Effect of Amino Acid Substitution on Cell Adhesion Properties of Octa-arginine

Abstract

Octa-arginine (R8) is a cell-permeable peptide with excellent cell adhesion properties. Surface-immobilized R8 mediates cell attachment via cell surface receptors, such as heparan sulfate proteoglycans and integrin β1, and promotes cell spreading and proliferation. However, it is not clear how these properties are affected by specific peptide composition and if they could be improved. Here, we synthesized XR8 peptides, in which half of the original R8 arginine residues were replaced with another amino acid (X). We then aimed to investigate the effect of the substitution on cell adhesion and proliferation on XR8-conjugated agarose matrices. The XR8-matrix showed slightly better cell attachment when X was a hydrophobic or aromatic amino acid. However, hydrophobic XR8-matrices tended to promote cell proliferation to a less extent. Eventually, YR8-matrix most efficiently promoted cell adhesion, spreading, and proliferation among the XR8-matrices tested. Collectively, these observations indicate that the properties of residue X play a major role in the biological activity of XR8-matrices and shed light on the interaction between small peptides and the cell membrane. Further, YR8 is a promising cell-adhesive peptide for the development of cell culture substrates and biomaterials.

INTRODUCTION

Surfaces of positively-charged materials have high affinity for negatively-charged cell surfaces. Hence, basic molecules, such as poly-lysine and adhesamine, have been used as coatings for cell culture experiments.^1,2) In the past, we have shown that the basic octa-arginine peptide (R8), a well-known cell-penetrating peptide,^3,4) exhibits pronounced cell adhesion properties when immobilized on a matrix.⁵⁾ Therefore, R8 could potentially be used for the development of cell culture substrates and biomaterials. R8 interacts with the cell surface mainly through heparan sulfate proteoglycans (HSPGs),⁶⁾ including syndecans, and integrin β1, promoting cell spreading and proliferation.^5,7)

Consensus motifs, such as XBBXB and XBBBXXBX (B = arginine or lysine; X = neutral or hydrophobic amino acid), are present in the heparin-binding domain of heparin-binding proteins.^8,9) In heparin-binding proteins, the guanidino group of arginine residues and amino group of lysine residues are mainly present on the protein surface and bind to sulfate groups of heparin/heparan sulfate (HS).^10,11) Meanwhile, non-basic amino acids are oriented towards the inside of the protein and affect the affinity to heparin/HS. For example, the presence of flexible glycine or serine promotes binding of basic motifs to heparin/HS.¹²⁾ It has also been reported that phenylalanine residues in antithrombin III are involved in protein conformational change and are required for heparin binding.¹³⁾ In terms of cell adhesion, non-basic amino acids in heparin/HS-binding peptides are thought to affect peptide cell adhesion ability. This is because heparin binding directly affects HSPG-mediated cell adhesion and indirectly affects integrin-mediated adhesion, as HSPG signaling activates integrins.^14–16) In addition, non-basic amino acids in heparin/HS-binding peptides potentially affect binding affinity of the peptides to integrins.

In the current study, we aimed to investigate the effect of non-basic amino acids on the cell adhesion activity of R8-based peptides to identify a peptide that would be most suitable for cell adhesion and culture. XR8 peptides were designed by substitution of half the arginine residues of R8. The XR8 peptides were covalently conjugated with neutral agarose matrices, and their promotion of cell adhesion and proliferation properties were analyzed. We believe that the findings of our study shed light on peptide–cell surface interactions and will inform the development of new biomaterials for biotechnological and biomedical applications.

MATERIALS AND METHODS

Peptide Synthesis

All peptides were manually synthesized using the 9-fluorenylmethoxycarbonyl strategy, with a C-terminal amide. The resulting protected peptides were deprotected and cleaved from the resin using trifluoroacetic acid (TFA)/1,3-dimethoxybenzene/thioanisole/m-cresol/ethanedithiol/H₂O (85 : 3 : 3 : 3:3 : 3, v/v). Crude peptides were purified using reversed-phase HPLC on a COSMOSIL 5C₁₈-AR-II column (Nacalai Tesque, Kyoto, Japan) using a gradient elution with water/acetonitrile containing 0.1% TFA. Peptide purity and mass were confirmed using analytical HPLC and electrospray ionization mass spectrometry at the Central Analysis Center, Tokyo University of Pharmacy and Life Sciences.

Peptide Conjugation with Aldehyde-Functionalized Agarose Matrix

Aldehyde-functionalized agarose was synthesized from agarose (Agarose S, NIPPON GENE, Tokyo, Japan) using (2,2,6,6-tetramethylpiperidin-1-yl)oxyl oxidation reaction, as previously reported.¹⁷⁾ The aldehyde content of aldehyde-agarose was 19.2% agarobiose unit. Aldehyde-agarose was dissolved in water at 100 µg/mL, transferred to treated 96-well tissue culture plates (AGC Techno Glass, Shizuoka, Japan; 100 µL/well), and allowed to dry for the preparation of aldehyde-agarose matrix. Subsequently, cysteine-containing peptides in 100 mM acetate buffer at pH 5 (100 µL/well) were added, and the plates were incubated for 2 h. The wells were then washed with phosphate-buffered saline (PBS) and used for assays.

Cell Culture

Human dermal fibroblasts (HDFs) (Kurabo, Tokyo, Japan) were maintained in low-glucose Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific, Waltham, MA, U.S.A.) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific), 100 U/mL penicillin, and 100 µg/mL streptomycin (Thermo Fisher Scientific). HeLa and A549 cells (Japanese Collection of Research Bioresources Cell Bank, Osaka, Japan) were maintained in high-glucose DMEM containing 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin.

Cell Attachment Assay

HDFs were detached from culture plates using 0.05% trypsin–ethylenediaminetetraacetic acid (EDTA) solution and suspended in 0.1% bovine serum albumin (BSA)/DMEM. Subsequently, the cells were seeded in peptide-agarose matrices (5 × 10³ cells/100 µL/well). For the inhibition assay, the cells were seeded in the presence or absence of 10 µg/mL heparin, 5 mM EDTA, or 10 µg/mL mouse monoclonal antibody against human integrin β1 (AIIB2). The cells were incubated at 37 °C for 1 h to evaluate the number of attached cells or for 2 h to evaluate cell spreading. After incubation, the attached cells were fixed and stained with 0.2% aqueous solution of crystal violet containing 20% methanol and imaged using a BZ-X810 microscope (Keyence, Osaka, Japan). The attached cells in nine central fields of view (0.77 mm² each) were counted and averaged using BZ-X800 Analyzer software (Keyence). The area of the attached cells was measured using the BZ-X800 Analyzer software.

Immunostaining

HDFs in 0.1% BSA/DMEM were added to peptide-agarose matrices (5 × 10³ cells/100 µL/well) and incubated for 2 h. The attached cells were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized using 0.1% Triton X-100 for 15 min, and blocked with 1% BSA in PBS for 1 h. The cells were then incubated with Alexa Fluor^® 488-labeled phalloidin (Thermo Fisher Scientific, 1 : 100) for 60 min in the dark. The nuclei were stained with Hoechst 33258 (Thermo Fisher Scientific, 1 : 10000) in PBS for 15 min. The cells were washed with PBS and imaged using a BZ-X810 microscope.

Immunoblotting for Focal Adhesion Kinase (FAK) Signaling

HDFs (2 × 10⁴ cells/well) were incubated on the peptide-matrices at 37 °C for 2 h. After incubation, the cells were lysed with 20 µL of sodium dodecyl sulfate (SDS) sample buffer, resolved by 7.5% SDS-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 3% BSA, incubated with either an anti phospho-FAK (Tyr397) or anti FAK polyclonal antibody (1 : 1000, Cell Signaling, Danvers, MA, U.S.A.) overnight at 4 °C, and detected using a horseradish peroxidase-conjugated secondary antibody (1 : 2000, GE Healthcare, Piscataway, NJ, U.S.A.) with an ECL kit (GE Healthcare).

LIVE/DEAD Cell Viability Assay

Cell viability was assessed using the LIVE/DEAD Cell Viability kit (Thermo Fisher Scientific). After cell culture on peptide-agarose matrices with serum-free DMEM for 2 d, the media was replaced with DMEM containing 1 µM calcein-AM and 1 µg/mL propidium iodide. After incubation at 37 °C for 30 min, live and dead cells were visualized using a BZ-X810 microscope. The percentage of live and dead cells were obtained by counting the cells in four central fields of three wells.

Cell Proliferation Assay

Cells in 10% FBS/DMEM were seeded into peptide-agarose matrices (5 × 10³ cells/100 µL/well). After 2 d, cell viability was determined using the cell-counting kit-8 (CCK8; Dojindo, Kumamoto, Japan). CCK8 reagent was mixed with DMEM (1 : 10 v/v), added to the cells (100 µL/well), and incubated for 1 h at 37 °C. Sample absorbance at 450 nm was then measured using Multiskan GO microplate spectrophotometer (Thermo Fisher Scientific).

Circular Dichroism (CD) Spectroscopy

CD spectroscopy of peptides was performed using a J-1500 CD spectropolarimeter (JASCO, Tokyo, Japan). Peptides were suspended in PBS containing 2 mM Tris(2-carboxyethyl)phosphine to prevent disulfide formation. Wavelength-scan spectra were collected using a 1 mm path-length cell. The mean residue ellipticity [θ] was calculated from the equation [θ] = (θ_obs/10lcr), where θ_obs is the measured ellipticity in millidegrees, l is the cell path-length in cm, c is the molar concentration, and r is the number of residues in a peptide.

Statistical Analysis

Data were analyzed using one-way ANOVA with Tukey’s multiple-comparison test. A confidence level of >95% (p < 0.05) was considered significant.

RESULTS AND DISCUSSION

Cell Attachment Activity of XR8-Matrices

All peptides were synthesized with an N-terminal CGG sequence (Table 1). The two glycine residues were used as a spacer. The peptides were bound to a matrix of aldehyde-functionalized agarose via thiazolidine formation, as previously reported.¹⁷⁾ First, we prepared three peptides, LR8, LR8-db, and LR8-cs to examine effects of the X positions on cell attachment activity of XR8 peptides. Here, leucine was used as X because leucine is most frequently present in the heparin-binding consensus XBBBXXBX motif, as previously reported.⁸⁾ LR8 (CGG-LRLRLRLR) has repeats of LR and thus, arginine residues are uniformly placed on the peptide. LR8-db (CGG-RRRRLLLL) is a diblock peptide. In LR8-cs (CGG-LRRRLLRL), leucine and arginine residues are placed according to the XBBBXXBX motif. We compared their cell attachment activity using HDFs (Fig. 1A), and found that the LR8- and LR8-db-matrices have stronger activity than the LR8-cs-matrix, indicating that the X positions of XR8 peptides may affect their cell attachment activity. In subsequent experiments, the CGG-XRXRXRXR sequence was used for the XR8 peptides.

Table 1. Sequences of XR8 Peptides

Peptide	Sequencea)
R8	CGGRRRRRRRR
ER8	CGGERERERER
GR8	CGGGRGRGRGR
AR8	CGGARARARAR
NR8	CGGNRNRNRNR
QR8	CGGQRQRQRQR
SR8	CGGSRSRSRSR
TR8	CGGTRTRTRTR
VR8	CGGVRVRVRVR
IR8	CGGIRIRIRIR
LR8	CGGLRLRLRLR
LR8-db	CGGRRRRLLLL
LR8-cs	CGGLRRRLRLL
YR8	CGGYRYRYRYR
FR8	CGGFRFRFRFR
WR8	CGGWRWRWRWR

a) All peptides were synthesized with a C-terminal amide.

Fig. 1. Cell Attachment Activity of Peptide-Matrices

Cell attachment activity of (A) LR8-, LR8-db, and LR8-cs-matrices and (B) XR8-matrices. Serially diluted peptides were conjugated with aldehyde-agarose matrices. HDFs in 0.1% BSA/DMEM (5 × 10³ cells/well) were seeded into the sample wells and incubated for 1 h at 37 °C. Attached cells per 0.77 mm² were then counted. Values are shown as the mean of three independent experiments.

Next, thirteen different XR8 peptides (CGG-XRXRXRXR, X = E, G, A, N, Q, S, T, V, I, L, Y, F, or W) were prepared. Cell attachment to the XR8-matrices and the R8-matrix was compared (Fig. 1B). No cells attached to the ER8-matrix because the original positive charge of R8 was cancelled in ER8. The cells adhered to the other XR8-matrices, but not as strongly as to the R8-matrix because of the loss of half of the arginine residues. Cell attachment activity of the IR8-, LR8-, YR8-, FR8-, and WR8-matrices was slightly stronger than that to the other XR8-matrices, indicating that hydrophobic or aromatic amino acids are relatively suitable as the X residue for cell adhesion.

Morphology of Cells Attached to XR8-Matrices

Next, we observed cell morphology on the XR8-matrices (Fig. 2). The R8-matrix promoted extensive cell spreading as previously reported.⁵⁾ Cell shapes on the YR8-, FR8-, and WR8-matrices appeared similar to that on the R8-matrix. However, cell spreading on the other XR8-matrices was relatively weak. These results indicate that aromatic amino acids in the XR8 peptides contributed to the cell spreading. In this experiment, we also examined the effect of heparin and EDTA on cell attachment and spreading. Cell attachment to the XR8-matrices was completely inhibited by heparin, as was also observed for the R8-matrix. This indicates that all XR8-matrices, except for the ER8-matrix, continued to mediate cell attachment mainly via HSPGs. The cells remained attached to the XR8-matrices and the R8-matrix in the presence of EDTA, however, EDTA changed the cell morphology. In Fig. 3, the effect of EDTA and anti-integrin β1 antibody on the cell morphology was quantitatively evaluated by measuring area of the attached cells. Cell area of the attached cells on the XR8-matrices, except for the IR8-matrix, was significantly reduced by EDTA and anti-integrin β1 antibody, indicating that cell spreading on these matrices as well as that on the R8-matrix was mediated by integrin β1. However, in the case of the IR8-matrix, EDTA and the antibody did not show inhibitory effects on cell morphology. Actin staining revealed that the cells on the IR8-matrix showed a rounded morphology with membrane ruffling, and that the cell morphology was not affected by EDTA, suggesting only a small contribution of integrin β1 to the morphology (Fig. 4A). These findings demonstrate that substitution of half the arginine residues of R8 may impact the involvement of integrin β1 in cell adhesion. Meanwhile, the cells on the YR8-matrix as well as that on the R8-matrix formed actin fibers. EDTA inhibited the actin fiber formation, resulting in a significant decrease in the attached cell area. We also analyzed the phosphorylation of FAK, a key mediator of integrin signaling, in HDFs cultured on the R8-, IR8-, and YR8-matrices (Figs. 4B, C). FAK phosphorylation on the IR8-matrix was weaker than that on the R8-matrix. However, FAK phosphorylation was promoted on the YR8-matrix. These results indicate that integrin β1 largely contributes to cell spreading on the YR8-matrix. It has been reported that aromatic amino acid-modified cellulose scaffolds promote cell spreading via integrin.¹⁸⁾ In that report, the authors speculated that the cell spreading was due to the high adsorption of fibronectin in serum due to the aromatic amino acids. However, since serum was not included in the medium in this study, fibronectin adsorption was unlikely to be the cause of cell spreading on the YR8-, FR8-, and WR8-matrices. Further studies are needed to determine why XR8 with aromatic amino acids strongly promotes integrin-mediated cell spreading like R8.

Fig. 2. Cell Morphology on XR8-Matrices and the Effects of Heparin and EDTA on Cell Adhesion

XR8 peptides (100 µM) were conjugated with aldehyde-agarose matrices in 96-well plates. HDFs in 0.1% BSA/DMEM (5 × 10³ cells/well) were seeded into the sample wells in the presence of either 10 µg/mL heparin or 5 mM EDTA and incubated for 2 h. Scale bar = 100 µm.

Fig. 3. Effects of EDTA and Anti-integrin β1 Antibody on Cell Area on XR8-Matrices

XR8 peptides (100 µM) were conjugated with aldehyde-agarose matrices in 96-well plates. HDFs in 0.1% BSA/DMEM (5 × 10³ cells/well) were seeded into the sample wells in the presence or absence of 5 mM EDTA or 10 µg/mL anti-integrin β1 antibody and incubated for 2 h. Area of the attached cells was measured. Values are shown as the mean of more than 30 measurements ± standard deviation (S.D.). * p < 0.05, ** p < 0.0001 vs. None.

Fig. 4. Cytoskeletal Organization and FAK Phosphorylation in Cells on R8-, IR8-, and YR8-Matrices

(A) Actin cytoskeleton of cells on the R8-, IR8-, and YR8-matrices. The peptides (100 µM) were conjugated with aldehyde–agarose matrices in 96-well plates. HDFs in 0.1% BSA/DMEM (5 × 10³ cells/well) were seeded into the sample wells in the presence or absence of 5 mM EDTA and incubated for 2 h. Actin (green) and nuclei (blue) were stained by using Alexa Fluor 488-labeled phalloidin and Hoechst 33258, respectively. Scale bar = 100 µm. (B) FAK Tyr397 phosphorylation in HDFs on the R8-, IR8-, and YR8-matrices. The HDFs were incubated on the matrices (2 × 10⁴ cells/well) for 2 h and were then lysed and assessed by Western blotting. (C) Quantitation of the results of B. The relative phosphorylation of FAK was assessed using image J software. Values are shown as the mean of quadruplicate experiments ± S.D. * p < 0.05.

Cytocompatibility of XR8-Matrices

To examine the cytocompatibility of the XR8-matrices, HDFs were cultured on the matrices in the absence of serum for 2 d, and then live and dead cells were quantified (Fig. 5). None of the XR8-matrices were cytotoxic, and more than 95% of cells survived on the XR8-matrices. However, the R8-matrix was cytotoxic, with 60% of the cultured cells dead. It has been reported that positively-charged scaffolds disrupt cell membranes and/or induce apoptosis, and cells on the R8-matrix might have also been affected in this way.¹⁹⁾ In the case of XR8, substitutions of arginine residues reduced the net positive charge, which may have reduced the matrix cytotoxicity. These findings suggest that the XR8-matrices are superior for long-term culture to the R8-matrix.

Fig. 5. Cytocompatibility of XR8-Matrices

XR8 peptides (100 µM) were conjugated with aldehyde-agarose matrices. HDFs in serum-free DMEM (5 × 10³ cells/well) were seeded into the sample wells and incubated for 2 d. The percentages of live (white) and dead cells (gray) were determined using calcein-AM and propidium iodide, respectively. Values are shown as the mean of three independent experiments ± standard error (S.E.).

Cell Proliferation on XR8-Matrices

Cell proliferation on the XR8-matrices was evaluated in the presence of serum (Fig. 6A). As previously reported, HDFs proliferated on the R8-matrix.⁵⁾ This was despite the observation that the R8-matrix was cytotoxic in the absence of serum (Fig. 5), indicating that the presence of serum reduced cytotoxicity. Proliferation of HDFs on the GR8-, AR8-, NR8-, QR8-, SR8-, TR8-, and VR8-matrices was comparable to that on the R8-matrix. Further, the YR8-matrix showed significantly higher cell proliferation than the R8-matrix. In contrast, the LR8- and WR8-matrices showed slightly weaker cell proliferation than the R8-matrix, and the ER8-, IR8-, and FR8-matrices did not promote cell proliferation. The low cell proliferation on the ER8-matrix could be explained by the lack of cell adhesion to the matrix. Meanwhile, the IR8- and FR8-matrices had strong cell attachment activity (Fig. 1B). The IR8- and FR8-matrices did not promote cell spreading in the presence of serum (Fig. 6B), although the FR8-matrix promoted integrin-mediated spreading under serum-free conditions (Fig. 2). These results indicated that the promotion of cell proliferation correlates with cell spreading. As a common feature, isoleucine and phenylalanine have a bulky hydrophobic side chain, and therefore, the associated hydrophobicity might reduce integrin-mediated cell spreading under serum-containing conditions, resulting in low cell proliferation. However, the low cell proliferation on the IR8-matrix was not observed in the case of HeLa and A549 cells (Supplementary Fig. S1), indicating that the effect of hydrophobicity on cell proliferation depends on cell type. Meanwhile, the proliferation of both cell types on the YR8-matrix was increased relative to that on the R8-matrix, indicating that the improvement of cell proliferation by tyrosine residues is not limited to HDFs. An EdU uptake assay revealed that DNA synthesis was suppressed on the IR8-matrix compared to that on the R8- and YR8-matrices, indicating that the X residue affects cell cycle progression (Supplementary Fig. S2). These findings indicate that neutral polar amino acids, but not hydrophobic amino acids, are suitable for cell proliferation under serum-containing conditions, as with the X residue of XR8 peptides.

Fig. 6. Cell Proliferation on XR8-Matrices in the Presence of Serum

(A) XR8 peptides (100 µM) were conjugated with aldehyde-agarose matrices. An aldehyde-agarose matrix with no peptide was used as a negative control (None). HDFs in 10% FBS/DMEM (5 × 10³ cells/well) were seeded into the sample wells and incubated for 2 d. Cell viability was measured using CCK8 assay. Relative cell viability vs. that on the R8-matrix was calculated. Values are shown as the mean of three independent experiments ± S.E. * p < 0.05, ** p < 0.0001 vs. R8-matrix. Dashed line indicates cell viability on a tissue culture plate on day 0. (B) Morphology of HDFs cultured on the R8-, IR8-, YR8-, and FR8-matrices in 10% FBS/DMEM for 2 h. Scale bar = 200 µm.

Secondary Structures of XR8 Peptides

The secondary structures of R8 and XR8 peptides were analyzed using CD spectroscopy (Fig. 7). R8, AR8, NR8, QR8, SR8, TR8, VR8, and LR8 exhibited a random coil structure. The IR8 spectrum indicated the presence of a β-sheet structure. A comparison of the IR8- and LR8-matrices indicated that the secondary structure of the peptides might affect the influence of the XR8-matrices on cell proliferation. The secondary structures of GR8, YR8, FR8, and WR8 were not determined, although the YR8 and WR8 spectra contained a peak at approximately 230 nm, which indicated the involvement of stacking interactions.^20–22)

Fig. 7. CD Spectra of XR8 Peptides

XR8 peptides were suspended at a final concentration of 100 µM in PBS. CD spectra were acquired at 37 °C.

In summary, we show here that biological activities of the XR8-matrices are different, and that the differences are associated with the nature of the incorporated amino acid X. The results indicated that the hydrophobicity and aromaticity of the XR8 peptides affect cell adhesion properties of the matrices. Hydrophobicity of the XR8 peptides tended to increase cell attachment activity but reduced cell proliferation under serum-containing conditions. Aromaticity of the XR8 peptides increased not only the cell attachment activity but also integrin β1-mediated cell spreading. Tyrosine is aromatic but less hydrophobic than phenylalanine and tryptophan. Therefore, the YR8-matrix most efficiently promoted cell adhesion, spreading, and proliferation among the XR8-matrices tested. These findings add to the understanding of cell adhesion mechanism of positively-charged peptides. Further, we anticipate that the YR8-matrix will be used for future applications in cell culture and biomaterial development.

Acknowledgments

This work was supported by JSPS KAKENHI, Grant Nos. JP20K20204, JP20K07622, and JP21K06563.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

REFERENCES

• 1) Yavin E, Yavin Z. Attachment and culture of dissociated cells from rat embryo cerebral hemispheres on polylysine-coated surface. J. Cell Biol., 62, 540–546 (1974).
• 2) Hoshino M, Tsujimoto T, Yamazoe S, Uesugi M, Terada S. Adhesamine, a new synthetic molecule, accelerates differentiation and prolongs survival of primary cultured mouse hippocampal neurons. Biochem. J., 427, 297–304 (2010).
• 3) Nakase I, Niwa M, Takeuchi T, Sonomura K, Kawabata N, Koike Y, Takehashi M, Tanaka S, Ueda K, Simpson JC, Jones AT, Sugiura Y, Futaki S. Cellular uptake of arginine-rich peptides: roles for macropinocytosis and actin rearrangement. Mol. Ther., 10, 1011–1022 (2004).
• 4) Futaki S, Nakase I, Tadokoro A, Takeuchi T, Jones AT. Arginine-rich peptides and their internalization mechanisms. Biochem. Soc. Trans., 35, 784–787 (2007).
• 5) Yamada Y, Onda T, Hamada K, Kikkawa Y, Nomizu M. Octa-arginine and octa-lysine promote cell adhesion through heparan sulfate proteoglycans and integrins. Biol. Pharm. Bull., 45, 207–212 (2022).
• 6) Kawaguchi Y, Takeuchi T, Kuwata K, Chiba J, Hatanaka Y, Nakase I, Futaki S. Syndecan-4 is a receptor for clathrin-mediated endocytosis of arginine-rich cell-penetrating peptides. Bioconjug. Chem., 27, 1119–1130 (2016).
• 7) Vogel BE, Lee SJ, Hildebrand A, Craig W, Pierschbacher MD, Wong-Staal F, Ruoslahti E. A novel integrin specificity exemplified by binding of the alpha v beta 5 integrin to the basic domain of the HIV Tat protein and vitronectin. J. Cell Biol., 121, 461–468 (1993).
• 8) Cardin AD, Weintraub HJ. Molecular modeling of protein-glycosaminoglycan interactions. Arteriosclerosis, 9, 21–32 (1989).
• 9) Kern A, Schmidt K, Leder C, Muller OJ, Wobus CE, Bettinger K, Von der Lieth CW, King JA, Kleinschmidt JA. Identification of a heparin-binding motif on adeno-associated virus type 2 capsids. J. Virol., 77, 11072–11081 (2003).
• 10) Fromm JR, Hileman RE, Caldwell EE, Weiler JM, Linhardt RJ. Differences in the interaction of heparin with arginine and lysine and the importance of these basic amino acids in the binding of heparin to acidic fibroblast growth factor. Arch. Biochem. Biophys., 323, 279–287 (1995).
• 11) Meneghetti MC, Hughes AJ, Rudd TR, Nader HB, Powell AK, Yates EA, Lima MA. Heparan sulfate and heparin interactions with proteins. J. R. Soc. Interface, 12, 20150589 (2015).
• 12) Caldwell EE, Nadkarni VD, Fromm JR, Linhardt RJ, Weiler JM. Importance of specific amino acids in protein binding sites for heparin and heparan sulfate. Int. J. Biochem. Cell Biol., 28, 203–216 (1996).
• 13) Jairajpuri MA, Lu A, Desai U, Olson ST, Bjork I, Bock SC. Antithrombin III phenylalanines 122 and 121 contribute to its high affinity for heparin and its conformational activation. J. Biol. Chem., 278, 15941–15950 (2003).
• 14) Dovas A, Yoneda A, Couchman JR. PKCbeta-dependent activation of RhoA by syndecan-4 during focal adhesion formation. J. Cell Sci., 119, 2837–2846 (2006).
• 15) Bass MD, Roach KA, Morgan MR, Mostafavi-Pour Z, Schoen T, Muramatsu T, Mayer U, Ballestrem C, Spatz JP, Humphries MJ. Syndecan-4-dependent Rac1 regulation determines directional migration in response to the extracellular matrix. J. Cell Biol., 177, 527–538 (2007).
• 16) Morgan MR, Humphries MJ, Bass MD. Synergistic control of cell adhesion by integrins and syndecans. Nat. Rev. Mol. Cell Biol., 8, 957–969 (2007).
• 17) Yamada Y, Yoshida C, Hamada K, Kikkawa Y, Nomizu M. Development of three-dimensional cell culture scaffolds using laminin peptide-conjugated agarose microgels. Biomacromolecules, 21, 3765–3771 (2020).
• 18) Kalaskar DM, Gough JE, Ulijn RV, Sampson WW, Scurr DJ, Rutten FJ, Alexander MR, Merry CLR, Eichhorn SJ. Controlling cell morphology on amino acid-modified cellulose. Soft Matter, 4, 1059–1065 (2008).
• 19) Yamada Y, Fichman G, Schneider JP. Serum protein adsorption modulates the toxicity of highly positively charged hydrogel surfaces. ACS Appl. Mater. Interfaces, 13, 8006–8014 (2021).
• 20) Yorita H, Otomo K, Hiramatsu H, Toyama A, Miura T, Takeuchi H. Evidence for the cation-pi interaction between Cu2+ and tryptophan. J. Am. Chem. Soc., 130, 15266–15267 (2008).
• 21) Peter B, Polyansky AA, Fanucchi S, Dirr HW. A Lys-Trp cation-pi interaction mediates the dimerization and function of the chloride intracellular channel protein 1 transmembrane domain. Biochemistry, 53, 57–67 (2014).
• 22) Andrushchenko VV, Vogel HJ, Prenner EJ. Solvent-dependent structure of two tryptophan-rich antimicrobial peptides and their analogs studied by FTIR and CD spectroscopy. Biochim. Biophys. Acta BBABiomembr., 1758, 1596–1608 (2006).