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Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics
Protein-based therapeutics with cytosolic targets are capable of exhibiting their therapeutic effect once they have escaped from the endosomes or lysosomes. In this study, the reporters—horseradish peroxidase (HRP), Alexa Fluor 488 ((Alexa)) and ricin A-chain (RTA)—were investigated for their capaci...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4052257/ https://www.ncbi.nlm.nih.gov/pubmed/24859158 http://dx.doi.org/10.3390/toxins6051644 |
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author | Gilabert-Oriol, Roger Thakur, Mayank von Mallinckrodt, Benedicta Bhargava, Cheenu Wiesner, Burkhard Eichhorst, Jenny Melzig, Matthias F. Fuchs, Hendrik Weng, Alexander |
author_facet | Gilabert-Oriol, Roger Thakur, Mayank von Mallinckrodt, Benedicta Bhargava, Cheenu Wiesner, Burkhard Eichhorst, Jenny Melzig, Matthias F. Fuchs, Hendrik Weng, Alexander |
author_sort | Gilabert-Oriol, Roger |
collection | PubMed |
description | Protein-based therapeutics with cytosolic targets are capable of exhibiting their therapeutic effect once they have escaped from the endosomes or lysosomes. In this study, the reporters—horseradish peroxidase (HRP), Alexa Fluor 488 ((Alexa)) and ricin A-chain (RTA)—were investigated for their capacity to monitor the endo/lysosomal escape of the ribosome-inactivating protein, saporin. The conjugates—saporin-HRP, (Alexa)saporin and saporin-KQ-RTA—were constructed, and the endo/lysosomal escape of these conjugates alone (lack of endo/lysosomal release) or in combination with certain structurally-specific triterpenoidal saponins (efficient endo/lysosomal escape) was characterized. HRP failed in reporting the endo/lysosomal escape of saporin. Contrastingly, Alexa Fluor 488 successfully allowed the report of the process at a toxin concentration of 1000 nM. In addition, single endo/lysosome analysis facilitated the determination of the amount of (Alexa)saporin released from each vesicle. RTA was also successful in reporting the endo/lysosomal escape of the enzymatically inactive mutant, saporin-KQ, but in this case, the sensitivity of the method reached a toxin concentration of 10 nM. In conclusion, the simultaneous usage of Alexa Fluor 488 and RTA as reporters may provide the possibility of monitoring the endo/lysosomal escape of protein-based therapeutics in the concentration range of 10–1000 nM. |
format | Online Article Text |
id | pubmed-4052257 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-40522572014-06-11 Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics Gilabert-Oriol, Roger Thakur, Mayank von Mallinckrodt, Benedicta Bhargava, Cheenu Wiesner, Burkhard Eichhorst, Jenny Melzig, Matthias F. Fuchs, Hendrik Weng, Alexander Toxins (Basel) Article Protein-based therapeutics with cytosolic targets are capable of exhibiting their therapeutic effect once they have escaped from the endosomes or lysosomes. In this study, the reporters—horseradish peroxidase (HRP), Alexa Fluor 488 ((Alexa)) and ricin A-chain (RTA)—were investigated for their capacity to monitor the endo/lysosomal escape of the ribosome-inactivating protein, saporin. The conjugates—saporin-HRP, (Alexa)saporin and saporin-KQ-RTA—were constructed, and the endo/lysosomal escape of these conjugates alone (lack of endo/lysosomal release) or in combination with certain structurally-specific triterpenoidal saponins (efficient endo/lysosomal escape) was characterized. HRP failed in reporting the endo/lysosomal escape of saporin. Contrastingly, Alexa Fluor 488 successfully allowed the report of the process at a toxin concentration of 1000 nM. In addition, single endo/lysosome analysis facilitated the determination of the amount of (Alexa)saporin released from each vesicle. RTA was also successful in reporting the endo/lysosomal escape of the enzymatically inactive mutant, saporin-KQ, but in this case, the sensitivity of the method reached a toxin concentration of 10 nM. In conclusion, the simultaneous usage of Alexa Fluor 488 and RTA as reporters may provide the possibility of monitoring the endo/lysosomal escape of protein-based therapeutics in the concentration range of 10–1000 nM. MDPI 2014-05-22 /pmc/articles/PMC4052257/ /pubmed/24859158 http://dx.doi.org/10.3390/toxins6051644 Text en © 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). |
spellingShingle | Article Gilabert-Oriol, Roger Thakur, Mayank von Mallinckrodt, Benedicta Bhargava, Cheenu Wiesner, Burkhard Eichhorst, Jenny Melzig, Matthias F. Fuchs, Hendrik Weng, Alexander Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics |
title | Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics |
title_full | Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics |
title_fullStr | Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics |
title_full_unstemmed | Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics |
title_short | Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics |
title_sort | reporter assay for endo/lysosomal escape of toxin-based therapeutics |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4052257/ https://www.ncbi.nlm.nih.gov/pubmed/24859158 http://dx.doi.org/10.3390/toxins6051644 |
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