Design of LipidâProtein Conjugates Using Amphiphilic Peptide

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Design of Lipid-Protein Conjugates Using Amphiphilic Peptide Substrates of Microbial Transglutaminase Mari Takahara, Rie Wakabayashi, Kosuke Minamihata, Masahiro Goto, and Noriho Kamiya ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00271 • Publication Date (Web): 30 Oct 2018 Downloaded from http://pubs.acs.org on November 3, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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H

N ACS Applied Bio Materials

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N O N H

H N

O N H

O

O

H N

H N

N H

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O NH2 OH

NH HN

NH2

NH2

C14-MRHKGS (X = 0) H N S N O N H

H N n

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O N H OH

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H N O

O NH2 OH

NH HN

NH2

NH2

C14-GnS-MRHKGS (X = GnS, n = 3-6)

(B)

Microbial transglutaminase (MTG) L L Q G

NH2

L L Q G O NH

O NH2

Q-tagged Protein fused with Q-tag (LLQG)

M R H Q K G S

ACS Paragon Environment Lipid-fused K-tag Plus peptides C14-X-MRHKGS

M R H Q K G S

Lipid-protein conjugate

+ NH3

ACS Applied Bio Materials

30

C14-MRHKGS C14-PG3S-MRHKGS

θ×10-3 (deg cm2 dmol-1)

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C14-G3S-MRHKGS

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1 µM C14-X-EGFP, 5 µM C14-X-MRHKGS 5 µM C14-X-EGFP, 25 µM C14-X-MRHKGS

Fluorescence/cell

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1) LQ-EGFP alone [M+H]+ 28513.26

()

8.93

2)( LQ-EGFP ) + C14-G3S-MRHKGS 0 5 6. +MTG (30 min)

(

14.17

2) LQ-EGFP +C14-G3S-MRHKGS +MTG (30 min)

)

(D) 100

[M+H]+ 29643.38

80 60 40

linkerC14-MRHKGS GGGS C14-G3S-MRHKGS

20 0

100 80 60 40


20 0

0

10

20

30

40

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Δ = 1130.12

14.17 min (C14-G3S-EGFP)

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13.4 min (EGFP)

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13.42

3)(Purified C14-G3S-EGFP 1 6 7 8.0

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[M+H]+ 29688.62

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Retention time (min)

20

Conversion rate (%)

8.09

Conversion rate (%)

Absorbance at 280 nm

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2 34-

1243.

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30000 m/z

Ratio of [C14-(G3S)-MRHKGS]/[LQ-EGFP]


Ratio of [C14-(G3S)-MRHKGS]/[FQ-EGFP]

60

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Design of Lipid-Protein Conjugates Using Amphiphilic Peptide Substrates of Microbial Transglutaminase Mari Takahara,†,‡ Rie Wakabayashi,† Kosuke Minamihata,† Masahiro Goto,†,§ and Noriho Kamiya.†,§,* †

Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan ‡ National Institute of Technology, Kitakyushu College, Department of Materials Science & Chemical Engineering, 5-20-1 Shii, Kokuraminamiku, Kitakyushu, Fukuoka, Japan § Division of Biotechnology, Center for Future Chemistry, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan Keywords: Lipid, peptide, self-assembly, lipid-protein conjugate, microbial transglutaminase ABSTRACT: Lipid-modification of proteins plays a significant role in regulating the cellular environment. Mimicking natural lipidated proteins is a key technique for assessing the function of proteins modified with lipids and also to render self-assembly of lipids to a target protein. Herein, we report a facile method of conjugating proteins with lipid-fused peptides under homogeneous physiological conditions by using the microbial transglutaminase (MTG) reaction. MTG catalyzes the cross-linking reaction between a specific glutamine (Q) in a protein and a lysine (K) in newly designed lipid-fused peptides. The water-soluble peptide substrates for lipid modification, C14-X-MRHKGS, were newly synthesized, where C14, X and MRHKGS represent myristic acid, linker peptides composed of G, P or S, and MTG-reactive K surrounded with basic amino acids, respectively. The MTG-mediated cross-linking reaction between a protein fused with LLQG at the C-terminus and C14-X-MRHKGS (5 molar eq.) dissolved in a phosphate saline solution resulted in lipid-protein conjugates with yields of 70 to 100%. The anchoring ability of the obtained lipidprotein conjugates to cell membranes was dependent on the number of G residues in the GnS linker, suggesting that self-assembly and hydrophobicity of the GnS motif serves to enhance membrane anchoring of lipid-protein conjugates.

INTRODUCTION Lipidated proteins anchored to cell membranes regulate cellular functions, including cell signaling, proliferation and apoptosis.1-5 Myristoylation is a well-known lipid-modification6 that is catalyzed by N-myristoyl transferase, and this modification plays a significant role in cell survival; however, excess myristoylation of Src family kinases is implicated in carcinogenesis.7-9 Additionally, orientation of lipid-protein conjugates has been exploited to immobilize receptor-recognizing proteins for targeting of drug delivery carriers10,11 represented by myristoylated liposomes.12 In drug delivery systems, lipidmodification of proteins or peptides leads to enhanced stability and prolonged half-lives in blood because of the affinity of the lipid towards serum albumin.13,14 For example, several lipidfused insulin analogues such as degludec15 and liraglutide16 are commercially available. Thus, mimicking natural lipidated proteins is a key technique to assess the function of lipidconjugated proteins in disease or metabolic pathways, and to stabilize a protein on a drug delivery carrier by lipid affinity. However, efficient synthesis of lipid-conjugated proteins without partial loss of activity has been limited mainly because of the hydrophobicity of lipids and the selectivity of the coupling site. As a major problem, the hydrophobicity of lipids can potentially impair the function of the target protein by attachment of the lipid at an undesired site, and exposure to organic solvents

is required for solubility.17 Chemical coupling strategies that target lysine, cysteine or tyrosine residues18 of the protein of interest (POI) have been developed. However, some of the obtained protein conjugates suffered from altered activities after exposure to excess amount of fatty acid derivatives dispersed in organic solvents. Therefore, to avoid denaturation of the POI, site-specific conjugation in a homogeneous aqueous phase is vital. Native chemical ligation has overcome the aforementioned problems by coupling a recombinant protein thioester and cysteine at the C-terminus of lipid-fused peptides; although, incorporation of the relatively large size of the intein domain, restriction of C-terminal attachment and slow reaction rates limit production of the modified POI.19,20 Recently, copper-free click chemistry has developed for lipidprotein conjugation using the small chemical handle of tirazole moiety.14 The artificial triazole moiety is useful for stability in vivo, however, lack of biocompatibility inhibit the intracellular enzymatic digestion that works for immune response via drug delivery of antigens.21 Alternatively, enzymatic ligation has been employed to render the biocompatible handle in conjugation although the recombinant expression of POI is required, and the insertion of extra peptide sequence may affect the function of POI. As a significant advantage, enzymes efficiently catalyze the coupling reactions between substrates with specific peptide sequence under mild conditions. For example, the sortase A-catalyzed conjugation method has been established in a site-specific manner using a LPETG-tag fused pro-


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tein and peptides containing various kinds of fatty acids and cholesterol; however, despite the insertion of relatively short peptide sequence without the denaturation of POI, this approach has the drawbacks of reversible covalent bond formation, the presence of a surfactant in the reaction and insufficient yields that did not reach 100%.22 Herein we report a novel method of conjugating proteins with lipid-fused peptides under organic solvent free conditions using the microbial transglutaminase (MTG) reaction.23-25 MTG catalyzes the cross-linking reaction between specific glutamine (Q) and lysine (K) residues in proteins or peptides. We have reported previously MTG-mediated lipidation of a recombinant protein fused with the MRHKGS amino acid sequence (K-tag; underlined K is MTG-reactive);26 however, use of the hydrophobic lipid-G3S-LLQG peptide substrate and the requirement of a surfactant limits the applicability to target proteins that possess rigid structures. Therefore, design of water-soluble lipid-fused peptides is required for the development of MTG-mediated conjugation. In this context, we focused on the reverse combination of MTG substrates, i.e., a protein fused with the amino acid sequence MTG-reactive Q (Q-tag) was conjugated with lipid-fused peptides containing the MRHKGS sequence (Figures 1A and S1). In this peptide design, basic and hydrophilic R and K support the solubility of lipid substrates (Figure 1B). The peptide substrates for lipid modification were designed as the C14-X-MRHKGS backbone, where C14 (myristic acid) and the K-tag sequence (MRHKGS) are coupled via the peptidyl linker X composed of G, P or S, resulting in improved solubility in a phosphate saline solution up to 250 µM. The model Q-tagged proteins used were enhanced green fluorescent proteins (EGFPs) fused with LLQG (LQ-EGFP)27 or FYPLQMRG (FQ-EGFP)28 at the C-terminus. By flipping the MTG-reactive substrates, the MTG-mediated cross-linking reaction between a Q-tagged EGFP and C14-XMRHKGS (5 molar eq.) in a phosphate saline solution proceeded without any additives, yielding lipid-modified EGFPs up to 100% conversion. The obtained lipid-protein conjugates showed biocompatibility and were capable of anchoring to a cell membrane. The anchoring ability of the lipidated-EGFP increased in accordance with the number of G residues in the linker GnS motif used as the X moiety. The mechanism of enhanced anchoring ability that was dependent on the number of glycine residues reflects the self-assembly nature of the GnS motif. RESULTS AND DISCUSSION Design of MTG-Reactive Lipid-Fused Peptides. The lipid-fused peptides, C14-X-MRHKGS peptides, were synthesized by Fmoc solid-phase peptide synthesis. Synthesis of all peptides was confirmed by MALDI-TOF-MS and reversedphase (RP)-HPLC. The purity of the lipid-fused peptides was determined to be >90%, except for C14-SG2S-MRHKGS (88%) (Figures S2–S4). The newly designed lipid-fused peptides were soluble to 250 µM in PBS, whereas the previously reported C14-G3S-LLQG (100 µM) showed minimal solubility in PBS (Figure S5). This enhanced solubility in PBS was achieved by using the sequence MRHKGS, which contains more hydrophilic basic amino acids R and K than the previously reported hydrophobic lipid-G3S-LLQG construct.

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NH2

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O NH2


+ NH3

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Lipid-fused K-tag peptides C14-X-MRHKGS

M R H Q K G S

Lipid-protein conjugate

Figure 1. (A) Chemical structures of C14-X-MRHKGS. (X = GnS, n = 3–6). (B) Schematic illustration of MTG-mediated cross-linking between C14-X-MRHKGS and Q-tagged EGFP.

Selection of a Q-Tagged Protein for C14-(G3S)-MRHKGS Labeling Using the MTG Reaction. The Q-tag (LLQG26 or FYPLQMRG27) was selected to prepare LQ-EGFP or FQEGFP to test the MTG reactivity toward C14-MRHKGS or C14G3S-MRHKGS. Optimal lipidation of the Q-tag was carried out through a time-course analysis and by examining the effect of the substrate ratio of [K]/[Q] on lipidation. Q-tagged EGFP (10 µM) and C14-(G3S)-MRHKGS (50 µM, 5 molar eq.) were cross-linked by MTG (0.1 U/mL) in PBS (pH 7.4) at 37 °C for 60 min to yield C14-(G3S)-MRHKGSmodified EGFP (C14-(G3S)-EGFP). Additionally, to assess the effect of the substrate ratios between C14-(G3S)-MRHKGS and Q-tagged EGFP ([K]/[Q] = 1, 2, 5, 10, 20), Q-tagged EGFPs (10 µM) were modified with different molar ratios of C14-G3SMRHKGS under the same conditions but for 30 min. The identification of the reaction product was carried out by MALDI-TOF-MS (Figure S6). The samples were assessed using RP-HPLC with EGFPs and C14-(G3S)-EGFP eluting at 13.4 and 14.2 min, respectively (Figure 2A). The purified C14G3S-EGFP was used for identification in both RP-HPLC and MS results where the main peak at 14.2 min corresponded to the molecular weight of one LQ-EGFP with single C14-G3SMRHKGS (Δtheor = 1151.64) (Figure 2B). The conversion rate was calculated from the integrated peak areas as follows: Conversion rate (%) = Area14.2 × 100/(Area14.2 + Area13.4) The MTG reaction was saturated after 10 min for FQ-EGFP using C14-G3S-MRHKGS, 10 min for LQ-EGFP using C14MRHKGS and 30 min for LQ-EGFP using C14-G3SMRHKGS (Figure 2C, D). The maximum conversion rate of FQ-EGFP using C14-MRHKGS or C14-G3S-MRHKGS was ~80%, whereas that of LQ-EGFP and C14-MRHKGS reached almost 100%. The conversion rate of LQ-EGFP using C14G3S-MRHKGS was 80%. The different conversion rates of LQ-EGFP with C14-MRHKGS or C14-G3S-MRHKGS can be explained by the presence of the hydrophobic G3S moiety, which potentially inhibits access of MTG to the lipid substrate.



(C)

1) LQ-EGFP alone

()

8.93

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[M+H]+ 29643.38

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[M+H]+ 29688.62

Δ = 1175.35 9.59

13.42

28000 8

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16

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20

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8.09

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2 34-

)

2)( LQ-EGFP ) + C14-G3S-MRHKGS 0 5 6. +MTG (30 min)

(D) 100

[M+H]+ 28513.26

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13.4 min (EGFP)

Conversion rate (%)

(B)

1) LQ-EGFP alone

1243.

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Absorbance at 280 nm

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30000 m/z

Ratio of [C14-(G3S)-MRHKGS]/[LQ-EGFP]

Ratio of [C14-(G3S)-MRHKGS]/[FQ-EGFP]

Figure 2. (A) RP-HPLC chromatograms and (B) MALDI-TOF-MS analysis of 1) unreacted LQ-EGFP, 2) LQ-EGFP with C14-G3SMRHKGS in the presence of MTG, 3) purified C14-G3S-EGFP. Analysis of the MTG reaction between (C) LQ-EGFP or (D) FQEGFP and C14-G3S-MRHKGS. (Top) Time-course analysis: C14-MRHKGS (red), C14-G3S-MRHKGS (blue). (Bottom) Effect of the [K]/[Q] ratio; [K] = C14-G3S-MRHKGS. [Q] = Q-tagged EGFP. In the bottom graph, the blue column or pink column represents C14-MRHKGS or C14-G3S-MRHKGS as the substrate, respectively.

Furthermore, the conversion rate between the Q-tagged EGFPs and the lipid-fused peptide increased up to 100% at [K]/[Q] = 20 in LQ-EGFP (Figure 2D). Thus, MTG-mediated lipid modification of LQ-EGFP depends on the [K]/[Q] ratio, whereas conversion of FQ-EGFP was saturated at [K]/[Q] = 2 (80% yield, C14-G3S-MRHKGS). In contrast to the conversion of FQ-EGFP being independent of the [K]/[Q] ratio in the MTG reaction, LQ-EGFP exhibited a higher conversion ratio (100% for C14-MRHKGS or 80% C14-G3S-MRHKGS) than FQ-EGFP ( G4S > G6S > G3S > X = 0. Despite the low conversion rate, C14-G5SEGFP lipidated with C14-G5S-MRHKGS, which displayed the lowest CMC and β-sheet structure, showed the highest anchoring ability. This tendency coincides with the trade-off relationships of conversion rate, CMC and CC50, which are related to aggregation and hydrophobicity of the lipid-fused peptides. Additionally, C14-GnS-EGFP (n = 1, 2) and C14-X-EGFP (X = SG2S, G2, G4, PG3S, PEG, PG3) showed the same fluorescence intensity as C14-EGFP (Figure S16). Without the GnS motif (n ≥ 3), or in the presence of P or S at the N-terminus of C14-XMRHKGS, C14-X-EGFP did not readily anchor to the cell membrane, in accordance with the relatively hydrophilic nature as depicted by the high CC50 and CMC values. Since C14GnS-EGFP (n = 3–6) bound to the cell membrane regardless of whether the peptide formed a β-sheet conformation, not only the secondary structure of lipid-fused peptides but also the hydrophobicity of GnS (the number of G residues) served as a driving force for membrane binding. Therefore, the GnS (n ≥ 3) moiety of the peptides played a significant role in membrane interaction through secondary structure or its hydrophobicity, in addition to the myristic acid moiety at the Nterminus. Although 5 µM C14-GnS-EGFP showed cell membrane anchoring ability, the treatment caused cytotoxicity toward SNU-1 cells (50–77% viability). Therefore, treatment of cells with 1 µM C14-GnS-EGFP was carried out, resulting in no cytotoxicity during the incubation period (Figure S17). For 1 µM C14-GnS-EGFP, C14-GnS-EGFP (n = 5, 6) had slightly lower fluorescence intensities when compared with equivalent data at 5 µM, whereas those of C14-GnS-EGFP (n = 3, 4) drastically decreased. This result suggests the hydrophobicity of

GnS had a more critical effect on the interaction with cell membranes than the secondary structure at the lower concentration. Equal anchoring ability of C14-X-MRHKGS (X = SG2S, G2, G4) when compared with that of C14-MRHKGS also supports the significance of the GnS motif (n = 3–6), especially when considering that C14-G2-MRHKGS or C14-G4-MRHKGS (X was composed of G alone) could not interact with membrane as much as C14-GnS-MRHKGS. We also attempted the purification26 of C14-G5S-EGFP, which showed the highest anchoring ability, and compared the anchoring ability with or without purification. After purification, the anchoring ability of C14-G5S-EGFP (1 µM, 85% conversion rate with the addition of a surfactant22, 26) was lower than the result without purification (64.1% conversion rate), regardless of their conversion rate (Figure S18). The enhanced anchoring ability in the presence of unreacted C14-G5SMRHKGS suggests that C14-G5S-EGFP interacts with selfassembled co-existing C14-G5S-MRHKGS. The different cellanchoring behavior was also explained by the smaller diameter of unpurified C14-G5S-EGFP in DLS measurements when compared with that of purified C14-G5S-EGFP, assuming a more compact micellar structure by the co-assembly of the free lipid-fused peptide (C14-G5S-MRHKGS) and the lipidized protein (C14-G5S-EGFP). The localization of C14-GnS-EGFP was then confirmed by confocal laser scanning microscopy (CLSM). The SNU-1 cells after C14-GnS-EGFP (n = 3–6) treatment showed fluorescence at the cell surface (Figure 5). The intensity of the EGFP fluorescence reached a maximum with the G5S linker, followed by G6S and G4S. The fluorescence of EGFP conjugated with any C14-GnS-MRHKGS remained primarily at the surface of cells, indicating that the C14-GnS-moiety did not support the incorporation of EGFP into the cell and just anchored EGFP to the cell membrane, although lipid moieties such as cholesterol or C22 have been shown to cause intracellular localization21. The above results showed that increasing the number of G residues in the C14-GnS-MRHKGS construct generally improved anchoring ability with the C14-G5S-MRHKGS peptide showing the highest membrane binding activity and the second 1 µM C14-X-EGFP, 5 µM C14-X-MRHKGS 5 µM C14-X-EGFP, 25 µM C14-X-MRHKGS

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Figure 4. Results of the anchored MTG reaction product containing C14-X -EGFP on SNU-1 cells (X = 0 or GnS, n = 3–6). Bars represent standard deviations. Blue column: 5 µM C14X-EGFP and 25 µM C14-X-MRHKGS. White column: 1 µM C14-X-EGFP and 5 µM C14-X-MRHKGS.



best peptide, C14-G6S-MRHKGS, displaying biocompatibility and MTG reactivity. However, sequential glycine coupling in peptide synthesis gives low yields and is therefore costly. In order to satisfy easy synthesis, high biocompatibility, MTG reactivity and membrane anchoring ability, the C14-G3SMRHKGS peptide is considered to be a well-balanced substrate for MTG-mediated lipidation of proteins. Based on C14G3S-MRHKGS as a minimum membrane anchoring peptide, our next approach is the design of a lipid moiety coupled with the G3S-MRHKGS peptidyl moiety (lipid-G3S-MRHKGS) to facilitate lipidation of a POI that functions alone on a cellular membrane. CONCLUSIONS We have achieved MTG-mediated lipidation between Qtagged EGFP and C14-GnS-MRHKGS (5 eq. molar) in a homogeneous PBS that does not include organic solvents. The enzymatic lipidation proceeded quantitatively, enabling us to validate how the linker sequence affects the characteristics of lipidated EGFPs. By changing the number of G residues in the peptidyl GnS moiety, the cell-membrane anchoring ability of C14-GnS-EGFP was modulated because of the self-assembling ability and hydrophobicity of the GnS motif. Among the C14GnS-MRHKGS peptides examined, C14-G5S-MRHKGS clearly EGFP

Bright field

Merge

LQ-EGFP alone 20 µm

C14-EGFP

C14-G3S-EGFP

C14-G4S-EGFP

C14-G5S-EGFP

C14-G6S-EGFP

Figure 5. CLSM observation of SNU-1 cells after treatment of EGFP or C14-GnS-EGFP (5 µM). Scale bar: 20 µm.

Page 10 of 13

formed a β-sheet, had the lowest CC50, a low CMC and high MTG reactivity, rendering this peptide with the highest membrane-anchoring ability following the MTG reaction. In contrast, attachment of C14-MRHKGS that lacks the GnS moiety with EGFP did not lead to sufficient interaction with the membrane even though this lipidated EGFP had high CC50, CMC and MTG reactivity values. The second-best membrane anchoring species, C14-G6S-MRHKGS, without a β-sheet supports the observation that the hydrophobicity of GnS also facilitates interaction with the membrane. Accumulated insights into C14-GnS-MRHKGS showed the trade-off relationships among membrane-anchoring ability, conversion rate in the MTG reaction and biocompatibility that associates with the self-assembly nature of C14-GnS-MRHKGS. Despite being a desirable anchoring lipid-fused substrate, synthesis cost and cytotoxicity of C14-G5S-MRHKGS preclude general use. Therefore, we have selected C14-G3S-MRHKGS as a minimum anchoring sequence for further study, and the design of a fatty acid moiety in (lipid)-G3S-MRHKGS is a work in progress. EXPERIMENTAL SECTION MTG Reaction of Q-Tagged EGFP with C14-XMRHKGS. Q-tagged EGFPs (LQ-EGFP, FQ-EGFP) were expressed in E. coli and purified following previous reports.26, 27 The concentrations of EGFPs were determined by the absorbance at 488 nm using a molar extinction coefficient of 55000 M–1 cm–1 with a NanoDrop ND-2000 device (Thermo Fisher Scientific, Waltham, MA). For time-course analysis of the MTG reaction, Q-tagged EGFPs (10 µM) were cross-linked with C14-X-MRHKGS (50 µM) through MTG (0.1 U/mL) catalysis in PBS at 37 °C for 60 min with a substrate ratio of [C14-X-MRHKGS]/[Q-tagged EGFP] = 5. Thirty microliter aliquots of the reaction were sampled at 5, 10, 15, 30 and 60 min in 150 µL reaction solution and the reaction terminated by the addition of 30 µL ACN (0.2% TFA). The effect of the substrate ratio on the MTG reaction was analyzed by incubating 30 µL of a PBS reaction mixture containing Q-tagged EGFPs (10 µM), C14-X-MRHKGS (10, 20, 50, 100, 200 µM) and MTG (0.1 U mL–1) at 37 °C for 60 min. The reaction solution was terminated by the addition of 30 µL ACN (0.2% TFA). The MTG reaction under various conditions was evaluated by RP-HPLC. Twenty microliters of terminated reaction solutions (5 µM lipidated-EGFP in PBS/ACN (50/50, 0.1% TFA) were injected into a Inertsil ODS-3 (4.6 × 250 mm) column with the following analysis conditions: flow rate, 1.0 mL/min; detection wavelength, 280 nm; mobile phase, ACN/water (0.1% TFA) = 20/80 to 0/100 over 40 min. The purity was determined by the ratio of integrated peaks of unreacted EFGP and C14-X-modified EGFP. MALDI-TOF-MS analysis was performed for the samples of LQ-EGFP alone, MTG reaction mixture of LQ-EGFPs (10 µM), C14-G3S-MRHKGS (50 µM) and MTG (0.1 U mL–1), and purified C14-G3S-EGFP after MTG reaction of LQ-EGFPs (10 µM), C14-G3S-MRHKGS (200 µM) and MTG (0.1 U mL–1) in PBS for 30 min at 37 °C terminated by 1 mM NEM. The purification using a Ni-NTA slurry was carried out following previous reports.22,26 The C14-X-modified EGFPs (C14-X-EGFPs) after the MTG reaction for 30 min were used for cell membrane anchoring without purification.


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Cell Culture. The SNU-1 cell line (CRL-5971) was purchased from the American Type Culture Collection (ATCC, Manassas, VA). Medium and buffers were purchased from Thermo Fisher Scientific. SNU-1 cells were grown in Roswell Park Memorial Institute medium (RPMI 1640) supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic at 37 °C in a humidified atmosphere of 5% CO2. SNU-1 cells were washed twice with Dulbecco’s phosphate-buffered saline (D-PBS) prior to an experiment. Cell Membrane Anchoring of C14-X-EGFP. For flowcytometry, SNU-1 cells (2.0 × 105 cells) were incubated with C14-X-EGFP (1 or 5 µM) in 50 µL D-PBS for 15 min at 37 °C. The incubation time and concentration of C14-GnS-EGFP used were optimized (Figure S16). Cells were then rinsed with 200 µL D-PBS and suspended in 400 µL D-PBS. The samples were passed through a 40-µm cell strainer (Corning Inc., Corning, NY) and analyzed by a cell analyzer EC800 (Sony, Tokyo, Japan) to detect EGFP bound to SNU-1 cells until the count of SNU-1 cells reached 1.0 × 104. The measurement was repeated in triplicate. For CLSM imaging, SNU-1 cells (5.0 × 104 cells) were incubated with 5 µM C14-X-EGFP in 50 µL D-PBS for 15 min at 37 °C. Cells were then rinsed with 100 µL D-PBS and cells suspended in 100 µL D-PBS were transferred into glass bottom dishes (Matsunami Glass, Osaka, Japan). The localization of C14-X-EGFP on SNU-1 cells was observed by CLSM (Carl Zeiss AG, Oberkochen, Germany) CD spectroscopy. C14-X-MRHKGS (100 µM) peptides were incubated in PBS prior to measurement. CD spectra of peptides were recorded on a J-820 CD spectropolarimeter (Jasco, Tokyo, Japan) using a 1 mm quartz cell at 37 °C with a scanning rate of 50 nm/min and a response time of 1 s. All spectra were background subtracted using PBS as the blank.

MALDI-TOF-MS and RP-HPLC analysis of C14-X-MRHKGS peptides, MALDI-TOF-MS analysis and RP-HPLC of C14-XEGFPs, CD spectra, estimated secondary structure components of peptides, measurement method of CMC and CC50, CMC plot, cell viability assay results, histograms from flow-cytometry analysis, dynamic light scattering results, time-course analysis of C14-XEGFP in cellular anchoring, analysis of C14-X-EGFP at different concentrations in cellular anchoring (PDF).

Table 1. Properties of C14-X-MRHKGS peptides

REFERENCES

C14-XMRHKGS

Conversion rate of LQ-EGFP

Fluorescence/ cell (a. u.)

CC50 (µM)

CMC (µM)

X=0

97.1

9.32

73.8

93.3

X = G3S

80.3

147

9.72

18.1

X = G4S

82.3

208

5.91

22.5

X = G5S

64.1

278

2.57

8.23

X = G6S

98.8

175

5.92

1.26

X = PG3S

100

20.0

14.6

55.5

X = G4

98.3

50.0

9.77

20.9

X = GS

100

10.1

79.7

88.8

X = G2S

97.6

12.9

31.3

32.0

X = G2

99.9

8.21

56.0

21.6

X = SG2S

85.5

17.8

36.9

88.9

X = PEG

94.6

8.72

17.1

57.9

X = PG3

83.7

10.2

28.6

41.9

ASSOCIATED CONTENT

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected], Tel: +81-(0)92802-2807; Fax: +81-(0)92-802-2810.

ORCID Mari Takahara: 0000-0002-5435-3536 Rie Wakabayashi: 0000-0003-0348-8091 Masahiro Goto: 0000-0002-2008-9351 Noriho Kamiya: 0000-0003-4898-6342

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was financially supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (No. 17H07320 to M. T., No. JP16H04581 to N. K.). A part of this work was conducted at Kyushu University and supported by the Nanotechnology Platform Program (Molecule and Material Synthesis) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. This study was funded by the Asahi Glass Foundation. We thank Professor Sonoda (National Institute of Technology, Kitakyushu Colleg) for supporting Fmoc solid-phase peptide synthesis. We thank the Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

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Supporting Information The Supporting Information is available free of charge on the ACS Publications website.

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Walsh, C. T., Posttranslational Modification of Proteins: Expanding Nature's Inventory. Englewood, Colo.: Roberts and Co. Publishers 2006, 171-201. Resh, M. D., Targeting Protein Lipidation in Disease. Trends Mol. Med. 2012, 18, 206-214. Resh, M. D., Covalent Lipid Modifications of Proteins. Curr. Biol. 2013, 23, R431-R435. Tate, E. W.; Kalesh, K. A.; Lanyon-Hogg, T.; Storck, E. M.; Thinon, E., Global Profiling of Protein Lipidation Using Chemical Proteomic Technologies. Curr. Opin. Chem. Biol. 2015, 24, 48-57. Kakugawa, S.; Langton, P. F.; Zebisch, M.; Howell, S.; Chang, T. H.; Liu, Y.; Feizi, T.; Bineva, G.; O'Reilly, N.; Snijders, A. P.; Jones, E. Y.; Vincent, J. P., Notum Deacylates Wnt Proteins to Suppress Signalling Activity. Nature 2015, 519, 187-192. Boutin, J. A.; Myristoylation. Cell Signal. 1997, 9(1), 15-35. Sakurai, N.; Utsumi, T., Posttranslational N-myristoylation Is Required for the Anti-apoptotic Activity of Human tGelsolin, the C-terminal Caspase Cleavage Product of Human Gelsolin. J. Biol. Chem. 2006, 281 (20), 14288-14295. Patwardhan, P.; Resh, M. D., Myristoylation and Membrane Binding Regulate c-Src Stability and Kinase Activity. Mol. Cell Biol. 2010, 30, 4094-4107. Wright, M. H.; Heal, W. P.; Mann, D. J.; Tate, E. W., Protein Myristoylation in Health and Disease. J. Chem. Biol. 2010, 3, 19-35. Gaber, M.; Medhat, W.; Hany, M.; Saher, N.; Fang, J. Y.; Elzoghby, A., Protein-lipid Nanohybrids as Emerging Plat-



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Insert Table of Contents artwork here (TOC graphic) Microbial transglutaminase (MTG)

H 2N

O

H N O OH

NH2 O

NH

N O

N H

O

H N

N H

O

S O

H N O

N H HO

O

H N O

N H

n

HN

H N

O

NH H 2N

S

NH

N O

L L Q G

12

N H

H N n

O

O N H OH

H N

O

H N

N H

O

O

O N H

H N O

O

+ NH3

L L Q G

NH2 OH

NH HN


w/o GnS

NH2

NH2

Lipid-fused K-tag peptides C14-GnS-MRHKGS n=3

n=4

C14-GnS-protein conjugate

n=5

n=6

20 µm

Anchoring ability of C14-GnS dependent on the number of G (maximum: n = 5)

TOC graphic


8

12

Design of LipidâProtein Conjugates Using Amphiphilic Peptide

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