosaminoglycan‐dependent endocytosis

Abstract

Deciphering the structural requirements and mechanisms for internalization of cell-penetrating peptides (CPPs) is required to improve their delivery efficiency. Herein, a unique role of tryptophan (Trp) residues in the interaction and structuring of cationic CPP sequences with glycosaminoglycans (GAGs) has been characterized, in relation with cell internalization. Using isothermal titration calorimetry, circular dichroism, NMR, mass spectrometry, and phase-contrast microscopy, we compared the interaction of 7 basic CPPs with 5 classes of GAGs. We found that the affinity of CPPs for GAGs increases linearly with the number of Trp residues, from 30 nM for a penetratin analog with 1 Trp residue to 1.5 nM for a penetratin analog with 6 Trp residues for heparin (HI); peptides with Trp residues adopt a predominantly β-strand structure in complex with HI and form large, stable β-sheet aggregates with GAGs; and in the absence of any cytotoxicity effect, the quantity of peptide internalized into CHO cells increased 2 times with 1 Trp residue, 10 times with 2 Trp residues, and 20 times with 3 Trp residues, compared with +6 peptides with no Trp residues. Therefore, Trp residues represent molecular determinants in basic peptide sequences not only for direct membrane translocation but also for efficient endocytosis through GAGs.

Neuroprotective peptides fused to arginine-rich cell penetrating peptides: Neuroprotective mechanism likely mediated by peptide endocytic properties

“…Consistent with our proposed endocytic neuroprotective mechanism, we have demonstrated that poly-lysine (K10) is only weakly neuroprotective in a cortical neuronal glutamic acid excitotoxicity model (Meloni et al, 2015). It is also likely that other amino acids can influence the endocytic properties of cationic peptides in both a positive and negative manner as has been demonstrated for tryptophan (W; Rydberg et al, 2012;Bechara et al, 2013) and alanine (A; Yang et al, 2014), respectively. Indeed, we have now confirmed that tryptophan and alanine amino acids within arginine-rich peptides respectively increase and decrease neuroprotective efficacy in a glutamic acid excitotoxicity model (Fig.…”

“…Additionally, there is evidence that tryptophan residues within basic peptides can also promote proteoglycan binding and endocytosis (Bechara et al, 2013;Rydberg et al, 2012), while alanine resides have been shown to impede peptide-proteoglycan binding . Interestingly, the replacement of 1 to 3 arginine residues in poly-arginine-6 with the equivalent number of tryptophan residues increases the ability of the peptide to block NMDA receptor activity in amphibian oocytes (Ferrer-Montiel et al, 1988).…”

“…Neuroprotective efficacy, at least for poly-arginine peptides (Meloni et al, 2015), appears to correlate with peptide transduction efficacy (Mitchell et al, 2000), a process known to occur by endocytosis (Appelbaum et al, 2012;Bechara et al, 2013;El-Sayed et al, 2009). Furthermore, it is important to note that the rapid and transient (lasting up to 4 h with peptide pre-treatment) nature of the neuroprotection induced by poly-arginine peptides (Meloni et al, 2015) corresponds closely to the timeframes of endocytosis and endosomal receptor re-cycling (Gundelfinger et al, 2003;Maxfield & McGraw, 2004;Yashunsky et al, 2009).…”

Penetratin-Mediated Transepithelial Insulin Permeation: Importance of Cationic Residues and pH for Complexation and Permeation

“…A recent study underlines the importance of hydrophobic Trp residues for the cell-penetrating ability of a CPP (21), which was found to be augmented by an increased number of Trp residues that promote enhanced affinity in the interaction with cellsurface GAGs (6). Therefore, the promising results obtained in the present study with administration of PenShufinsulin complexes in vitro may well be a result of the specific positioning of the Trp residues giving rise to an enhanced interaction with the surface of the Caco-2 epithelium leading to enhanced transepithelial insulin permeation.…”

New Amphiphilic Amino Acid Derivatives for Efficient DNA Transfection <i>in Vitro</i>

“…Meanwhile, the interaction of tryptophane residues with GAG (glycosaminoglycan) cell membranes promote the endocytosis of DNA [38] [39]. The design of these amphiphiles is new.…”