生物機能中分子の細胞導入の分子基盤：...

生物機能中分子の細胞導入の分子基盤：

細胞内送達ペプチドHAadの設計と評価

Introduction

京都大学化学研究所二木史朗

Development of systems that effectively bring macromolecules into cytosolshould be beneficial for the understanding of biological significance of cytosoliccomponents by modulating their functions using macromolecules, and fordeveloping therapeutic systems based on these understandings. Our laboratorydeveloped a peptide “L17E” that delivers macromolecules into cytosol, by modifyingthe sequence of a hemolytic spider venom peptide M-lycotoxin (Akishiba, M., et

al., Nat. Chem. (2017)). L17E is capable of efficiently delivering various bioactiveproteins, including IgG, into cytosol. Specific recognition of cytosolic proteins andsuppression of glucocorticoid receptor-mediated transcription were thus achievedusing the targeting IgGs in the presence of L17E.

Results & Discussion

[Cells; HeLa, L17E; 40 μM,

Alexa488-hIgG; 1mg/mL,

treatment; 1hr in α-MEM(-

)]

A. Substitution of histidine to alanine residues

*cytotoxic

peptide name sequence

IWLTALKFLGK AAK AKQQLSKL-amide


IWLTALKFLGK AAK AKQ LSKL-amide


no peptide

DIC

Ale

xa

48

8

cell death

cell death

52.3%

B. Introduction of acidic amino acid with longer side chain


IWLTALKFLGKHAAKH AKQQLSKL-amide






ce

lls y

ield

ing

cyto

so

lic

de

xtr

an

sig

na

ls (

%)

0

25

50

75

100

40μM 15μM 20μM 30μM 40μM

L17E HAad

X; L-2-aminoadipic acid (Aad)

C. Combination of A and B

13.3% 69.9%0.90%

52.3%

DIC

Ale

xa

48

8 24.5% 62.6%

D. Leakage assay

F. HAad-mediated intracellular delivery of functional protein

DsRed EGFP

DsRed

EGFP

DsRed

EGFP

Cre + peptide

Cre-mediated

recombination

loxP STOP

40 mM 40 mM

52.3% 74.4%

[Cells; HeLa, peptides; 40 μM, Alexa488-dextran (10 kDa); 200 mg/mL,

treatment; 1 hr in serum-free a-MEM]

[Cells; HeLa, peptides; 40 μM, Alexa488-dextran (10 kDa); 200 mg/mL,

treatment; 1 hr in serum-free a-MEM]

[Cells; HeLa, peptides; 40 μM, Alexa488-dextran (10 kDa); 200 mg/mL, treatment; 1 hr in serum-free a-MEM. Results are presented as mean ± SE

(n=3).]

✓ His-to-Ala substitution yielded

an improved intracellular macromolecule-delivering activity.

✓ has no pH selectivity.

✓ has negatively-charged lipid-selective

and low pH-selective membrane destabilizing

activity.

no peptide

DIC

EG

FP

DsR

ed

me

rge

no peptide

Cre 5 mM Cre 10 mM

EG

FP

-po

sitiv

e r

ep

ort

er-

tra

nsfe

cte

d c

ells

(%

)0

20

40

60

80

100

no peptide L17E HAad no peptide L17E HAad

+ Cre 5 μM + Cre 10 μM

[Cells; HeLa expressing loxP-DsRed-STOP-

losP-EGFP reporter, peptides; 40 μM, Cre

recombinase; 5 or 10 μM, treatment; 1 hr in

serum-free a-MEM. Results are presented as mean ± SE (n=3).]

A. Substitution of

His to Ala

B. Substitution of

Glu to L-2-aminoadipic acid

CH3CH3 CH3CH3

✓ Substitution of Glu to Aad also yielded

an improved intracellular macromolecule-delivering activity.

CH3CH3

✓ By combination of both substitutions

HAad was obtained to display the highest efficacy.

-20

0

20

40

60

80

100

120

0.0001 0.001 0.01 0.1 1

L17E

PC 100% LUV, pH 7.4PC 75%/PG 25 % LUV, pH 7.4PC 100% LUV, pH 5.0PC 75%/PG 25 % LUV, pH 5.0

P/L

-20

0

20

40

60

80

100

120

0.0001 0.001 0.01 0.1 1

HAad

PC 100% LUV, pH 7.4PC 75%/PG 25 % LUV, pH 7.4PC 100% LUV, pH 5.0PC 75%/PG 25 % LUV, pH 5.0

P/L

PC, pH7.4

PC, pH5.0

PC, pH7.4

PC, pH5.0

PC/PG,

pH5.0

PC/PG,

pH7.4

PC/PG,

pH5.0

PC/PG,

pH7.4

L-2-aminoadipic

acid (Aad)GluAsp

[lipid concentration; 200μM, lipid composition; 100% POPC or 75% POPC and 25% POPG (mol:mol) encapsulating 12.5mM ANTS/45.0mM DPX, pH; 7.4 or 5.0,

incubation; 1hr at 37℃, leakage (%)=(Fobs-Fblank)/(Fmax-Fblank)*100]

[peptide concentration; 20μM, lipid concentration; 2mM, lipid composition; 100% POPC or 75% POPC and 25% POPG (mol:mol), pH; 7.4 or 5.0, temperature;

25°C,]

E. CD spectroscopy

✓ does not have a marked helical

structure even in the presence of LUVs.

✓ showed almost complete helical

structure in the presence of POPG at pH 5.0.-4x10

4

-3x104

-2x104

-1x104

0

1x104

200 210 220 230 240 250 260

L17E

POPC only LUV (pH 7.4)


POPC/POPG mix LUV (pH 7.4)


WL (nm)

-4x104

-3x104

-2x104

-1x104

0

1x104

200 210 220 230 240 250 260

HAad





WL (nm)

PC, pH7.4PC, pH5.0

PC/PG,

pH5.0PC/PG,

pH7.4

PC, pH7.4PC, pH5.0

PC/PG,

pH5.0

PC/PG,

pH7.4

L17E HAadno peptidebright

field

EG

FP

DsR

ed

co

rona

l se

ctio

n

cut

Cre (5 mM) +

peptide (40 mM)

7 days

G. Intracerebroventrical administration of Cre and HAad

EGFP

DsRed

DsRed

Cre-mediated

recombination

EGFP DsRed

EGFP

loxP

H. Mode of action of HAad

L17E HAadno peptide

Ga

lectin

3

LLOMe

(positive control)

0

25

50

75

100

NH4Cl (-) NH4Cl (+) NH4Cl (-) NH4Cl (+)

L17E HAad

Cel

ls w

ith

cyt

oso

lic

dex

tran

sig

nal

s (%

)

0

25

50

75

100

CQ(-) CQ(+) CQ(-) CQ(+)

L17E HAad

Cel

ls w

ith

cyt

oso

lic

dex

tran

sig

nal

s (%

)*** ***

n.s.

******

n.s.

[mouse; adult female R26GGR mouse expressing loxP-

EGFP-STOP-loxP-DsRed reporter, peptide; 40 μM, Cre

recombinase; 5 μM, treatment; i.c.v. administration for 7

days]

STOP

➢ Immunocytochemistry of Galectin-3

➢ Inhibition of endosomal acidification

✓ In HAad-treated cells Galectin-3 was strongly localized

to disrupted endosomes (red puncta signal).

◆ We are deeply grateful to Dr. Sugiyama and his lab members (Laboratory Animal Science, Tsukuba Univ.) to perform this in vivo experiment.

intact endosome disrupted endosome

b-galactosides

Galectin-3

endosomolysis

Galectin-3 recognizing

b-galactosides

[Cells; HeLa, L17E; 40 μM, HAad; 40 μM, LLOMe; 1 mM, treatment; 1 hr in serum-free a-MEM. 1st antibody; mouse

anti-Galectin 3 antibody [A3A12], 2nd antibody; Alexa Fluor 488 goat anti-mouse IgG (H+L).]

[Cells; HeLa, L17E or HAad; 40 μM, Alexa488-dextran; 200 mg/mL,

NH4Cl: 25 mM, chloroquine (CQ); 100 mM, pretreatment; 30 min w/ or

w/ o NH4Cl or CQ, treatment; 1 hr in serum-free a-MEM w/ or w/o BafA1 or CQ. Results are presented as mean ± SE (n = 3).]

✓ In the presence of endosomal acidification inhibitors, the cytosolic

macromolecule-delivering activity of HAad was attenuated

to the same level of L17E.

✓ The increment of the activity from L17E to HAad would be due to the

obtained/enhanced endosomolytic activity.

To improve the cytosolic delivery activity of L17E, we focused on the helical structure formation of the peptidesdependent on endosomal pH decrease to more interact with endosomal membranes to improve the endosomolytic activity.We have succeeded in obtaining a peptide named “HAad”, by stepwise remodeling of L17E. HAad attained a significantlyimproved cytosolic delivery of various macromolecules (polydextran and Cre recombinase).

In detailed analysis, we have found that L17E has an unique mechanismof action: L17E stimulates polymerization of actin followed by induction ofmembrane ruffling. At that site L17E transiently permeabilize the ruffledplasma membrane and macromolecules enters into cytosol within 5 min.This mode of action is thought to be the crucial mechanism for cytosolicdelivery of macromolecules rather than endosomal destabilization.

Akishiba, M. and Futaki, S., Mol., Pharmaceutics (2019).

Injection to right

lateral ventricle

DA

PI_

DsR

ed

cro

ss s

ection

✓ HAad delivered Cre

even in vivo.

生物機能中分子の細胞導入の分子基盤：...

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