Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency disease affecting cell morphology and signal transduction in hematopoietic cells. The function of Wiskott-Aldrich syndrome protein (WASp) and its partners in protein interaction have been studied intensively in mice; however, detailed biochemical characterization of its signal transduction and assessment of its functional consequence in human WASp-deficient lymphocytes remain difficult. In this study, we generated Nalm-6 cells in which the WAS protein gene (WASP) was disrupted by homologous recombination-based gene targeting and a cell-permeable form of recombinant WASp for functional study. The WASP -/- cells showed impaired adhesive capacity and polarization to plate-bound antiCD47 mAb, anti-CD9 mAb, or to fibronectin. The defective morphological changes were accompanied by impaired intracellular signaling. In addition, the WASp-deficient cells displayed augmented apoptosis induced by CD24 cross-linking. A recombinant fusion protein composed of Hph-1 cell-permeable peptide and WASp prepared in Escherichia coli. Hph-1-WASp was efficiently transduced and expressed in WASP -/- Nalm-6 cells in a dosedependent manner. The wild-type WASp, but not the mutant restored adhesion capacity, spreading morphology, and cytoskeletal reorganization. Additionally, the recombinant protein was successfully transduced into normal lymphocytes. These findings suggest that gene-disrupted model cell lines and cell-permeable recombinant proteins may serve as important tools for the detailed analysis of intracellular molecules involved in PID.
Bibliographical noteFunding Information:
Acknowledgments We thank Drs. Kiyokawa, Clark and Molden-hauer for providing us with antibody and Dr. Erdyni Tsitsikov for valuable comments. We thank Dr. Terada-Takahashi, Mrs. Minegishi, Mr. Ochiai, Ms. Kurihara, Ms. Kumaki, Dr. Kimura-Sato and Mrs. Kutami for technical assistance. This work was supported by a Grant-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), a Grant-in-Aids from the Ministry of Health, Labour, and Welfare, and grant from JSPS-KOSEF Joint Research Program to T.M. This work was supported in part by Creative Research Initiatives, a National Research Foundation of Korea Grant funded by the Korean Government (2010-0000733), and the Brain Korea 21 (BK21) Program to Sang-Kyou Lee.
All Science Journal Classification (ASJC) codes