Light Chain LC and TAT-EGFP-HCS of Botulinum Toxin Expression and Biological Function in vitro and in vivo

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

Journal Name: Current Proteomics

Volume 16 , Issue 3 , 2019

Become EABM
Become Reviewer

Graphical Abstract:


Abstract:

Objective: To verify whether the botulinum toxin heavy chain HCS has specific neuronal targeting function and to confirm whether TAT-EGFP-LC has hydrolyzable SNAP-25 and has transmembrane biological activity.

Methods: We constructed the pET-28a-TAT-EGFP-HCS/LC plasmid. After the plasmid is expressed and purified, we co-cultured it with nerve cells or tumors. In addition, we used Western-Blot to identify whether protein LC and TAT-EGFP-LC can digest the protein SNAP-25.

Results: Fluorescence imaging showed that PC12, BV2, C6 and HeLa cells all showed green fluorescence, and TAT-EGFP-HCS had the strongest fluorescence. Moreover, TAT-EGFP-LC can hydrolyze intracellular SNAP-25 in PC12 cells, C6 cells, BV2 cells and HeLa, whereas LC alone cannot. In addition, the in vivo protein TAT-EGFP-HCS can penetrate the blood-brain barrier and enter mouse brain tissue.

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 HSC, CNS, in vitro, in vivo, drugs.

[1]
Umland, T.C.; Wingert, L.M.; Swaminathan, S.; Furey, W.F.; Schmidt, J.J.; Sax, M. Structure of the receptor binding fragment HC of tetanus neurotoxin. Nat. Struct. Biol., 1997, 4(10), 788-792.
[2]
Rossetto, O.; Pirazzini, M.; Bolognese, P.; Rigoni, M.; Montecucco, C. An update on the mechanism of action of tetanus and botulinum neurotoxins. Acta Chim. Slov., 2011, 58(4), 702-707.
[3]
Ko, E.C.; Wang, X.; Ferrone, S. Immunotherapy of malignant diseases. Challenges and strategies. Int. Arch. Allergy Immunol., 2003, 132(4), 294-309.
[4]
Silvaggi, N.R.; Boldt, G.E.; Hixon, M.S.; Kennedy, J.P.; Tzipori, S.; Janda, K.D.; Allen, K.N. Structures of Clostridium botulinum neurotoxin serotype A light chain complexed with small-molecule inhibitors highlight active-site flexibility. Chem. Biol., 2007, 14(5), 533-542.
[5]
Bade, S.; Rummel, A.; Reisinger, C.; Karnath, T.; Ahnert-Hilger, G.; Bigalke, H.; Binz, T. Botulinum neurotoxin type D enables cytosolic delivery of enzymatically active cargo proteins to neurones via unfolded translocation intermediates. J. Neurochem., 2010, 91(6), 1461-1472.
[6]
Lacy, D.B.; Tepp, W.; Cohen, A.C.; Dasgupta, B.R.; Stevens, R.C. Crystal structure of botulinum neurotoxin type A and implications fortoxicity. Nat. Struct. Biol., 1998, 5(10), 898-902.
[7]
Rawat, R.; Ashraf, A.S.; Swaminathan, S. High level expression of the light chain of botulinum neurotoxin serotype C1 and an efficient HPLC assay to monitor its proteolytic activity. Protein Expr. Purif., 2008, 60(2), 165-169.
[8]
Lönn, P.; Dowdy, S.F. Cationic TAT peptide transduction domain enters cells by micropinocytosis. J. Control. Release, 2005, 190, 56.
[9]
Suzuki, T.; Futaki, S.; Niwa, M.; Tanaka, S.; Ueda, K.; Sugiura, Y. Possible existence of common internalization mechanisms among arginine-rich peptides. J. Biol. Chem., 2002, 277(4), 2437-2443.
[10]
Console, S.; Marty, C.; Garcíaecheverría, C.; Schwendener, R.; Ballmerhofer, K. Antennapedia and HIV transactivator of transcription (TAT) “protein transduction domains” promote endocytosis of high molecular weight cargo upon binding to cell surface glycosaminoglycans. J. Biol. Chem., 2003, 278(37), 35109-35114.
[11]
Kline, C.N.; Joseph, N.M.; Grenert, J.P.; Van, Z.J.; Talevich, E.; Onodera, C.; Aboian, M.; Cha, S.; Raleigh, D.R.; Braunstein, S. Targeted next-generation sequencing of pediatric neuro-oncology patients improves diagnosis, identifies pathogenic germline mutations, and directs targeted therapy. Neuro-oncol., 2017, 19(5), 699-709.
[12]
Qiao, Y.; Ma, N.; Wang, X.; Hui, Y.; Li, F.; Xiang, Y.; Zhou, J.; Zou, C.; Jin, J.; Lv, G. MiR-483-5p controls angiogenesis in vitro and targets serum response factor. FEBS Lett., 2011, 585(19), 3095-3100.
[13]
Jiang, X.W.; Bai, J.P.; Zhang, Q.; Hu, X.L.; Tian, X.; Zhu, J.; Liu, J.; Meng, W.H.; Zhao, Q.C. Caffeoylquinic acid derivatives protect SH-SY5Y neuroblastoma cells from hydrogen peroxide-induced injury through modulating oxidative status. Cell. Mol. Neurobiol., 2017, 37(3), 1-11.
[14]
Guleng, B.; Ren, J.L.; Jazag, A.; Yang, X.N.; Liu, J.J.; Xiao, C.X.; Chen, X.; Li, Y.Y.; Xiao, L.; Li, P. Fibrinogen alpha chain acts as a HBsAg binding protein and their interaction promotes HepG2 cell apoptosis. Curr. Proteomics, 2014, 11(1), 48-54.
[15]
Sutton, J.M.; Wayne, J.; Scott-Tucker, A.; O’Brien, S.M.; Marks, P.M.H.; Alexander, F.C.G.; Shone, C.C.; Chaddock, J.A. Preparation of specifically activatable endopeptidase derivatives of Clostridium botulinum toxins type A, B, and C and their applications. Protein Expr. Purif., 2005, 40(1), 31-41.
[16]
Sharma, S.; Zhou, Y.; Singh, B.R. Cloning, expression, and purification of C-terminal quarter of the heavy chain of botulinum neurotoxin type A. Protein Expr. Purif., 2006, 45(2), 288-295.
[17]
Du, L.; Kayali, R.; Bertoni, C.; Fike, F.; Hu, H.; Iversen, P.L.; Gatti, R.A. Arginine-rich cell-penetrating peptide dramatically enhances AMO-mediated ATM aberrant splicing correction and enables delivery to brain and cerebellum. Hum. Mol. Genet., 2011, 20(16), 3151-3160.
[18]
Chacko, A.M.; Li, C.; Pryma, D.A.; Brem, S.; Coukos, G.; Muzykantov, V. Targeted delivery of antibody-based therapeutic and imaging agents to CNS tumors: Crossing the blood-brain barrier divide. Expert Opin. Drug Deliv., 2013, 10(7), 907-926.
[19]
Van-Meir, E.G.; Hadjipanayis, C.G.; Norden, A.D.; Shu, H.K.; Wen, P.Y.; Olson, J.J. Exciting new advances in neuro-oncology: The avenue to a cure for malignant glioma. CA Cancer J. Clin., 2010, 60(3), 166-193.
[20]
Rahman, R.; Smith, S.; Rahman, C.; Grundy, R. Antiangiogenic therapy and mechanisms of tumor resistance in malignant glioma. J. Oncol., 2010, 2010, 251231.


Rights & PermissionsPrintExport Cite as

Article Details

VOLUME: 16
ISSUE: 3
Year: 2019
Page: [175 - 180]
Pages: 6
DOI: 10.2174/1570164615666180817100248
Price: $58

Article Metrics

PDF: 32
HTML: 5
EPUB: 2