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

Daisuke Shiokawa,Tokiyoshi Matsushita,Yukari Shika,Mamoru Shimizu,Masahiro Maeda,Sei-Ichi Tanuma

doi:10.1074/jbc.m610428200

Daisuke Shiokawa, Tokiyoshi Matsushita + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m610428200

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Abstract

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, beta-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.

Highlights

DNase I is a secretory endonuclease that hydrolyzes DNA to 3Ј-hydroxyl oligonucleotides in the presence of divalent metal ions, such as Ca2ϩ, Mg2ϩ, and Mn2ϩ [1,2,3]
We have demonstrated that DNase X hydrolyzes endocytosed extracellular DNA, thereby protecting cells from invasion by foreign genes
DNase X Is Expressed during Myogenic Differentiation of RD Cells—To examine DNase X expression at the protein level, we performed an immunohistochemical analysis of several human tissues using the newly developed anti-DNase X monoclonal antibody (mAb) 1B1

Summary

Introduction

DNase I is a secretory endonuclease that hydrolyzes DNA to 3Ј-hydroxyl oligonucleotides in the presence of divalent metal ions, such as Ca2ϩ, Mg2ϩ, and Mn2ϩ [1,2,3]. We obtained essentially the same results with the endogenous DNase X of RD myotubes (Fig. 3C); PI-PLC effected the release of DNase X without solubilizing cadherin, a membrane-integrated protein responsible for stable contact between cells [13].

Results

Conclusion