Use of Membrane Potential to Achieve Transmembrane Modification

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Use of Membrane Potential to Achieve Transmembrane Modification with an Artificial Receptor Wataru Hatanaka,† Miki Kawaguchi,‡ Xizheng Sun,‡ Yusuke Nagao,‡ Hiroyuki Ohshima,# Mitsuru Hashida,∇ Yuriko Higuchi,∇ Akihiro Kishimura,†,‡,§,∥ Yoshiki Katayama,†,‡,§,∥,⊥ and Takeshi Mori*,†,‡,§ †

Department of Applied Chemistry, Faculty of Engineering, ‡Graduate School of Systems Life Sciences, §Center for Future Chemistry, ∥International Research Center for Molecular Systems, and ⊥Center for Advanced Medical Innovation Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan # Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8501, Japan ∇ Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan S Supporting Information *

ABSTRACT: We developed a strategy to modify cell membranes with an artificial transmembrane receptor. Coulomb force on the receptor, caused by the membrane potential, was used to achieve membrane penetration. A hydrophobically modified cationic peptide was used as a membrane potential sensitive region that was connected to biotin through a transmembrane oligoethylene glycol (OEG) chain. This artificial receptor gradually disappeared from the cell membrane via penetration despite the presence of a hydrophilic OEG chain. However, when the receptor was bound to streptavidin (SA), it remained on the cell membrane because of the large and hydrophilic nature of SA.

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artificial receptor consists of a transmembrane domain, which has been modified with a membrane-penetrating domain and a ligand-binding domain on each terminus. The membranepenetrating domain is a cationic and relatively hydrophobic domain that can penetrate the hydrophobic cell membrane via the Coulomb force that arises from the membrane potential. The plasma membranes of mammalian cells have negative membrane potentials (−10 to −90 mV) that are dependent on the differences in the potassium concentrations on the outside and inside of the cell.9 Given that the membrane potential only exists on the cell membrane, the Coulomb force can work on the membrane-penetrating domain during the membranepenetration process. Once the membrane-penetrating domain reaches the inner surface of the cell membrane, it would no longer be affected by Coulomb force, thereby allowing for the retention of the artificial receptor on the cell membrane and avoiding further migration into the cytosol.

he chemical modification of the outer layer of a cell surface with conjugate molecules has recently attracted considerable attention because the resulting conjugates can function as receptors and ligands, thereby expanding on the existing repertoire of cellular functions.1 The modification techniques reported to date can be categorized into two approaches: modification via a covalent bond to the extracellular domains of membrane proteins2,3 or modification via hydrophobic interaction with the outer leaflet of the bilayer cell membrane.4−8 Conjugated molecules function on the outer surface of the cell to recognize target molecules. However, we have observed that natural transmembrane proteins report the recognition of target molecules by their extracellular domains to the inside of the cell through their cytoplasmic domains to induce intracellular signal transduction. This system enables more-complicated and sophisticated cellular responses than exterior recognition alone. However, it can be challenging to mimic the structure and function of transmembrane proteins via the conjugation of artificial molecules to the surface of a cell membrane. The hydrophobic lipid membrane does not allow for the penetration of hydrophilic molecules to achieve transmembrane modification. Here, we report for the first time the development of a new strategy to achieve a transmembrane structure by the chemical modification of a cell membrane with an artificial receptor. The concept of our strategy and the molecular design of the artificial receptor are shown in Figure 1. In terms of its structure, the © XXXX American Chemical Society

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RESULTS AND DISCUSSION First, we designed a membrane-penetrating domain for the artificial receptor. It was reported that hydrophobic peptides and peptide analogs bearing a cationic arginine10−12 or guanidinium Received: August 12, 2016 Revised: December 11, 2016 Published: January 4, 2017 A

DOI: 10.1021/acs.bioconjchem.6b00449 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

Figure 1. Modification of a cell membrane by an artificial receptor via Coulomb force and the recognition of the receptor by a ligand.

Scheme 1. Chemical Structures of the Membrane-Penetrating Peptides 1 and 2 and Receptor 3

group13 display membrane penetration via Coulomb’s force, generated by the membrane potential.14 We therefore designed two peptides that satisfy these conditions. These peptides contained cationic arginine residues as well as lysine residues that had been modified with hydrophobic groups (Scheme 1). A pair of different hydrophobic groups were evaluated in the current study, including lithocholoyl (peptide 1) and palmitoyl

(peptide 2) groups, which were two representative endogenous lipids. Number of the hydrophobic groups was selected to be two because more than two hydrophobic groups results in quite low solubility in aqueous media.15 These peptides were labeled with a nitrobenzofurazan (NBD) fluorophore on their N-termini. The modification of a peptide with a hydrophobic group can lead to a reduction in its aqueous solubility. Therefore, we used B

DOI: 10.1021/acs.bioconjchem.6b00449 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry methyl β-cyclodextrin (M-β-CD) to improve the solubility of the modified peptides because M-β-CD has been reported to solubilize hydrophobic molecules, including alkyl chains and bile acids via the formation of inclusion complexes.16−19 For example, dissociation constants (Kd) of inclusion complex formation between β-CD and bile acids were reported to be 10−3−10−5 M.18 These moderate Kd values will facilitate the transfer of peptides 1 and 2 from aqueous media to the cell membrane. Actually, cyclodextins were utilized for the modification of hydrophobic lipids to the cell membrane.19 The solubilities of peptides 1 and 2 were evaluated using the filtration method.5 Figure 2 shows absorption spectra of

As shown in Figure 3, peptides 1 and 2 showed clear differences in their intracellular distribution. For example, peptide 2

Figure 3. Fluorescence images of K562 cells treated with peptides 1 and 2 (2 μM). The two smaller panels shown below the main images are magnified images of the regions highlighted in the white squares.

showed fluorescence almost exclusively on the cell surface, whereas peptide 1 was mostly located within the cell, demonstrating successful membrane penetration by peptide 1 but not by peptide 2. The cellular localization of peptide 2 is similar to those of previously reported membrane anchoring molecules, which were anchored on the outer leaflet of the cell membrane.5 The membrane penetration of peptide 2 may be too slow to be observed during the period of observation. We clarified the intracellular distribution of peptide 1 using fluorescent markers for each organelle. As shown in Figure 4, peptide 1 co-localized well with the mitochondrial marker MitoTracker red rather than the endosomal/lysosomal marker LysoTracker red. The co-localization ratios of peptide 1 with the different organelle marker were calculated to be 32% and 72% for the endosomal and lysosomal and the mitochondrial markers, respectively. The high localization of peptide 1 in the mitochondria provided strong evidence of the membrane penetrating ability of peptide 1 via Coulomb force, which would be generated by the negative membrane potential inside of the mitochondria.20 Similar mitochondrial accumulations have been reported for other membrane-penetrating molecules, which were designed according to a similar concept to our peptides.12,13 The differences in the membrane penetrating abilities of peptides 1 and 2 can be attributed to their relative hydrophobicity. Kelley’s group clearly showed that the extent of peptide accumulation in the mitochondria is proportional to the hydrophobicity of the peptide.12 The palmitoyl group of peptide 2 does not appear to be sufficiently hydrophobic to penetrate the cell membrane. We further examined the effect of membrane potential on the membrane penetration of peptide 1 by using K+PBS as a medium for the modification of peptide 1 (Figure S4). As a result, the amount of peptide 1 that penetrates the cell membrane became smaller than Figure 3, indicating the importance of the membrane potential on the membrane penetration of peptide 1. We subsequently examined the cytotoxicity of peptide 1 toward K562 and did not detect any discernible toxicity after 48 h of incubation at 37 °C at a concentration of 2 μM (Figure S1). Next, we designed transmembrane and ligand-binding domains for the artificial receptor. Here, we chose oligoethylene glycol

Figure 2. Evaluation of the aqueous solubilities of peptides 1 and 2 and receptor 3. The peptide or receptor was dispersed in aqueous media (10 mM HEPES buffer containing 300 mM mannitol) in the presence or absence of M-β-CD. The resulting dispersion was filtered (0.22 μm), and the absorption spectrum of the filtrate was recorded. The absorption spectra of the filtrates obtained in the presence (dashed line) or absence of M-β-CD (dotted line) are shown in the figure along with the absorption spectra of peptides and receptor (2.0 μM) completely solubilized in DMSO (solid line).

aqueous solutions of peptides 1 and 2 in the presence and absence of M-β-CD after the removal of any insoluble peptide aggregates by filtration (0.22 μm). Peptide 1 was almost insoluble in aqueous medium in the absence of M-β-CD. However, in the presence of M-β-CD, about 70% of peptide 1 was solubilized (Figure 2). A similar improvement in the solubility was also observed for peptide 2, but the extent of this improvement was less pronounced than that of peptide 1, reflecting the differences in the association constants of the bile acid and alkyl moieties of peptides 1 and 2 with M-β-CD. The peptides (2 μM) were added to K562 cells using M-βCD as a solubilizer. This modification process was conducted at 37 °C for 5 min, followed by the removal of any unbound peptide by twice washing the cells with serum-containing medium. C

DOI: 10.1021/acs.bioconjchem.6b00449 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

Figure 4. Evaluation of the intracellular distribution of peptide 1. The upper and lower panels show the localization in the endosome and lysosome and the mitochondria, respectively.

membrane, most likely because of the hydrophilicity and large size of SA (6.8 nm along its longest axis).23,24 In contrast, the results for procedure 2 (Figure 5Biii) revealed that the fluorescence resulting from the receptor 3−SA complex was much weaker than that observed in panels i and ii of Figure 5B. This result clearly indicated that most of the receptor 3 molecules had disappeared from the cell membrane via their penetration into the cytosol before the addition of SA during the extended 3 h incubation. The quantitative evaluation of the fluorescence of SA by imaging cytometry also showed that the fluorescence in panel iii was much weaker than the fluorescence in panel ii (Figure 5C). The results shown in Figure 5B are schematically illustrated in Figure 6. Receptor 3 penetrated the cell membrane into the cytosol even though the Coulomb force would not work when the membrane-penetrating domain reached to the inner surface of the cell membrane. One of the possible explanations of the translocation of receptor 3 to the cytosol may be due to the pulling of receptor 3 from the cell membrane to the cytosol by some cytosolic proteins that interact with receptor 3 via hydrophobic interaction. The subsequent binding of receptor 3 to SA suppressed the penetration of receptor 3 because of the large and hydrophilic nature of SA. Taken together, these results indicated that the receptor 3−SA complex was taken up by the cell via endocytosis.

(OEG) as the transmembrane domain because of its amphiphilic characteristics; OEG is not only soluble in aqueous media but also is soluble in organic solvents, which would allow the distribution of the OEG domain across the hydrophobic cell membrane. In fact, OEG and some types of lipids are miscible enough to form homogeneous aqueous solutions.21 The OEG domain used in this study was 8.2 nm in length in its extended conformation, which was twice as long as the thickness of the lipid bilayer of the plasma membrane (4.25 nm).22 Biotin was modified as a ligand-binding molecule on the terminal of the OEG domain. The resulting receptor 3 (Scheme 1) was synthesized using traditional Fmoc solid-phase peptide synthesis. The aqueous solubility of receptor 3 was improved by the addition of M-β-CD (Figure 2), which was subsequently used to solubilize receptor 3 to investigate its effects in cells. We designed two experiments with different procedures, which are shown in Figure 5A. After the modification of K562 cells with receptor 3, the modified cells were immediately treated with SA (procedure 1) or after 3 h of incubation at 37 °C (procedure 2). As for procedure 1, receptor 3 and SA co-localized on the cell membrane (Figure 5Bi), indicating the successful recognition of receptor 3 by SA. Some SA was found in the cytosol adjacent to the nucleus, which is typical of the accumulation of endosomes in the microtubule organizing center (MTOC).23 This result therefore suggested that the receptor 3−SA complex was taken up by endocytosis. We confirmed that the receptor 3−SA complex actually localized in endosome or lysosome (Figure S5). Some of the cytosolic receptor 3 was not co-localized with SA in MTOC. This isolated material was attributed to the penetration of the cell membrane in the cytosol despite the hydrophilic nature of the OEG domain. Figure 5Bii shows an image of the cells after 3 h of incubation from the cells shown in Figure 5Bi. Notably, this image still exhibited strong fluorescence from the receptor 3−SA complex on the cell membrane. This result demonstrated that the receptor 3−SA complex no longer penetrated the cell

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CONCLUSIONS We have developed a new strategy for the modification of cell membranes using a transmembrane molecule through the use of the Coulomb force that arises from the membrane potential. Coulomb force allowed for the penetration of a cell membrane using cationic peptide 1, which was modified with several hydrophobic groups. Receptor 3 bearing an OEG chain penetrated the cell membrane into the cytosol. However, the binding of receptor 3 to SA suppressed its ability to penetrate the cell membrane because of the large and hydrophilic nature of SA. To the best of our knowledge, this strategy represents D

DOI: 10.1021/acs.bioconjchem.6b00449 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

Figure 5. (A) Pair of different experimental procedures for the modification of receptor 3 on the cell surface and the subsequent recognition of the receptor by SA. Fluorescence images (B) and the results of imaging cytometry (C) for the cells obtained by the two experiments. The panels to the right of the main images shown in (B) are magnified images of regions highlighted with white squares.

extracellular domain of the artificial receptor. Applicable agonist or antagonist molecules, without interrupting the membrane penetration of the artificial receptor, will be small in size with little charge; it will be possible to find satisfactory molecules from a huge library of previously reported agonist and antagonist molecules.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00449. Experimental details for the preparation of peptides 1 and 2 and receptor 3 as well as their use for the modification of cell surfaces. Images showing analysis of K652 cells. (PDF)

Figure 6. Summary of the behavior of receptor 3 before and after the formation of a complex with SA.

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AUTHOR INFORMATION

Corresponding Author

the first reported account of the transmembrane modification of a membrane with an artificial receptor that is a structural mimic of a transmembrane protein. If an agonist or an antagonist molecule for cytoplasmic signaling proteins is modified as a cytoplasmic domain of the artificial receptor, we may be able to induce cellular response signaling to ligand recognition on an

*E-mail: [email protected]. Phone/Fax: +8192-802-2849. ORCID

Akihiro Kishimura: 0000-0002-0503-1418 Takeshi Mori: 0000-0002-1821-5427 E

DOI: 10.1021/acs.bioconjchem.6b00449 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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(18) Yu, Y., Chipot, C., Cai, W., and Shao, X. (2006) Molecular Dynamics Study of the Inclusion of Cholesterol into Cyclodextrins. J. Phys. Chem. B 110, 6372−6378. (19) Koivusalo, M., Jansen, M., Somerharju, P., and Ikonen, E. (2007) Endocytic Trafficking of Sphingomyelin Depends on Its Acyl Chain Length. Mol. Biol. Cell 18, 5113−5123. (20) Chen, L. B. (1988) Mitochondrial Membrane Potential in Living Cells. Annu. Rev. Cell Biol. 4, 155−181. (21) Prajapati, H. N., Dalrymple, D. M., and Serajuddin, A. T. M. (2012) A Comparative Evaluation of Mono-, Di- and Triglyceride of Medium Chain Fatty Acids by Lipid/Surfactant/Water Phase Diagram, Solubility Determination and Dispersion Testing for Application in Pharmaceutical Dosage Form Development. Pharm. Res. 29, 285−305. (22) Mitra, K., Ubarretxena-Belandia, I., Taguchi, T., Warren, G., and Engelman, D. M. (2004) Modulation of the bilayer thickness of exocytic pathway membranes by membrane proteins rather than cholesterol. Proc. Natl. Acad. Sci. U. S. A. 101, 4083−4088. (23) Tobinaga, K., Li, C., Takeo, M., Matsuda, M., Nagai, H., Niidome, T., Yamamoto, T., Kishimura, A., Mori, T., and Katayama, Y. (2014) Rapid and serum-insensitive endocytotic delivery of proteins using biotinylated polymers attached via multivalent hydrophobic anchors. J. Controlled Release 177, 27−33. (24) Weber, P. C., Ohlendorf, D. H., Wendoloski, J. J., and Salemme, F. R. (1989) Structural origins of high-affinity biotin binding to streptavidin. Science 243, 85−88.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology of Japan (grant no. 16H04167).

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ABBREVIATIONS SA, streptavidin; M-β-CD, methyl β-cyclodextrin REFERENCES

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DOI: 10.1021/acs.bioconjchem.6b00449 Bioconjugate Chem. XXXX, XXX, XXX−XXX