Tài liệu Báo cáo khoa học: Inhibitory effects of nontoxic protein volvatoxin A1 on pore-forming

Inhibitory effects of nontoxic protein volvatoxin A1

on pore-forming cardiotoxic protein volvatoxin A2

by interaction with amphipathic a-helix

Pei-Tzu Wu1

, Su-Chang Lin2

, Chyong-Ing Hsu1

, Yen-Chywan Liaw2 and Jung-Yaw Lin1

1 Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan

2 Institute of Molecular Biology Academia Sinica, Taipei, Taiwan

Volvatoxin A (VVA) has been isolated from Volvari￾ella volvacea, and consists of volvatoxin A2 (VVA2)

and volvatoxin A1 (VVA1) [1]. VVA has several biolo￾gical activities, such as: (a) lysis of human red blood

cells; (b) swelling tumor cells and the mitochondria

of liver cells; (c) inhibition of protein biosynthesis;

and (d) causing cardiac arrest via activation of the

Ca2+-dependent ATPase enzyme in the ventricular

microsomal fraction [1–3]. The hemolytic activity of

VVA2 is totally inhibited by VVA1 at a molar ratio of

2 [4,5]. Previous studies have shown that VVA2 is a

b-pore-forming toxin, with a heparin-binding site

(HBS) encoded within the C-terminal b-strands (b6, b7

and b8). This HBS structure is indispensable for the

Keywords

amphipathic a-helix; co-pull-down

experiment; tandem repeat protein;

volvatoxin A1; volvatoxin A2

Correspondence

J.-Y. Lin, Institute of Biochemistry and

Molecular Biology, College of Medicine,

National Taiwan University, F9, no. 1,

Section 1, Jen-Ai Road, Taipei 10051,

Taiwan

Fax: +886 2 23415334

Tel: +886 2 23123456 (ext. 8206 ⁄ 8207)

E-mail: [email protected]

Database

The nucleotide sequence reported in this

paper has been submitted to the

DDBJ ⁄ EMBL ⁄ GenBank databases under the

accession number AY952461

(Received 22 March 2006, revised 1 May

2006, accepted 17 May 2006)

doi:10.1111/j.1742-4658.2006.05325.x

Volvatoxin A2, a pore-forming cardiotoxic protein, was isolated from the

edible mushroom Volvariella volvacea. Previous studies have demonstrated

that volvatoxin A consists of volvatoxin A2 and volvatoxin A1, and the

hemolytic activity of volvatoxin A2 is completely abolished by volvatoxin

A1 at a volvatoxin A2 ⁄ volvatoxin A1 molar ratio of 2. In this study, we

investigated the molecular mechanism by which volvatoxin A1 inhibits the

cytotoxicity of volvatoxin A2. Volvatoxin A1 by itself was found to be

nontoxic, and furthermore, it inhibited the hemolytic and cytotoxic activit￾ies of volvatoxin A2 at molar ratios of 2 or lower. Interestingly, volvatoxin

A1 contains 393 amino acid residues that closely resemble a tandem repeat

of volvatoxin A2. Volvatoxin A1 contains two pairs of amphipathic a-heli￾ces but it lacks a heparin-binding site. This suggests that volvatoxin A1

may interact with volvatoxin A2 but not with the cell membrane. By using

confocal microscopy, it was demonstrated that volvatoxin A1 could not

bind to the cell membrane; however, volvatoxin A1 could inhibit binding

of volvatoxin A2 to the cell membrane at a molar ratio of 2. Via peptide

competition assay and in conjunction with pull-down and co-pull-down

experiments, we demonstrated that volvatoxin A1 and volvatoxin A2 may

form a complex. Our results suggest that this occurs via the interaction of

one molecule of volvatoxin A1, which contains two amphipathic a-helices,

with two molecules of volvatoxin A2, each of which contains one amphi￾pathic a-helix. Taken together, the results of this study reveal a novel

mechanism by which volvatoxin A1 regulates the cytotoxicity of volvatoxin

A2 via direct interaction, and potentially provide an exciting new strategy

for chemotherapy.

Abbreviations

FITC, fluorescein isothiocyanate; GSH, glutathione; GSP, gene-specific primer; HBS, heparin-binding site; RBC, red blood cell; VVA,

volvatoxin A; VVA1, volvatoxin A1; VVA2, volvatoxin A2; VVA1-CTD, volvatoxin A1 C-terminal domain (198–391 residues); VVA1-NTD,

volvatoxin A1 N-terminal domain (1–197 residues).

3160 FEBS Journal 273 (2006) 3160–3171 ª 2006 The Authors Journal compilation ª 2006 FEBS