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J. Biol. Chem., Vol. 282, Issue 23, 17132-17140, June 8, 2007

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DNase X Is a Glycosylphosphatidylinositol-anchored Membrane Enzyme That Provides a Barrier to Endocytosis-mediated Transfer of a Foreign Gene^*

Daisuke Shiokawa¹, Tokiyoshi Matsushita, Yukari Shika, Mamoru Shimizu, Masahiro Maeda, and Sei-ichi Tanuma^¶2

From the Faculty of Pharmaceutical Sciences, Department of Biochemistry, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan, Research and Development Department, Immuno-Biological Laboratories Co., Ltd., 1091-1 Naka, Fujioka, Gunma 375-0005, Japan, and ^¶Genome and Drug Research Center, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan

DNase X is the first mammalian DNase to be isolated that is homologous to DNase I. In this study, we have examined its function using a novel monoclonal antibody and showed it to be expressed on the cell surface as a glycosylphosphatidylinositolanchored membrane protein. High level expression was observed in human muscular tissues and in myotubes obtained in vitro from RD rhabdomyosarcoma cells. We observed that RD myotubes incorporated a foreign gene, lacZ, by endocytosis but that expression of the encoded coding product, -galactosidase, was strongly inhibited. Overexpression of DNase X inhibited endocytosis-mediated gene transfer, whereas knockdown of DNase X with small interfering RNA had the opposite effect. These results reveal that DNase X provides a cell surface barrier to endocytosis-mediated gene transfer.

Received for publication, November 8, 2006 , and in revised form, April 3, 2007.

^* This work was funded by a grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ Present address: Radiobiology Div., National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.

² To whom correspondence should be addressed: Dept. of Biochemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan. Tel.: 81-4-7124-1501; Fax: 81-4-7121-3620; E-mail: tanuma{at}rs.noda.tus.ac.jp.

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