Impaired cell adhesion, apoptosis, and signaling in WASP gene-disrupted Nalm-6 pre-B cells and recovery of cell adhesion using a transducible form of WASp.
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 anti-CD47 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 dose-dependent 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.
Department of Pediatrics and Developmental Biology, Tokyo Medical and Dental University Graduate School of Medicine, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.
Rikiya
R
Sato
Susumu
S
Iiizumi
Eun-Sung
ES
Kim
Fumiko
F
Honda
Sang-Kyou
SK
Lee
Noritaka
N
Adachi
Hideki
H
Koyama
Shuki
S
Mizutani
Tomohiro
T
Morio
D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D017209:Apoptosis / Q000235:genetics*; D051927:CD24 Antigen / Q000378:metabolism; D051928:CD47 Antigen / Q000276:immunology / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002448:Cell Adhesion / Q000235:genetics; D002460:Cell Line; D016764:Cell Polarity / Q000187:drug effects; D005353:Fibronectins / Q000494:pharmacology; D023061:Gene Order; D018390:Gene Targeting; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D063150:Polycomb Repressive Complex 1; D054448:Precursor Cells, B-Lymphoid / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D060245:Tetraspanin 29 / Q000037:antagonists & inhibitors / Q000276:immunology; D051316:Wiskott-Aldrich Syndrome Protein / Q000235:genetics* / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D051927:CD24 Antigen; D051928:CD47 Antigen; D002352:Carrier Proteins; D005353:Fibronectins; C491239:PHC1 protein, human; D011993:Recombinant Fusion Proteins; D060245:Tetraspanin 29; D051316:Wiskott-Aldrich Syndrome Protein; D063150:Polycomb Repressive Complex 1