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J. Biol. Chem., Vol. 278, Issue 11, 9972-9978, March 14, 2003
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From the Pierisin-1, a cytotoxic protein found
naturally in the cabbage butterfly, induces apoptosis of mammalian
cells. Our recent studies suggest that pierisin-1 consists of an
N-terminal ADP-ribosyltransferase domain, and a C-terminal region that
binds to receptors on the surfaces of target cells and incorporates the
protein into cells. The present study was undertaken to identify
receptors for pierisin-1. The cross-linking and cloning experiments
suggested that the proteins on cell membrane had no binding ability to
pierisin-1. Inhibitory assays of fractionated lipids from human
cervical carcinoma HeLa cells, which are highly sensitive to
pierisin-1, indicated neutral glycosphingolipids on the cell surface to
show receptor activity. Inhibitory assays and TLC immunostaining using
anti-pierisin-1 antibodies demonstrated two neutral glycosphingolipids
as active components. Analysis of their structures with
glycosphingolipid-specific antibodies and negative secondary ion mass
spectrometry identified them as globotriaosylceramide (Gb3) and
globotetraosylceramide (Gb4). The receptor activities of Gb3 and Gb4
for pierisin-1 were also confirmed with these authentic compounds.
Pierisin-1-insensitive mouse melanoma MEB4 cells were found to lack
pierisin-1 receptors, including Gb3 and Gb4, but pretreatment of the
cells with glycosphingolipid Gb3 or Gb4 enhanced their sensitivity to
pierisin-1. Thus, Gb3 and Gb4 were proven to serve as pierisin-1
receptors. The C-terminal region of pierisin-1 consists of possible
lectin domains of a ricin B-chain, containing QXW
sequences, which are essential for its structural organization.
Alteration of QXW by site-directed mutagenesis caused
marked reduction of pierisin-1 cytotoxicity. Thus, our results suggest
that pierisin-1 binds to Gb3 and Gb4 receptors at the C-terminal
region, in a manner similar to ricin, and then exhibits cytotoxicity
after incorporation into the cell.
Identification of Glycosphingolipid Receptors for Pierisin-1,
a Guanine-specific ADP-ribosylating Toxin from the Cabbage
Butterfly*
§,,
,,,, and
Cancer Prevention Division, National Cancer
Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, the ¶ Department of Biochemistry, University of Shizuoka School of
Pharmaceutical Sciences, 52-1 Yada, Shizuoka-shi 422-8526, and the
Instrumental Analysis Research Center for Life Science,
Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo
113-8510, Japan
*
This study was supported by a grant-in-aid for scientific
research from the Japan Society for the Promotion of Science, a grant-in-aid for cancer research from the Ministry of Health, Labor and
Welfare, Japan, and a research grant from the Princess Takamatsu Cancer
Research Fund.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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