Focused Screening of ECM-Selective Adhesion Peptides on Cellulose-Bound Peptide Microarrays

Abstract

The coating of surfaces with bio-functional proteins is a promising strategy for the creation of highly biocompatible medical implants. Bio-functional proteins from the extracellular matrix (ECM) provide effective surface functions for controlling cellular behavior. We have previously screened bio-functional tripeptides for feasibility of mass production with the aim of identifying those that are medically useful, such as cell-selective peptides. In this work, we focused on the screening of tripeptides that selectively accumulate collagen type IV (Col IV), an ECM protein that accelerates the re-endothelialization of medical implants. A SPOT peptide microarray was selected for screening owing to its unique cellulose membrane platform, which can mimic fibrous scaffolds used in regenerative medicine. However, since the library size on the SPOT microarray was limited, physicochemical clustering was used to provide broader variation than that of random peptide selection. Using the custom focused microarray of 500 selected peptides, we assayed the relative binding rates of tripeptides to Col IV, collagen type I (Col I), and albumin. We discovered a cluster of Col IV-selective adhesion peptides that exhibit bio-safety with endothelial cells. The results from this study can be used to improve the screening of regeneration-enhancing peptides.

Increasing the activity of cell adherent cyclic NGR peptides by optimizing the peptide length and amino acid character

“…We have also reported the sequence-function relationships of various peptides by analysis based on physical characteristics of amino acids, positions of amino acids, and peptide activity data. [25][26][27][28][29] For example, by analyzing an amino acid substitution library, we revealed that the cell death-inducing peptide (WELVVLGKL), which works inside cells, has some rules related to the properties of amino acids with peptide activity. The amino acid substitutions at the second and fourth residues from the N-terminus showed higher activity, especially when the residues were hydrophilic amino acids.…”

“…A cell adhesion assay was conducted on SPOT arrays according to a previously described method with minor modifications 27,28 . Each spot from the synthesized peptide array, corresponding to a different peptide, was punched out as a disk and embedded in a 96‐well plate.…”

“…A cell adhesion assay was conducted on SPOT arrays according to a previously described method with minor modifications. 27,28 Each spot from the synthesized peptide array, corresponding to a different pep- We selected cCAANGRAAC as a model sequence owing to its These results suggest that His residues, which are next to the NGR sequence, improved CD13 binding activity. His is classified as an aromatic and cationic amino acid.…”

“…The cell adhesion activity was calculated based on the fluorescence values of each peptide. All the peptides were compared with the cellulose membrane, which was not a synthesized peptide, and the relative cell adhesion ratio for each peptide binding to the cells was calculated (Figure 1B) 28. Four peptides (cCANGRC, cCAANGRAC, CAAANGRAC, and cCAANGRAAC) showed higher cell adhesion ratio than the average plus the SD value of a previously reported cCVLNGRMEC peptide.…”

“…Thus, the peptide array system is a powerful tool to evaluate the relationship between peptide sequences and functions. We have also reported the sequence–function relationships of various peptides by analysis based on physical characteristics of amino acids, positions of amino acids, and peptide activity data 25–29 . For example, by analyzing an amino acid substitution library, we revealed that the cell death‐inducing peptide (WELVVLGKL), which works inside cells, has some rules related to the properties of amino acids with peptide activity.…”