Effect of Linker and Spacer on the Design of a Fibronectin-Mimetic Peptide Evaluated via Cell Studies and AFM Adhesion Forces

Abstract

The design of a fibronectin-mimetic peptide that specifically binds to the alpha 5beta 1 integrin has been widely studied because of this integrin's participation in many physiological and pathological processes. A promising design for such a peptide includes both the primary binding site RGD and the synergy site PHSRN connected by a linker and extended off of a surface by a spacer. Our original hypothesis was that the degree of hydrophobicity/hydrophilicity between the two sequences (RGD and PHSRN) in fibronectin is an important parameter in designing a fibronectin-mimetic peptide (Mardilovich, A.; Kokkoli, E. Biomacromolecules 2004, 5, 950-957). A peptide-amphiphile, PR_b, that was previously designed in our laboratory employed a hydrophobic tail connected to the N terminus of a peptide headgroup that was composed of a spacer, the synergy site sequence, a linker mimicking both the distance and hydrophobicity/hydrophilicity present in the native protein fibronectin (thus presenting an overall "neutral" linker), and finally the primary binding sequence. Even though our previous work (Mardilovich, A.; Craig, J. A.; McCammon, M. Q.; Garg, A.; Kokkoli, E. Langmuir 2006, 22, 3259-3264) demonstrated that PR_b is a promising sequence compared to fibronectin, this is the first study that tests our hypothesis by comparing PR_b to other peptides with hydrophobic or hydrophilic linkers. Furthermore, different peptide-amphiphiles were designed that could be used to study the effect of building blocks systematically, such as the peptide headgroup linker length and hydrophobicity/hydrophilicity as well as the headgroup spacer length on integrin adhesion. Circular dichroism spectroscopy was first employed, and the collected spectra demonstrated that only one peptide-amphiphile exhibited a secondary structure. Their surface topography was evaluated by taking atomic force microscopy (AFM) images of Langmuir-Blodgett peptide-amphiphile membranes supported on mica. Their adhesion was first evaluated with AFM force measurements between the different sequences and an AFM tip functionalized with purified integrins. The amphiphiles were further characterized via 1-12 h cell studies that examined human umbilical vein endothelial cell adhesion and extracellular matrix fibronectin production. The AFM studies were in good agreement with the cell studies. Overall, the adhesion studies validated our hypothesis and demonstrated for the first time that a "neutral" linker, which more closely mimics the cell adhesion domain of fibronectin, supports higher levels of adhesion compared to other peptide designs with a hydrophobic or hydrophilic linker or even fibronectin. Neutral linker lengths that were within the distance found between PHSRN and RGD in fibronectin performed equally well. However, the 10 amino acid neutral linker gave slightly better cell adhesion than did the control fibronectin at all times. Also, a short spacer was shown to give higher adhesion than other sequences with no spacer or a longer spacer, suggesting that a short spac...

A synthetic substrate to support early mesodermal differentiation of human embryonic stem cells

“…A fibronectin-derived peptide ligand for α5β1 integrin, PHSRN-(SG) 5 -RGDSP (Lα5β1), and a CCN1-derived peptide ligand for α6β1 integrin, TTSWSQ (Lα6β1), were each fused to a coiled-coil A to yield polypeptides for modifying substrates [42-44]. The polypeptides cysA-Lα5β1 and cysA-Lα6β1 each have an N-terminal cysteine residue to allow polypeptide immobilization on gold-coated slides through the thiol-gold reaction (Fig.…”

“…However, the two major components in Matrigel, laminin and type IV collagen, both contain the RGD peptide [49,54,55]. This suggests that although a ligand having both the RGD peptide and the synergy peptide PHSRN was used in this study to mimic the ligand in fibronectin that specifically binds to α5β1 integrin [44], it is possible that the RGD peptide alone is sufficient. Another possibility is that redundant mechanisms support early mesodermal differentiation and other components in Matrigel elicit similar biological responses as the fibronectin-derived Lα5β1.…”

Nanoscale engineering of extracellular matrix-mimetic bioadhesive surfaces and implants for tissue engineering

“…Other attempts to mimic FN’s bioactivity have featured peptide designs in which RGD and PHSRN were co-presented by either immobilizing a combination of RGD and PHSRN peptides to a substrate [117], or by incorporating the RGD and PHSRN sequences on the same molecule separated by polyglycine linkers [118–120], a PEG spacer [121] or a 3.7 nm spacer [122, 123] . However, many of these studies have failed to show cell adhesion and integrin specificity characteristics that are equivalent to FN.…”

A General Approach for Generating Fluorescent Probes to Visualize Piconewton Forces at the Cell Surface

“…These peptide ligands recapitulate many of the cell responses in cell-cell and cell-ECM adhesions. 14,15 These ligands included: 1) cyclic RGDfK (cRGDfK) and linear GRGDS peptides derived from the fibronectin-III repeat 10 (FN-III10), which primarily bind the α5β1 and αvβ3 integrins, 16 2) the synergy site PHSRN derived from FN-III9, which is reported to increase cell adhesion when combined with the RGD sequence, 3) the PHSRN(SG) 4 RGDS peptide, which includes a spacer between the PHSRN and GRGDS peptides and better mimics the distance between the two binding domains in FN 17 and 4) the SHAVSS and LRAHAVDING peptides, which bind the E-cadherin and N-cadherin receptors, respectively. 15,18 Standard SPPS protocols were used to generate these α-thioester linear peptides, except for the cRGDfK sequence for which we used a protocol adapted from Xiao et al 19 Standard NCL conditions were used to couple the peptide to 1 (Figure S4).…”