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CNS & Neurological Disorders - Drug Targets

Editor-in-Chief

ISSN (Print): 1871-5273
ISSN (Online): 1996-3181

Research Article

A Novel Botulinum Toxin TAT-EGFP-HCS Fusion Protein Capable of Specific Delivery Through the Blood-brain Barrier to the Central Nervous System

Author(s): Fengjin Hao, Yueqin Feng and Yifu Guan*

Volume 18, Issue 1, 2019

Page: [37 - 43] Pages: 7

DOI: 10.2174/1871527317666181011113215

Price: $65

Abstract

Objective: Botulinum toxin has many applications in the treatment of central diseases, as biological macromolecules, it is difficult to pass through the blood-brain barrier which greatly limits their application. In this paper, we verified whether the botulinum toxin heavy chain HCS has a specific neural guidance function.

Methods: We have constructed a fusion protein with botulinum toxin heavy chain and a membrane penetrating peptide TAT (TAT-EGFP-HCS). Recombinant plasmid of botulinum toxin light chain (LC) and TAT were also constructed. The biological activity of HCS, LC, TAT-EGFP-HCS and TAT-EGFP-LC were measured by its ability to cleave protein SNAP-25. The intracellular expression efficiency was evaluated by detecting the fluorescence intensity of EGFP in the cells by fluorescence microscopy and FACS. In addition, we also determined the effect of the above plasmid expression on the apoptosis of PC12 cells. Finally, the tissue specificity of TAT-EGFP-HCS in vivo experiments was also examined.

Results: In the present study, we have constructed a fusion protein with botulinum toxin heavy chain and a membrane penetrating peptide TAT which can lead the entire molecule through the blood-brain barrier and reach the central nervous system. Moreover, we also examined the biological activities of this recombinant biological macromolecule and its physiological effects on nerve cells in vitro and in vivo.

Conclusion: TAT-EGFP-HSC expressed in vitro has neural guidance function and can carry large proteins across the cell membrane without influencing the biological activity.

Keywords: Botulinum toxin, Heavy chain, TAT, transmembrane, CNS delivery, central disease.

Graphical Abstract

[1]
Kuroiwa M, Ikeda H, Hongo T, et al. Effects of recombinant human endostatin on a human neuroblastoma xenograft. Int J Mol Med 2001; 8(4): 391-6.
[2]
Arvold ND, Armstrong TS, Warren KE, et al. Corticosteroid use endpoints in neuro-oncology: Response assessment in neuro-oncology working group. Neuro-oncol 2018; 20(7): 897-906.
[3]
Peck MW. Biology and genomic analysis of Clostridium botulinum. Adv Microb Physiol 2009; 55: 183-265.
[4]
Rossetto O, Pirazzini M, Montecucco C. Botulinum neurotoxins: Genetic, structural and mechanistic insights. Nat Rev Microbiol 2014; 12(8): 535-49.
[5]
Silvaggi NR, Boldt GE, Hixon MS, et al. Structures of Clostridium botulinum neurotoxin serotype a light chain complexed with small-molecule inhibitors highlight active-site flexibility. Chem Biol 2007; 14(5): 533-42.
[6]
Bade S, Rummel A, Reisinger C, et al. Botulinum neurotoxin type D enables cytosolic delivery of enzymatically active cargo proteins to neurones via unfolded translocation intermediates. J Neurochem 2004; 91(6): 1461-72.
[7]
Safarpour Y, Jabbari B. Botulinum toxin for the treatment of movement disorders. Curr Treat Neurol 2012; 12(4): 399-409.
[8]
Hackett G, Moore K, Burgin D, et al. Purification and characterization of recombinant botulinum neurotoxin serotype FA, also known as serotype H. Toxicon 2018; 123: 36.
[9]
Pellett S, Bradshaw M, Tepp WH, et al. The light chain defines the duration of action of botulinum toxin serotype a subtypes. Mol Biol 2018; 9(2): e00089-18.
[10]
Ko EC, Wang X, Ferrone S. Immunotherapy of malignant diseases. Challenges and strategies. Int Arch Allergy Immunol 2003; 132(4): 294-309.
[11]
Silvaggi NR, Boldt GE, Hixon MS, et al. Structures of clostridium botulinum neurotoxin serotype a light chain complexed with small-molecule inhibitors highlight active-site flexibility. Chem Biol 2007; 14(5): 533-42.
[12]
Bade S, Rummel A, Reisinger C, et al. Botulinum neurotoxin type D enables cytosolic delivery of enzymatically active cargo proteins to neurones via unfolded translocation intermediates. J Neurochem 2010; 91(6): 1461-72.
[13]
Binz T, Sikorra S. Structural and functional insights into the interaction of BoNT/A light chain with SNAP-25 and SNAP-23. Toxicon 2016; 123: 6.
[14]
Mesngon M, Mcnutt P. Alpha-latrotoxin rescues snap-25 from bont/a-mediated proteolysis in embryonic stem cell-derived neurons. Toxins 2011; 3(5): 489-503.
[15]
Wang M, Zhi D, Wang H, et al. TAT-HSA-α-MSH fusion protein with extended half-life inhibits tumor necrosis factor-α in brain inflammation of mice. Appl Microbiol Biotechnol 2016; 100(12): 5353-61.
[16]
Liu P, Liu X, Xing J, et al. The neuroprotective mechanism of erythropoietin-tat fusion protein against neurodegeneration from ischemic brain injury. CNS Neurol Disord Drug Targets 2014; 13(8): 1465-74.
[17]
Zhu Y, Bu Q, Liu X, Hu W, Wang Y. Neuroprotective effect of TAT-14-3-3ε fusion protein against cerebral ischemia/reperfusion injury in rats. PLoS One 2014; 9(3): e93334.
[18]
Doeppner TR, Aanbouri ME, Dietz GPH, Weise J, Schwarting S, Bähr M. Transplantation of TAT-Bcl-x-transduced neural precursor cells: Long-term neuroprotection after stroke. Neurobiol Dis 2010; 40(1): 265-76.
[19]
Cai B, Lin Y, Xue XH, Fang L, Wang N, Wu ZY. TAT-mediated delivery of neuroglobin protects against focal cerebral ischemia in mice. Exp Neurol 2011; 227(1): 224-31.
[20]
Jeong HJ, Kim DW, Kim MJ, et al. Protective effects of transduced Tat-DJ-1 protein against oxidative stress and ischemic brain injury. Exp Mol Med 2012; 44(10): 586-93.

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