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Syndecan-4 Is a Receptor for Clathrin-mediated Endocytosis of Arginine-rich Cell-penetrating Peptides Yoshimasa Kawaguchi, Toshihide Takeuchi, Keiko Kuwata, Junya Chiba, Yasumaru Hatanaka, Ikuhiko Nakase, and Shiroh Futaki Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/ acs.bioconjchem.6b00082 • Publication Date (Web): 28 Mar 2016 Downloaded from http://pubs.acs.org on March 30, 2016

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Bioconjugate Chemistry

Table of contents (5.1 cm × 9 cm) Photoaffinity probe of cell-penetrating peptide (R8) CPP Affinity tag Photoaffinity group

Cleavable linker CPP

Membrane

?

CPP

Photocrosslinking approach

Receptor

Syndecan-4

Endocytosis

Clathrin-mediated endocytosis

ACS Paragon Plus Environment

1


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Title: Syndecan-4 Is a Receptor for Clathrin-mediated Endocytosis of Arginine-rich Cell-penetrating Peptides

Authors: Yoshimasa Kawaguchi,† Toshihide Takeuchi,† Keiko Kuwata,‡ Junya Chiba,§ Yasumaru Hatanaka,¶ Ikuhiko Nakase,# and Shiroh Futaki†,*

Author affiliations: †

Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan

‡

Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Furo-cho, Chikusa-ku,

Nagoya 464-8602, Japan §

Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani, Toyama

930-0194, Japan ¶

University Office, University of Toyama, Gofuku 3190, Toyama 930-8555, Japan

#

Nanoscience and Nanotechnology Research Center, Research Organization for the 21st Century,

Osaka Prefecture University, Naka-ku, Sakai, Osaka 599-8570, Japan

Corresponding author: Shiroh Futaki Institute for Chemical Research, Kyoto University Gokasho, Uji, Kyoto 611-0011, Japan Tel: +81-774-38-3210 Fax: +81-774-32-3038 E-mail: [email protected]

1 ACS Paragon Plus Environment

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Abstract Arginine-rich cell-penetrating peptides (CPPs) such as Tat and oligoarginine peptides have been widely used as carriers for intracellular delivery of bioactive molecules. Despite accumulating evidence for involvement of endocytosis in the cellular uptake of arginine-rich CPPs, the primary cell-surface receptors for these peptide carriers that would initiate endocytic processes leading to intracellular delivery of bioactive cargoes have remained poorly understood. Our previous attempt to identify membrane receptors for octa-arginine (R8) peptide, one of the representative arginine-rich CPPs, using the photocrosslinking probe bearing a photoreactive diazirine was not successful due to considerable amounts of cellular proteins non-specifically bound to the affinity beads. To address this issue, here we developed a photocrosslinking probe in which a cleavable linker of a diazobenzene moiety was employed to allow selective elution of crosslinked proteins by reducing agent-mediated cleavage. We demonstrated that introduction of the diazobenzene moiety into the photoaffinity probe enables efficient purification of crosslinked proteins with significant reduction of non-specific binding proteins, leading to successful identification of 17 membrane-associated proteins that would interact with R8 peptide. RNAi-mediated knockdown experiments in combination with the pharmacological inhibitors revealed that, among the proteins identified, syndecan-4, one of the heparan sulfate proteoglycans, is an endogenous membrane-associated receptor for the cellular uptake of R8 peptide via clathrin-mediated endocytosis. This syndecan-4-dependent pathway was also involved in the intracellular delivery of bioactive proteins mediated by R8 peptide. These results reveal that syndecan-4 is a primary cell-surface target for R8 peptide that allows intracellular delivery of bioactive cargo molecules via clathrin-mediated endocytosis.



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INTRODUCTION Cell-penetrating peptides (CPPs, or protein transduction domains) are a group of short peptides that can be efficiently internalized into cells, and have been widely used as carriers for intracellular delivery of bioactive molecules.1,2 Among them, arginine-rich CPPs such as oligoarginine and HIV-1 Tat (48-60) peptides show high efficiency of internalization, facilitating intracellular delivery of a broad range of bioactive cargoes including proteins, nucleic acids and hydrophilic therapeutic compounds, all of which would be otherwise difficult to enter cells.3,4 Thus, arginine-rich CPPs have attracted much attention as one of the promising delivery carriers for in vitro/in vivo applications including tumor targeting and diagnosis.5-7 Despite their applicability and effectiveness as delivery carriers, the molecular mechanisms by which arginine-rich CPPs enter cells have remained elucidated. It has been suggested that endocytosis has a major role in the cellular uptake of these peptides. We and others have reported that octa-arginine (R8) and Tat peptides, as well as their fusion proteins, are internalized into cells via macropinocytosis, one class of the endocytosis pathways,8-10 while another classes of endocytosis pathways such as clathrin-mediated endocytosis11-14 and caveolae-mediated endocytosis15,16 are also suggested to be involved. In addition, direct penetration of these peptides through plasma membranes has been suggested, as it observed dependently on peptide concentrations and incubation conditions.17-19 Proteoglycans, membrane-associated proteins with glycosaminoglycans such as heparan sulfates and chondroitin sulfates, are one of the key molecules responsible for the internalization processes of these peptides.12,18,20-22 Although there is accumulating evidence for involvement of endocytosis pathways in the cellular uptake of arginine-rich CPPs, the primary cell-surface targets for these peptide carriers that would initiate endocytic processes leading to intracellular delivery of bioactive cargoes have remained poorly understood. Crosslinking reaction using a photoreactive probe has been widely used for identification of proteins and ligands that would interact with proteins of interest.23-25 Various photoreactive groups, such as diazirines, benzophenones and aryl azides, have been utilized for this reaction. Among them,


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diazirines show superior photoreactivity as they generate carbene upon UV irradiation, a highly reactive intermediate species that allows rapid formation of covalent crosslinking to nearby molecules.23,26 Diazirines are, however, less toxic to cells because carbene is produced by relatively short exposure of UV light and is readily quenched by reaction with surrounding H2O unless they react with other adjacent molecules,27-29 suggesting that diazirines are one of the ideal photoreactive groups with both high reactivity and low toxicity. We previously performed a crosslinking experiment using a photoaffinity probe of dodeca-arginine (R12) peptide, one of the arginine-rich CPPs, bearing trifluoromethyl diazirine as a photoreactive group and a biotin as a tag for affinity purification, and successfully identified a membrane-associated chemokine receptor CXCR4 as a cell-surface receptor responsible for the cellular uptake of R12 peptide via macropinocytosis.30 Similarly, we also identified LanCL1, a cytosolic protein that would be involved in the cellular uptake of R8 peptide: however, no membrane-associated proteins have been obtained because considerable amounts of cellular proteins non-specifically bound to affinity beads hampered to isolate a limited amount of cell-surface proteins that were crosslinked with the photoaffinity probe.31 These results have led us to consider that, although diazirine functions as an excellent photocrosslinker, technical improvement in separation of crosslinked proteins from the affinity beads is required for identification of membrane-associated receptors responsible for the cellular uptake of R8 peptide and other arginine-rich CPPs. To address this issue, in this study, we synthesized a newly-designed photoaffinity probe of R8 peptide bearing a diazobenzene moiety,32,33 functioning as a chemo-selective cleavable linker that would facilitate separation of the crosslinked proteins from the affinity beads and prevent elution of non-specific binding proteins. Using this cleavable probe, we identified syndecan-4, one of the heparan sulfate proteoglycans, as an endogenous membrane-associated receptor for the cellular uptake of

R8

peptide

via

clathrin-mediated

endocytosis.

We

also

demonstrated

that

this

syndecan-4-dependent pathway is involved in the intracellular delivery of bioactive proteins mediated by R8 peptide. Our study reveals that syndecan-4 is a primary cell-surface target for R8 peptide that allows intracellular delivery of bioactive cargo molecules via clathrin-mediated endocytosis.



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RESULTS Design, Synthesis and Characterization of PhotoR8CL, a Photoreactive R8 Probe Bearing a Chemo-selective Cleavable Linker. To identify membrane proteins that are involved in the cellular uptake of R8 peptide, we designed PhotoR8CL, a photoaffinity probe that consists of four functional moieties, including trifluoromethyl benzoyl diazirine, R8, diazobenzene, and biotin (Figure 1A). Trifluoromethyl benzoyl diazirine is one of the diazirine derivatives, which allow an irreversible covalent linkage with nearby proteins upon UV irradiation.34-36 Diazobenzene is a building block that can be cleaved by treatment of reducing agents such as Na2S2O4 (Figure 1B), and can be readily incorporated into peptide chains using the Fmoc-protected diazobenzene derivative.32 The peptide chain of PhotoR8CL, bearing the trifluoromethyl benzoyl diazirine at the N-terminus, was constructed by the Fmoc solid-phase peptide synthesis (See Supporting Information Figure S1). The biotin moiety, which functions as a tag for affinity purification, was introduced into the ε-amino group of the C-terminal Lys, after removal of its protecting group, using biotinamidohexanoic acid N-hydroxysuccinimide ester. PhotoR8CL was thus synthesized without difficulty and purified by HPLC. The structure of the peptide was then confirmed by MALDI-TOFMS. The photoreactivity and cleavage activity of PhotoR8CL were confirmed using HPLC and MALDI-TOFMS. It is reported that diazirine activated upon UV irradiation reacts not only with nearby molecules such as proteins, but also with H2O,25 and that the water-adducted product can be easily detected by mass spectrometry. Irradiation of PhotoR8CL at 365 nm for 5 min yielded a product with [M+H]+ 2203.01 Da (SI Figure S2A), which is corresponding to that of PhotoR8CL reacted with H2O. This result indicates that PhotoR8CL is photoreactive upon UV irradiation. The cleavability of diazobenzene linker of PhotoR8CL was next examined. Incubation of water-adducted PhotoR8CL with Na2S2O4 gave two products with [M+H]+ 604.14 and 1602.25, which are corresponding to those of two fragments derived from PhotoR8CL that would be yielded by cleavage at diazobenzene linker by Na2S2O4 (SI Figure S2B). These results indicate that, as expected, PhotoR8CL is photoreactive


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upon UV irradiation and can be cleaved by Na2S2O4, and that UV irradiation has no influence on Na2S2O4-mediated cleavage of the diazobenzene linker.

(A) O N

N

F3C

N

O

H N

N H 8

O

O

NH2

O

N H

N H

NH O

N

H

S

H N

H HN

O

OH

NH H2N

NH

photoreactive R8 moiety (Tmd)

diazobenzene linker

(B) Tmd

spacer

diazobenzene linker

PhotoR8CL R8

biotin

Endogeneuos biotinylated protein

biotin

crosslinking (i) UV irradiation

(ii) extraction

Membrane protein (iii) isolation

(v) SDS-PAGE

(iv) cleavage and elution

(vi) in-gel digestion Streptavidin bead

LC-MS/MS analysis

Figure 1. Strategy for identification of cell-surface receptors bound to R8 using photocrosslinking reaction. (A) The chemical structure of PhotoR8CL, a photoaffinity probe of R8 equipped with a diazirine-based photoreactive moiety (Tmd) and a cleavable linker (diazobenzene). A biotin is attached for affinity purification using streptavidin beads. (B) Experimental scheme of protein identification. (i) Cell-surface proteins that are potentially bound to R8 were crosslinked with PhotoR8CL upon UV irradiation. Membrane proteins were (ii) extracted, (iii) isolated using streptavidin beads, and then (iv) selectively eluted by chemo-selective linker-cleavage using Na2S2O4. The eluted proteins were (v) analyzed with SDS-PAGE and (vi) subjected to LC-MS/MS analysis for protein identification after in-gel digestion.



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Identification of Membrane Proteins That Interact with R8 Using PhotoR8CL. There are accumulating evidence that endocytosis pathways are involved in the cellular uptake of arginine-rich CPPs including oligoarginine and Tat peptides. However, direct penetration of these peptides has also been suggested as other forms of internalization, in which they directly enter the cells through the plasma membranes and readily make a diffuse distribution throughout the cells.2 We and others have reported that this form of internalization can be observed prominently when HeLa cells are incubated with oligoarginines at high administration concentrations, typically at 10 µM and the more,17,18,37 although this would be influenced by variable factors in each experiment such as cells, temperatures, and the characteristic of CPPs.2,19,38 These observations led us to hypothesize that the methods of internalization of arginine-rich CPPs are highly dependent on their administration concentrations and that endocytosis pathways would have major contributions to the cellular uptake of these peptides at lower concentrations. Considering this hypothesis, in this study we performed crosslinking experiments using a relatively low concentration of PhotoR8CL to find membrane-associated proteins that would interact with R8 peptide in the early stage of its endocytic uptake. HeLa cells were treated with 1 µM of PhotoR8CL and irradiated at 365 nm for crosslinking reaction. To avoid contamination of cytosolic and nuclear proteins, membrane proteins were isolated from the cell lysates by ultracentrifugation. Proteins that were crosslinked with PhotoR8CL were then collected with streptavidin beads from the membrane fractions of cell lysates, and were eluted by cleavage of the diazobenzene linker upon Na2S2O4 treatment32 (Figure 1B). Analysis of the eluted proteins by SDS-PAGE showed that 7 major, different bands were observed in the cells treated with PhotoR8CL compared with non-treated control cells (Figure 2A). In contrast, boiling of the affinity beads, after elution by Na2S2O4 treatment, in the SDS-PAGE sample buffer resulted in elution of large amounts of non-specific binding proteins left on the beads, which were similarly observed between PhotoR8CL-treated cells and non-treated cell (Figure 2B). These results suggest that not only the fractionation of the membrane proteins but also the Na2S2O4-mediated cleavage of the diazobenzene linker leads to efficient purification of photocrosslinked proteins with significant reduction of


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non-specific binding proteins. By LC-MS/MS analysis of the above protein bands, we identified 17 membrane proteins that would interact with R8 peptide, including proteoglycans, transporters, membrane traffic-related proteins, and others (Table 1).

(A)

(B)

a2S2O4

hotoR8CL –

250

+

PhotoR8CL –

1 2

150 100 75

SDS boil after Na2S2O4

250 150

3 4 5 6

100 75

50

50

37

37

25 20

25 20

15

+

7

15

Figure 2. Photocrosslinking using PhotoR8CL and selective elution by reductive cleavage of its diazobenzene linker enable to separate membrane proteins that would interact with R8. The proteins crosslinked with PhotoR8CL were eluted by Na2S2O4-mediated cleavage from streptavidin beads, and analyzed with SDS-PAGE and visualized by silver staining (A). The streptavidin beads after elution using Na2S2O4 were boiled in a SDS-PAGE sample buffer, and analyzed as above (B). Seven different bands were detected in (A), whereas large amounts of non-specific binding proteins left on the beads were detected in (B), suggesting that the Na2S2O4-mediated cleavage of the diazobenzene linker leads to selective release of crosslinked proteins from affinity beads and prevents elution of non-specific binding proteins.



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Table 1. Membrane proteins that were crosslinked with PhotoR8CL, identified by LC-MS/MS analysis. Band No.

Protein Names

Scores

Unique peptides

Description

1

Glypican-1 (GPC1) Syndecan-2 (SDC2) Syndecan-4 (SDC4) Chondriotin sulfate proteoglycan 4 (CSPG4) Transforming growth factor beta receptor type 3 (TGFR3) Collagen alpha-1(V) chain (COL5A1) Glypican-5 (GPC5) Myoferlin (MYOF) Laminin subunit gamma-1 (LAMC1) Zinc transporter ZIP10 (ZIP10) Transferrin receptor protein 1 (CD71) Semaphorin-3C (SEMA3C) 4F2 cell-surface antigen heavy chain (CD98) Choline transporter-like protein 2 (CTL2) Interferon-induced transmembrane protein 1 (IFITM1) Interferon-induced transmembrane protein 3 (IFITM3) Vesicle-associated membrane protein 1 (VAMP1)

882 120 216 546 56 96 20 27 22 100 27 115 104 35 793 497 28

25 4 5 22 2 6 1 1 1 6 1 5 5 1 15 9 1

Proteoglycan Proteoglycan Proteoglycan Proteoglycan Proteoglycan Collagen Proteoglycan Ferlin Laminin Transporter Receptor Semaphorin Solute carrier Transporter Antiviral protein Antiviral protein v-SNARE

2

3 4 5 6 7


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Syndecan-4 Is Involved in R8 Internalization. To examine whether the membrane proteins that were identified above are involved in the cellular processes of R8 internalization, we reduced the level of each protein by RNAi-mediated knockdown (SI Figure S3) and examined its effect on the cellular uptake of R8 peptide. Quantitative analysis of the cellular uptake of R8 peptide by flow cytometry revealed that knockdown of syndecan-4, one of the identified proteins that would interact with PhotoR8CL, resulted in a 28% decrease in the amounts of the internalized R8 peptide, compared with those in the cells transfected with the control siRNA (Figure 3A). Consistently, knockdown of syndecan-4 using another siRNA also reduced the cellular uptake of R8 peptide (SI Figure S4A). We confirmed that the protein expression levels on the cell surface, as well as the mRNA levels, of syndecan-4 were significantly reduced by RNAi-mediated knockdown using these siRNAs (SI Figures S3 and S4B). These results indicate that, among the proteins identified using PhotoR8CL, syndecan-4 is a key membrane protein for the cellular uptake of R8 peptide. In support of this finding, confocal microscopic analysis showed that syndecan-4 knockdown resulted in a significant decrease in punctate fluorescent signals of the internalized R8 peptide, which are suggested to be the endocytosis-mediated internalization of R8 peptide (Figure 3B). In addition, overexpression of syndecan-4 resulted in 29% increase in the cellular uptake of R8 peptide (Figure 3C). Furthermore, antibody-feeding assay using an anti-syndecan-4 antibody revealed that the intracellular localization of syndecan-4 that was internalized from cell surface was partially overlapped with that of R8 peptide (Figure 3D), suggesting that syndecan-4 is internalized into cells with R8 peptide. Thus, these results collectively indicate that syndecan-4 is involved in the cellular processes of R8 internalization. It is noted that contribution of syndecan-4 in the cellular uptake of R8 peptide is likely dependent on its extracellular concentration: we observed no significant knockdown effects of syndecan-4 on the cellular uptake of R8 peptide when HeLa cells were treated with 10 µM of R8 peptide (SI Figure S5). This result suggests that the cellular processes of R8 internalization are strongly affected by extracellular concentration of R8 peptide as previously suggested,17-19,37 and that syndecan-4 plays a major role in R8 internalization when concentration of R8 peptide is relatively low.



(A)

(B) 140

*

100

**

80

Alexa488

siNTC

120

DIC

*

*

60 40

siSDC4

Relative cellular uptake of R8-Alexa488 (%)

20 0

siRNA

(C)

(D)

**

140 120

SDC4 R8


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100 80 60 40 20

DIC

Merge

SDC4

R8-TAMRA

0 Mock

SDC4-HA

Figure 3. Syndecan-4 is involved in internalization of R8. (A) Quantitative analysis of R8 internalization into cells in which the expression level of the identified proteins was reduced by RNAi-mediated knockdown. HeLa cells were transfected with siRNAs targeting for the membrane proteins identified above, and treated with 1 µM of R8-Alexa488 at 37°C for 30 min, and subjected to flow cytometry analysis. Among the identified proteins, knockdown of syndecan-4 resulted in a decrease in R8 internalization. (B) Confocal microscopic images of the siNTC/siSDC4-transfected cells that were treated with 1 µM of R8-Alexa488 at 37°C for 30 min. Punctate signals, suggested to be endocytosis-mediated internalization of R8, were significantly reduced by syndecan-4 knockdown. (C)

Bar

graph

showing

R8

internalization

levels

in

the

mock/HA-tagged

syndecan-4

(SDC4-HA)-transfected cells that were treated with 1 µM of R8-Alexa488 at 37°C for 30 min. (D) Confocal microscopic images of the syndecan-4-labeled cells that were treated with 1 µM of Figure 3. R8-TAMRA

at 37°C for 30 min. Cell-surface syndecan-4 was labeled with anti-syndecan-4 antibody.

Punctate signals of R8 were colocalized with those of syndecan-4. siNTC, non-targeting control


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siRNA. siSDC4, siRNA targeting for syndecan-4. Data are represented as the mean ± SD of three independent experiments (*P < 0.05, **P < 0.01; Student’s t test). Scale bars: B and D, 20 µm.



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Syndecan-4 Is Involved in R8 Internalization via Clathrin-mediated Endocytosis. It is reported

that

endocytosis

pathways,

including

clathrin/caveolae-mediated

endocytosis

and

macropinocytosis, have major contributions to the cellular uptake of arginine-rich CPPs under physiological conditions.2 Indeed, when 1 µM of R8 peptide was added to HeLa cells in the presence of pharmacological inhibitors at the concentrations where these endocytic pathways were effectively suppressed (SI Figure S6), significant decreases in the amounts of the internalized R8 peptide were observed, except for the inhibitors for caveolae-mediated endocytosis such as filipin39 and nystatin40 (Figure 4A). This result indicates that clathrin-mediated endocytosis and macropinocytosis, but not caveolae-mediated endocytosis, make major contribution to the cellular uptake of R8 peptide in HeLa cells when its extracellular concentration is 1 µM. To investigate whether syndecan-4 is involved in R8 internalization via clathrin-mediated endocytosis or macropinocytosis, we performed knockdown of syndecan-4 by RNAi in the presence of inhibitors for these pathways and examined its effect on the cellular uptake of R8 peptide. Quantitative analysis of the cellular uptake of R8 peptide by flow cytometry revealed that syndecan-4 knockdown had no effects on the cellular uptake of R8 peptide when HeLa cells were treated with sucrose,41 an inhibitor for clathrin-mediated endocytosis (Figure 4B), whereas the cellular uptake of R8 peptide was significantly suppressed in syndecan-4-knockdown cells without treatment of inhibitors (Figures 3A and 3B, and SI Figure S4A). Similarly, no significant knockdown effects of syndecan-4 on R8 internalization were observed in the presence of monodansylcadaverine (MDC)42 and pitstop 2,43 another inhibitors for this pathway (Figures 4C and 4D). In contrast, knockdown of syndecan-4 in the presence of inhibitors for macropinocytosis including 5-(N-ethyl-N-isopropyl) amiloride (EIPA),44 2,2’-dihydroxy-1,1’-dinaphthyldisulfide (IPA-3),45 wortmannin46 and Gö6983,47 resulted in additional suppression of the cellular uptake of R8 peptide (Figures 4E-H). These results suggest that syndecan-4 is involved in the cellular processes of R8 internalization that are suppressed by inhibitors for clathrin-mediated endocytosis, but not for macropinocytosis. In agreement with this finding, knockdown of syndecan-4 had no effects on R8 internalization in the cells that were


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transfected with siRNA for clathrin heavy chain (CHC), which is responsible for clathrin-mediated endocytosis (Figure 4I and SI figure S7).48,49 Additionally, image correlation analysis of the overlapping fluorescent signals of the internalized R8 and syndecan-4 using Pearson’s correlation coefficient50 revealed that colocalization ratios between R8 peptide and syndecan-4 were significantly reduced by MDC treatment (Figures 4J and 4K), indicating that clathrin-mediated endocytosis has an important role in the cellular processes by which R8 peptide is internalized together with syndecan-4. Furthermore, live-cell imaging demonstrated that syndecan-4 signals that were colocalized with R8 peptide, trafficking from the cell surface to the interior, were overlapped with the signals of transferrin, a marker for clathrin-mediated endocytosis (Figure 4L). These results suggest that syndecan-4 is involved in R8 internalization via clathrin-mediated endocytosis.



100 80 60

**

40 20

0 siNTC 1 + siSDC4 – EIPA –

+ 2

– 3

+

+

–

n.s.

40 20

80 60 40 20

120

***

100 80 60

*

40 20 + 2 –

3–

+

+

+

(L)

+

+

+

(H) **

120 100 80

**

60 40 20

0 siNTC + 1 siSDC4 – wortmannin –

+ 2 –

+

3–

+ +

(I) **

120 100

***

80 60 40 20

0 siNTC + 1 siSDC4 – Gö6983 –

+ 2

3–

+

+

–

MDC

DIC 11 min

Merge 12 min

13 min

14 min

120 100

n.s.

80 60 40 20

0 siNTC + 1 siSDC4 – siCHC –

+

***

+ 2 – +

3– + +

***

0.6 0.5 0.4 0.3 0.2 0.1 0 DMSO

15 min

MDC

16 min

17 min

Tf-Alexa488

SDC4

R8-TAMRA

10 min

n.s.

20

pitstop 2 –

+

Pearson’s correlation coefficient

DMSO

Merge

40

+

MDC –

(K)

DIC

60

3–

+

+

80

2 + –

+

sucrose –

100

+

– 3

3–

***

120

0 1 siNTC + siSDC4 –

+ 2 –

+ 2 –

(J)

SDC4 R8

n.s.

100

0 siNTC + 1 siSDC4 –

0 siNTC + 1 siSDC4 –


60



80

(G)

0 siNTC 1 + siSDC4 – IPA-3 –

+

100

*

120



120

***

120

(D)

macropinocytosis

(F) **

Gö6983

nystatin

filipin

*** ***


(E)

caveolaemediated endocytosis

***

wortmannin

***


clathrinmediated endocytosis

(C)

**

***

pitstop 2

***

(B)

with inhibitor

IPA-3

n.s.

***

MDC

160 140 120 100 80 60 40 20 0

sucrose


no treatment

EIPA

( )


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Page 16 of 37

Figure 4. Syndecan-4 is involved in internalization of R8 via clathrin-mediated endocytosis. (A) Quantitative flow cytometry analysis of R8 internalization into cells that were treated with 1 µM of R8-Alexa488 Figure 4.

at 37°C for 30 min in the presence of pharmacological inhibitors targeting for

clathrin-mediated endocytosis (0.45 M sucrose, 50 µM monodansylcadaverine (MDC), and 30 µM


Page 17 of 37

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pitstop 2), for caveolae-mediated endocytosis (1 µg/mL filipin and 50 µM nystatin) and for macropinocytosis (100 µM EIPA, 20 µM IPA-3, 500 nM wortmannin, and 2 µM Gö6983). Internalization of R8 was significantly reduced by treatment of inhibitors for clathrin-mediated endocytosis and macropinocytosis, but not for caveolae-mediated endocytosis. (B-H) Bar graphs showing levels of R8 internalization in the siNTC/siSDC4-transfected cells that were treated with 1 µM of R8-Alexa488 at 37°C for 30 min in the presence of inhibitors including sucrose (B), MDC (C), pitstop 2 (D), EIPA (E), IPA-3 (F), wortmannin (G) and Gö6983 (H). R8 internalization was significantly affected by knockdown of syndecan-4 in the presence of EIPA, IPA-3, wortmannin and Gö6983, but not of sucrose, MDC and pitstop 2, suggesting that syndecan-4 is involved in the cellular pathways of R8 internalization that are blocked by inhibitors for clathrin-mediated endocytosis. (I) Bar graph showing R8 internalization levels in the double-knockdown cells of syndecan-4 and CHC that were treated with 1 µM of R8-Alexa488 at 37°C for 10 min. (J and K) Confocal microscopic images of the syndecan-4-labeled cells that were treated with 1 µM of R8-TAMRA at 37°C for 30 min in the presence of MDC (J) and bar graph showing Pearson’s coefficient values of colocalization between R8-TAMRA and syndecan-4 signals (K). (L) Time-lapse images of the syndecan-4-labeled cells that were treated with R8-TAMRA (1 µM) and transferrin-Alexa488 (Tf-Alexa488, 25 µg/mL). Fluorescent images were taken by focusing in the rectangle area of the left DIC image. The punctate fluorescent signals of R8-TAMRA and antibody-labeled syndecan-4 were colocalized and trafficking with the Tf-Alexa488 signals. Data are represented as the mean ± SD of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001; n.s., not significant; Student’s t test). Scale bars: J, 20 µm; L, 5 µm.



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Syndecan-4 Is Involved in Internalization of Cre Recombinase Fused with R8. We next explored the possibility that syndecan-4 would also be involved in internalization of proteins that are conjugated with R8. To test this possibility, we performed a Cre-loxP recombination assay using a recombinant protein of Cre recombinase that is fused with R8 (CreR8), and examined the effects of syndecan-4 knockdown on recombination efficiency mediated by Cre. We have established Cre reporter cells from a 293 cell line to have a gene encoding a loxP-DsRed-loxP-EGFP sequence. The reporter cells constitutively express red fluorescent protein (DsRed), but they switch to produce green fluorescent protein (EGFP) once Cre undergoes recombination reaction between the loxP sequences – this allows monitoring of intracellular activity of Cre recombinase by analyzing its fluorescent color change from red to green (Figure 5A). When we treated the reporter cells with 5 µM of CreR8, we observed a significant increase in the number of cells expressing EGFP, whereas Cre alone had no such effect (Figure 5B), demonstrating that conjugation of R8 is an effective way to enable the intracellular delivery of proteins with cellular functions. Quantitative analysis of EGFP-positive cells using flow cytometry revealed that knockdown of syndecan-4 resulted in a 35% decrease in the number of reporter cells expressing EGFP, compared with that of the control siRNA-transfected cells (Figure 5C). This result indicates that syndecan-4 is involved not only in the cellular uptake of R8 peptide, but also in that of Cre recombinase fused with R8.


Page 19 of 37

(A)

(B)

(C) –

STOP

loxP

CreR8

Relative EGFP-positive cells (%)

DsRed

Cre

EGFP loxP

DIC

Promoter

Merge

+ Cre recombinase

EGFP

Promoter

** 120 100 80 60

Cre

40

CreR8

20 0 siNTC siSDC4

loxP

***

100 80 60 40 20

0 sucrose

control sucrose – +

120

***


120

(F) 100 80 60 40 20

0 EIPA control –

(G) 100 80

n.s.

60 40 20

0 siNTC siSDC4 sucrose

EIPA +

**

120

+ 1 – –

+ 2 – +

– 3 + +


(E) Relative EGFP-positive cells (%)

(D) Relative EGFP-positive cells (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


***

120 100

**

80 60 40 20

0 siNTC siSDC4 EIPA

+ 1 – –

+ 2 – +

– 3 + +

Figure 5. Syndecan-4 is involved in R8-mediated intracellular delivery of Cre recombinase via clathrin-mediated endocytosis. (A) Schematic representation of Cre-loxP recombination assay. The Cre reporter cell line, expressing stably DsRed, starts to express EGFP once Cre recombinase catalyzes recombination reaction in the loxP sites around the DsRed gene. Recombination efficiency, reported as EGFP expression, is correlated with efficiency of intracellular delivery of Cre recombinase. (B) Confocal microscopic images of the reporter cells that were treated with Cre or CreR8 (5 µM). Remarkable EGFP fluorescent signals were observed in the CreR8-treated cells, suggesting that conjugation with R8 leads to efficient delivery of Cre recombinase into cells. (C) Bar graph showing ratios of the EGFP-positive cells in the siNTC/siSDC4-transfected cells that were treated with Cre or CreR8. Knockdown of syndecan-4 reduced recombination efficiency, indicating that syndecan-4 is involved in R8-mediated delivery of Cre recombinase. (D and E) Quantitative analysis of the EGFP-positive cells that were treated with CreR8 in the presence of sucrose (D) and EIPA (E). Both inhibitors suppressed recombination efficiency, indicating that clathrin-mediated endocytosis and macropinocytosis are involved in R8-mediated delivery of Cre recombinase. (F and G) Bar graphs showing ratios of the EGFP-positive cells in the siNTC/siSDC4-transfected cells that were treated



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Page 20 of 37

with CreR8 in the presence of sucrose (F) and EIPA (G). Recombination efficiency was affected by knockdown of syndecan-4 in the presence of EIPA, but not of sucrose, indicating that syndecan-4 is involved in R8-mediated delivery of Cre recombinase via clathrin-mediated endocytosis. Data are represented as the mean ± SD of three independent experiments (**P < 0.01, ***P < 0.001; n.s., not significant; Student’s t test).


Page 21 of 37

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Syndecan-4

Is

Involved

in

Internalization

of

R8-fused

Cre

Recombinase

via

Clathrin-mediated Endocytosis. To test whether syndecan-4 is involved in internalization of CreR8 via clathrin-mediated endocytosis, like as R8 peptide, we carried out the Cre-loxP recombination assay in the presence of endocytosis inhibitors, and examined the effects of syndecan-4 knockdown on Cre-mediated recombination. Quantitative analysis of EGFP-positive cells using flow cytometry revealed that the number of cells expressing EGFP was significantly reduced when the Cre reporter cells were treated with 5 µM of CreR8 in the presence of sucrose (Figure 5D) or EIPA (Figure 5E), suggesting that both clathrin-mediated endocytosis and macropinocytosis are involved in the CreR8-mediated recombination. Knockdown of syndecan-4 in the Cre reporter cells, however, resulted in no significant decrease in the number of EGFP-positive cells, compared with that of the control siRNA-transfected cells, when cells were treated with sucrose (Figure 5F). In contrast, syndecan-4 knockdown in the presence of EIPA resulted in additional reduction of the number of cells expressing EGFP (Figure 5G). These results strongly suggest that syndecan-4 is involved not only in the cellular uptake of R8 peptide, but also in R8-mediated intracellular delivery of Cre recombinase, via clathrin-mediated endocytosis.



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DISCUSSION In this study, we demonstrated that syndecan-4, one of the membrane-associated proteins identified by the cleavable photoaffinity probe for R8 (PhotoR8CL), is a primary cell-surface target for R8 peptide that is internalized via clathrin-mediated endocytosis. We further provided evidence that this endocytosis pathway initiated from syndecan-4 is utilized for the cellular uptake of R8-fused proteins to exert their biological activity in cells. Although the previous reports have suggested that the interaction with the membrane-associated proteoglycans is important for the endocytic uptake of arginine-rich CPPs, it has not been fully understood which proteoglycans would function as a receptor for the CPP-mediated delivery. Thus, the present study is the first report, to our knowledge, to identify the cell-surface receptor responsible for R8-mediated intracellular delivery via clathrin-mediated endocytosis. The photocrosslinking approach for identification of proteins that interact with molecules of interest in situ normally consists of three steps, including covalent capture of target proteins by photocrosslinking reaction, purification with affinity beads and identification by LC-MS/MS analysis. Although extensive studies on development of photocrosslinkers with excellent properties, e.g., high reactivity and short half life, to enable efficient covalent bond formation with nearby target molecules have been reported so far,23,25,26 the basic principle on the subsequent steps such as purification and identification has remained almost unchanged. Our previous study using the trifluoromethyl diazirine derivative revealed that, despite the excellent photoreactivity of the photocrosslinker, it is quite difficult to identify target proteins in the presence of large amounts of unrelated cellular proteins non-specifically bound to the affinity beads,31 demonstrating the definite need for technical improvement in the purification step to facilitate efficient identification of crosslinked proteins. Here we conducted the photocrosslinking experiment using the photoaffinity probe equipped with a diazobenzene moiety (Figure 1), one of the cleavable linkers that have been utilized for affinity purification of proteins bound to small molecule ligands, but not yet been applied to photocrosslinking approach.32,33,51 We demonstrated that introduction of the diazobenzene moiety in the photoaffinity


Page 23 of 37

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probe results in significant improvement in the purification step, which enables separation of crosslinked proteins from the affinity beads and prevents elution of non-specific binding proteins by its chemo-selective cleavage, leading to successful identification of 17 crosslinked proteins without difficulty (Figure 2, Table 1). Our study clearly shows that not only development of excellent photocrosslinkers but also use of cleavable linkers provides key improvements in protein identification using the photocrosslinking approach. Although potential involvement of a few membrane proteins (e.g., tumor necrosis factor (TNF) receptor14 and scavenger receptor52) have been implicated in the internalization of arginine-rich CPPs, there are still limited knowledge on specific proteins acting as a receptor responsible for their cellular uptake: the only exception is our previous work, in which a chemokine receptor CXCR4 was identified as a macropinocytic receptor of R12 peptide.30 The membrane proteins including vascular endothelial growth factor receptor 2 (VEGFR2),53 low-density lipoprotein receptor-related protein (LRP)54 and CXCR455 have been suggested to act as a receptor for the cellular uptake of full-length Tat protein, but in all cases the basic domain corresponding to the CPP sequence (position 48-60) does not act as a high-affinity ligand for their proteins. We previously demonstrated in HeLa cells that CXCR4 acts as a receptor to allow the cellular uptake of R12 peptide, one of the oligoarginine CPPs, via macropinocytosis.30 Interestingly, the CXCR4-mediated endocytosis pathway was not activated by treatment of other arginine-rich CPPs such as R8 and Tat peptides, indicating that these peptides would have different but distinct receptors, which may vary dependent on cell lines, for their internalization despite the similarity of their peptide sequences. Involvement of membrane-associated proteoglycans bearing glycosaminoglycan chains such as heparan sulfates and chondroitin sulfates have been implicated in the cellular uptake of arginine-rich CPPs such as oligoarginine and Tat peptides, as these peptides strongly interact with glycosaminoglycans,21,56,57 and deficiency for proteoglycans largely suppresses their cellular uptake.12,20,21 We also pointed out that interaction of oligoarginine and Tat peptides with proteoglycans leads to induction of actin re-organization and for their macropinocytic uptake.8,22 Furthermore, it has been reported that overexpression of syndecans



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Page 24 of 37

such as syndecan-2 and syndecan-4, which are one group of proteoglycan family proteins, leads to an increase in the cellular uptake of R8 and Tat peptides.22,58,59 However, it still remained unclear which molecular species of the proteoglycan families would be responsible receptors not only for the cellular uptake of arginine-rich CPPs, but also for the CPP-mediated intracellular delivery. Using the cleavable photocrosslinking probe for R8 peptide and subsequent mechanistic studies with RNAi-mediated knockdown and chemical inhibitors, here we demonstrated that syndecan-4 is a primary cell-surface receptor that initiates cellular processes responsible for the cellular uptake of R8 peptide via clathrin-mediated endocytosis (Figures 3 and 4). This finding is important not only because of the first report that identified a specific protein among the proteoglycan families as a receptor for the cellular uptake of arginine-rich CPPs, but also because of the first mechanistic elucidation of the internalization route through which proteins conjugated with CPPs exert their bioactivity in cells. Syndecan-4 is one of the four subtypes of the syndecan family that control adhesion, cell proliferation, differentiation and migration.60 Syndecans are known as multifunctional proteins, functioning as co-receptors for various growth factors, transducing signals via their intracellular domain by interacting with various effector molecules, and forming clusters in response to ligand binding to be internalized into cells via endocytosis.61 Recent study has suggested that syndecan-4 activates non-canonical Wnt/PCP signaling via clathrin-mediated endocytosis in response to the binding of roof plate-specific spondin 3 (R-spondin 3), a secreted protein of Wnt modulator.62 Considering that R-spondin 3, which has a basic domain at the C-terminus,63,64 binds syndecan-4 with high affinity, it would be quite interesting that R8 peptide shares similar internalization pathway as R-spondins through non-canonical Wnt signaling via syndecan-4, although this speculation needs to be examined. Our findings are also important for better understanding of the internalization mechanisms of arginine-rich CPPs, as it raises possibility that syndecan-4 would be utilized as a marker for clathrin-mediated endocytosis, providing information on intracellular trafficking and fate of these peptides as to how they are transported from cell surface to the interior, accumulated and degraded. Furthermore, detailed analysis on internalization of both arginine-rich CPPs and syndecan-4


Page 25 of 37

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would also lead to basic understanding of the molecular mechanisms as to when and where the internalized arginine-rich CPPs would get out of the endosomes before lysosomal degradation, one of the most important but challenging questions for intracellular delivery mediated by these peptides. Development of methods that enable efficient intracellular delivery of various cargo molecules in a cell/tissue-specific manner is one of the major goals in our laboratory and others. Here we demonstrated using the Cre/loxP recombination assay that the clathrin-mediated pathway initiated by syndecan-4 is involved in the intracellular delivery mediated by R8 peptide, through which R8-conjugated cargoes are internalized and exert their bioactivity in cells (Figure 5). Although the detailed cellular mechanisms as to how the internalized cargoes escape from endosomes and reach cytosol/nucleus remain to be elucidated at the present stage, our results suggest that the syndecan-4-mediated endocytosis pathway would be one of the key internalization routes for CPP-mediated intracellular delivery, raising the possibility that optimization of the molecular structures of arginine-rich CPPs into the ones that strongly activate this pathway would lead to development of the more sophisticated delivery carriers with high efficiency. On the other hand, syndecan-4 is known to be expressed ubiquitously, but detailed studies have pointed out the enhanced expression particularly in some cancer cells including breast and testicular germ cells65,66 and in the kidney cells of IgA nephropathy patients.67 The method for controlling CPP internalization using proteolytic cleavage mediated by extracellular proteases has been utilized for tissue-specific tumor imaging.5 Considering the importance of the syndecan-4-initiated pathway in the CPP-mediated delivery, efforts to develop peptide carriers that enable to control internalization by switching the binding affinity to syndecan-4 in a spatio-temporal manner, and thus enable cell/tissue-specific delivery of various cargo molecules, are now ongoing.



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Page 26 of 37

EXPERIMENTAL PROCEDURES Reagents. All reagents including salts, inhibitors and incubation media were obtained from Sigma-Aldrich and Wako unless otherwise specified. Anti-syndecan-4 antibody (5G9 mouse monoclonal) and siRNAs were obtained from Santa Cruz Biotechnology and Sigma-Aldrich. Fluorescently labeled secondary antibodies and transfection reagents were purchased from Thermo Fisher Scientific.

Synthesis of PhotoR8CL. Synthetic scheme of PhotoR8CL is shown in Supporting Information Figure

S1.

The

building

block

for

cleavable

linker,

a

diazobenzene

derivative

4-[5-(N-fluorenylmethyloxycarbonyl-2-aminoethyl)-2-hydroxyphenylazo]-benzoic

acid

(Fmoc-CL-OH; Fmoc, 9-fluorenylmethyloxycarbonyl; CL, cleavable linker), was synthesized as described

previously.32

The

peptide

chain

[Arg(Pbf)]8-CL-Lys(Mtt)-resin

(Pbf,

2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; Mtt, methyltrityl) was constructed on a TGS-RAM resin (Shimadzu) by Fmoc solid-phase peptide synthesis with the standard coupling system

using

N-hydroxybenzotriazole

(HOBt)/O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium (HBTU)/N,N-diisopropylethylamine

(DIEA).

A

hexafluorophosphate diazirine

derivative

4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzoic acid (TmdBz)34,36 was then incorporated at the N-terminus of the peptide chain using the HOBt/HBTU/DIEA coupling system to yield TmdBz-[Arg(Pbf)]8-CL-Lys(Mtt)-resin. For introduction of the biotin tag, the Mtt moiety of the peptide was removed by treatment with 1,1,1,3,3-hexafluoroisopropanol (HFIP)/dichloromethane (DCM) (1:4) at 25°C for 3 h, and reacted with biotinamidohexanoic acid N-hydroxysuccinimide ester in the presence of DIEA to yield TmdBz-[Arg(Pbf)]8-CL-Lys(biotinamidohexanoyl)-resin. The peptide was cleaved from the resin and deprotected by treatment with trifluoroacetic acid (TFA)/ethanedithiol (EDT) (95:5), and purified by reverse phase high-performance liquid chromatography (RP-HPLC). The product was confirmed by matrix-assisted laser desorption


Page 27 of 37

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ionization time-of-flight mass spectroscopy (MALDI-TOFMS). MALDI-TOFMS: 2214.6 [calculated for (M+H)+: 2214.6]. Retention time in HPLC: 27.5 min [column: Cosmosil 5C18-AR-II (4.6 × 150 mm) (Nacalai tesque); gradient: 5-85% B in A (A = H2O containing 0.1% TFA, B = CH3CN containing 0.1% TFA) over 80 min; flow: 1 mL/min; detection: 220 nm]. The photoreactivity and cleavage activity of PhotoR8CL were analyzed by HPLC [column: Cosmosil 5C18-AR-II (4.6 × 150 mm); gradient: 10-40% B in A (A = H2O containing 0.1% TFA, B = CH3CN containing 0.1% TFA) over 30 min; flow: 1 mL/min; detection: 220 nm] and MALDI-TOFMS (given in SI Figure S2).

Peptide Synthesis and Fluorescent Labeling. R8-Alexa488 and R8-TAMRA were chemically synthesized by Fmoc solid-phase peptide synthesis, and were fluorescently labeled by Alexa488 C5 maleimide sodium salt or tetramethylrhodamine-5-maleimide, as previously reported.22,57 Actual sequences

of

the

synthesized

peptides:

R8-Alexa488,

RRRRRRRRGC(Alexa488)-amide;

R8-TAMRA, RRRRRRRRGC(tetramethylrhodamine)-amide.

Cell Culture. HeLa cells were maintained in α-MEM supplemented with 10% heat-inactivated bovine serum. Cre reporter cells (see below) were cultured in DMEM supplemented with 10% heat-inactivated FBS and hygromycin B (100 µg/mL). All cells were maintained at 37°C in a humidified 5% CO2 atmosphere.

Photocrosslinking, Elution and Identification of Proteins. HeLa cells (1 x 106 cells/dish) were seeded on 100-mm dishes, which were cultured for 48 h (80-90% confluent). The cells (total 10 dishes) were treated with 1 µM of PhotoR8CL for 5 min at 4°C using the K+-rich buffer (20 mM HEPES, 43 mM KCl, 100 mM L-glutamic acid, 5 mM glucose, 1 mM MgCl2, 1 mM CaCl2, pH 7.4), and irradiated at 365 nm for 10 min at 4°C. After washing with PBS containing 0.5% (w/v) heparin, the cells were collected with a hypotonic buffer (10 mM Tris, 4 mM EDTA, pH7.4) and lysed by sonication. The cell lysates were then centrifuged sequentially at 1,000 × g for 10 min, at 17,000 × g



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Page 28 of 37

for 10 min, and at 100,000 × g for 1 h. The pellets were solubilized with a RIPA buffer [25 mM Tris, 150 mM NaCl, 0.1% (w/v) SDS, 1% (w/v) NP-40, 1% (w/v) sodium deoxycholate, 1 mM EDTA, pH 7.4], and centrifuged at 17,000 × g for 30 min to obtain the clear lysates of membrane fractions. The membrane lysates (450 µg) were added to Dynabeads MyOne Streptavidin T1 (Invitrogen) to collect the proteins that were crosslinked with PhotoR8CL, which were eluted with a phosphate buffer containing 25 mM Na2S2O4, pH 7.4. The eluted proteins were separated by SDS-PAGE, digested by trypsin, and subjected to LC-MS/MS analysis using TripleTOF 5600+ system (AB Sciex) equipped with ekspert nanoLC 400. For protein identification, Mascot analysis was performed using Mascot Server version 2.4.0.

Flow Cytometry. HeLa cells were cultured in 24-well microplates until 80-90% confluent. The cells were treated with 1 µM of R8-Alexa488 for 30 min at 37°C in the presence or absence of the pharmacological inhibitors. After washing with PBS containing 0.5% (w/v) heparin, the cells were incubated with 0.01% trypsin for 10 min at 37°C. The cells were collected and washed with PBS, and subjected to flow cytometry analysis using FACS Calibur (BD Biosciences). Each sample was analyzed for 10,000 events.

Confocal Microscopy. HeLa cells were cultured in 35-mm glass-bottomed dishes until 80-90% confluent. The cells were treated with 1 µM of R8-Alexa488 at 37°C. After washing with PBS containing 0.5% (w/v) heparin, intracellular distribution of the fluorescently labeled peptides was analyzed without fixing using a confocal microscope (FV1000, Olympus). For colocalization analysis of R8 and syndecan-4, cell-surface syndecan-4 was labeled by sequential pretreatment of HeLa cells with

anti-syndecan-4

antibody

(5G9

mouse

monoclonal,

1:40)

for

30

min

and

with

Alexa488-conjugated anti-mouse IgGs (1:200) as secondary antibody for 30 min in serum-free α-MEM containing 0.5% (w/v) bovine serum albumin. After washing with PBS, the cells were treated with 1 µM of R8-TAMRA for 30 min at 37°C, and was subjected to microscopic analysis.


Page 29 of 37

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Colocalization was analyzed by calculating Pearson’s correlation coefficients with Image J software (NIH). For time-lapse imaging of R8, syndecan-4 and transferrin, cell-surface syndecan-4 was labeled by sequential pretreatment of HeLa cells with anti-syndecan-4 antibody (5G9 mouse monoclonal, 1:40) and with Alexa405-conjugated anti-mouse IgGs (1:200) as described above. After washing with PBS, the cells were placed at 37°C in a microchamber (MI-IBC, Olympus) attached on the stage of the inverted microscope. The cells were then added with R8-TAMRA and transferrin-Alexa488 (final concentration, 1 µM and 25 µg/mL, respectively), and images were acquired every 30 sec for 10 min using the confocal microscopy.

Quantitative RT-PCR. Total RNA was isolated from cells using the RNeasy Mini Kit (Qiagen). cDNA was synthesized from total RNA using a PrimeScript RT reagent Kit (Takara), and real-time PCR was performed with 7300 Real-Time PCR System (Applied Biosystems) with Power SYBR Green PCR Master Mix (Applied Biosystems). The primer sequences are listed in Table S1.

Generation of Cre Reporter Cells. Cre reporter cells were generated using Flp-In system (Invitrogen)

according

to

the

manufacturer’s

instructions.

A

plasmid

vector

encoding

loxP-DsRed-loxP-EGFP sequence was created by subcloning corresponding DNA fragments of loxP-DsRed-loxP and EGFP from pMSCV-loxP-dsRed-loxP-eGFP-Puro-WPRE (plasmid 32702, Addgene) and pEGFP-N1 (Clontech), respectively, into the NheI/BamHI sites of pcDNA5/FRT. The resultant plasmid vector and Flp-recombinase expression vector (pOG44) (1:4) were transfected into the Flp-In-293 cell line using Lipofectamine 3000 (Invitrogen). After 48 h, the culture medium was changed to the one supplemented with 400 µg/mL hygromycin B. The antibiotics-resistant cells with stable expression of DsRed and high recombination efficiency after expression of Cre recombinase were selected after 3 weeks.



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Page 30 of 37

Preparation of Cre Recombinases. The recombinant proteins of Cre recombinases (Cre and CreR8) were prepared as Cre-(His)6 and CreR8-(His)6, containing a (His)6 sequence at the C-terminus. A plasmid vector encoding Cre-(His)6 was created by subcloning a DNA fragment of Cre from pCAG-Cre (plasmid 32702, Addgene) into the NdeI/EcoRI sites of pET-42b(+) (Novagen). For construction of a plasmid vector encoding CreR8-(His)6, the DNA fragment of Cre from pCAG-Cre was amplified using a forward primer coding a NdeI site and a reverse primer coding a EcoRI site and a (Arg)8 sequence, and inserted into the NdeI/EcoRI sites of pET-42b(+). The recombinant proteins of Cre and CreR8 were overexpressed in a soluble form in the E. coli strain BL21(DE3), and purified by ÄKTA start equipped with a HisTrap FF column (GE Healthcare). The purified proteins were dialyzed with a cell culture medium (DMEM) containing 10 mM HEPES and 500 mM NaCl using the Slide-A-Lyzer Dialysis Cassettes (molecular weight cut off, 20K), and stored at 4°C until use in Cre-loxP recombination assay. The purity of the proteins was confirmed by SDS-PAGE.

Cre-loxP Recombination Assay. The Cre reporter cells were treated with 5 µM of Cre or CreR8 in serum-free DMEM at 37°C for 30 min in the presence or absence of the pharmacological inhibitors. The cells were then harvested, replated in another dishes, and cultured with complete medium for 24 h. Efficiency of recombination was calculated by analyzing the number of the Cre reporter cells expressing EGFP using flow cytometry.


Page 31 of 37

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ACKNOWLEDGEMENTS This work was supported by a Grant-in-Aid of The FUGAKU TRUST FOR MEDICINAL RESEARCH to S.F. This work was also supported by the Collaborative Research Program of Institute for Chemical Research, Kyoto University. Y.K. is grateful for Japan Society for the Promotion of Science (JSPS) Fellowship for Young Scientists. ITbM is supported by the World Premier International Research Center Initiative (WPI), Japan.

Supporting Information The Supporting Information including a supplementary table and supplementary figures is available free of charge on the ACS Publications website at DOI: xx.xxxx.



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