Abstract
Aim Nogo extracellular peptide 1-40 (NEP1-40), a Nogo-66 antagonistic peptide, is one of the potential candidates for therapeutic intervention after central nervous system injury. This study is focused on the generation of TAT-NEP1-40 fusion protein and its transducible effects and biological activity. Methods TAT-NEP1-40 fusion protein was expressed in vitro. Transducible effects of TAT-NEP1-40 were analyzed by using immunofluorescence staining or Western blot in vitro and in vivo. The biological activity of TAT-NEP1-40 was assessed by its effects against oxygen and glucose deprivation (OGD)-induced PC12 cell damages. Results Our results showed that the TAT-NEP1-40 fusion protein was successfully expressed, purified, and refolded. Western blot analysis and immunofluorescence staining confirmed the delivery of TAT-NEP1-40 protein into PC12 cells and rat brains. OGD caused cell apoptosis or death, decreased cell viability, increased lactate dehydrogenase release in medium and the Bax/Bcl-2 ratio, all of which were prevented by the TAT-NEP1-40 fusion proteins when added exogenously to culture medium. In addition, TAT-NEP1-40 promoted neurite outgrowth of PC12 cells exposed to OGD. Conclusion These results demonstrate that the TAT-NEP1-40 can be successfully generated and efficiently transduced into PC12 cells and rat brains. The TAT-NEP1-40 can protect PC12 cells against OGD and promote neurite outgrowth. This finding suggests that the transducible TAT-NEP1-40 fusion protein offers a possibility of the development of novel therapy for cerebral injuries via delivery of the biologically active TAT-NEP1-40 fusion protein into injured sites.
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Acknowledgments
This work was supported by the National Natural Science Foundation of China (Grant 30371763), and in part by the Natural Science Foundation of Shaanxi Province (Grant 2006C254). Nogo-A cDNA (KIAA0886) was generously provided by the Kazusa DNA Research Institute. We wish to thank Dr. Steven F. Dowdy (Ph.D., Howard Hughes Medical Institute and Department of Cellular and Molecular Medicine, University of California San Diego, School of Medicine, La Jolla, CA 92093-0686, USA) for providing the pTAT-HA vector and pTAT-HA-β-galactosidase plasmids; and Dr. Yan Lu for his critical reading of the manuscript.
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Qiang Wang and Xingchun Gou contributed equally to this article.
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Wang, Q., Gou, X., Jin, W. et al. TAT-Mediated Protein Transduction of Nogo Extracellular Peptide 1-40 and its Biological Activity. Cell Mol Neurobiol 29, 97–108 (2009). https://doi.org/10.1007/s10571-008-9301-2
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DOI: https://doi.org/10.1007/s10571-008-9301-2