The <i>Clostridium botulinum</i> C2 Toxin Subunit C2IIa Delivers Enzymes with Positively Charged N-Termini into the Cytosol of Target Cells
The binary <i>Clostridium</i> (<i>C.</i>) <i>botulinum</i> C2 toxin consists of two non-linked proteins. The proteolytically activated binding/transport subunit C2IIa forms barrel-shaped homoheptamers, which bind to cell surface receptors, mediate endocytosis, and...
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| Format: | Article |
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MDPI AG
2023-06-01
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| Series: | Toxins |
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| Online Access: | https://www.mdpi.com/2072-6651/15/6/390 |
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| author | Sebastian Heber Joscha Borho Nicole Stadler Fanny Wondany Irina König Jens Michaelis Panagiotis Papatheodorou Holger Barth Maximilian Fellermann |
| author_facet | Sebastian Heber Joscha Borho Nicole Stadler Fanny Wondany Irina König Jens Michaelis Panagiotis Papatheodorou Holger Barth Maximilian Fellermann |
| author_sort | Sebastian Heber |
| collection | DOAJ |
| description | The binary <i>Clostridium</i> (<i>C.</i>) <i>botulinum</i> C2 toxin consists of two non-linked proteins. The proteolytically activated binding/transport subunit C2IIa forms barrel-shaped homoheptamers, which bind to cell surface receptors, mediate endocytosis, and translocate the enzyme subunit C2I into the cytosol of target cells. Here, we investigate whether C2IIa can be harnessed as a transporter for proteins/enzymes fused to polycationic tags, as earlier demonstrated for the related anthrax toxin transport subunit PA<sub>63</sub>. To test C2IIa-mediated transport in cultured cells, reporter enzymes are generated by fusing different polycationic tags to the N- or C-terminus of other bacterial toxins’ catalytic A subunits. C2IIa as well as PA<sub>63</sub> deliver N-terminally polyhistidine-tagged proteins more efficiently compared to C-terminally tagged ones. However, in contrast to PA<sub>63</sub>, C2IIa does not efficiently deliver polylysine-tagged proteins into the cytosol of target cells. Moreover, untagged enzymes with a native cationic N-terminus are efficiently transported by both C2IIa and PA<sub>63</sub>. In conclusion, the C2IIa-transporter serves as a transport system for enzymes that harbor positively charged amino acids at their N-terminus. The charge distribution at the N-terminus of cargo proteins and their ability to unfold in the endosome and subsequently refold in the cytosol determine transport feasibility and efficiency. |
| first_indexed | 2024-03-11T01:52:04Z |
| format | Article |
| id | doaj.art-66b5f7fb68b34416971626e785278fd1 |
| institution | Directory Open Access Journal |
| issn | 2072-6651 |
| language | English |
| last_indexed | 2024-03-11T01:52:04Z |
| publishDate | 2023-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Toxins |
| spelling | doaj.art-66b5f7fb68b34416971626e785278fd12023-11-18T12:55:43ZengMDPI AGToxins2072-66512023-06-0115639010.3390/toxins15060390The <i>Clostridium botulinum</i> C2 Toxin Subunit C2IIa Delivers Enzymes with Positively Charged N-Termini into the Cytosol of Target CellsSebastian Heber0Joscha Borho1Nicole Stadler2Fanny Wondany3Irina König4Jens Michaelis5Panagiotis Papatheodorou6Holger Barth7Maximilian Fellermann8Institute of Experimental and Clinical Pharmacology, Toxicology and Pharmacology of Natural Products, Ulm University Medical Center, 89081 Ulm, GermanyInstitute of Experimental and Clinical Pharmacology, Toxicology and Pharmacology of Natural Products, Ulm University Medical Center, 89081 Ulm, GermanyInstitute of Experimental and Clinical Pharmacology, Toxicology and Pharmacology of Natural Products, Ulm University Medical Center, 89081 Ulm, GermanyInstitute of Biophysics, Ulm University, 89081 Ulm, GermanyInstitute of Experimental and Clinical Pharmacology, Toxicology and Pharmacology of Natural Products, Ulm University Medical Center, 89081 Ulm, GermanyInstitute of Biophysics, Ulm University, 89081 Ulm, GermanyInstitute of Experimental and Clinical Pharmacology, Toxicology and Pharmacology of Natural Products, Ulm University Medical Center, 89081 Ulm, GermanyInstitute of Experimental and Clinical Pharmacology, Toxicology and Pharmacology of Natural Products, Ulm University Medical Center, 89081 Ulm, GermanyInstitute of Experimental and Clinical Pharmacology, Toxicology and Pharmacology of Natural Products, Ulm University Medical Center, 89081 Ulm, GermanyThe binary <i>Clostridium</i> (<i>C.</i>) <i>botulinum</i> C2 toxin consists of two non-linked proteins. The proteolytically activated binding/transport subunit C2IIa forms barrel-shaped homoheptamers, which bind to cell surface receptors, mediate endocytosis, and translocate the enzyme subunit C2I into the cytosol of target cells. Here, we investigate whether C2IIa can be harnessed as a transporter for proteins/enzymes fused to polycationic tags, as earlier demonstrated for the related anthrax toxin transport subunit PA<sub>63</sub>. To test C2IIa-mediated transport in cultured cells, reporter enzymes are generated by fusing different polycationic tags to the N- or C-terminus of other bacterial toxins’ catalytic A subunits. C2IIa as well as PA<sub>63</sub> deliver N-terminally polyhistidine-tagged proteins more efficiently compared to C-terminally tagged ones. However, in contrast to PA<sub>63</sub>, C2IIa does not efficiently deliver polylysine-tagged proteins into the cytosol of target cells. Moreover, untagged enzymes with a native cationic N-terminus are efficiently transported by both C2IIa and PA<sub>63</sub>. In conclusion, the C2IIa-transporter serves as a transport system for enzymes that harbor positively charged amino acids at their N-terminus. The charge distribution at the N-terminus of cargo proteins and their ability to unfold in the endosome and subsequently refold in the cytosol determine transport feasibility and efficiency.https://www.mdpi.com/2072-6651/15/6/390bacterial AB-type protein toxinscytosolic drug deliverymolecular Trojan horse<i>Clostridium botulinum</i> C2 toxinC2IIapolyhistidine-tag |
| spellingShingle | Sebastian Heber Joscha Borho Nicole Stadler Fanny Wondany Irina König Jens Michaelis Panagiotis Papatheodorou Holger Barth Maximilian Fellermann The <i>Clostridium botulinum</i> C2 Toxin Subunit C2IIa Delivers Enzymes with Positively Charged N-Termini into the Cytosol of Target Cells Toxins bacterial AB-type protein toxins cytosolic drug delivery molecular Trojan horse <i>Clostridium botulinum</i> C2 toxin C2IIa polyhistidine-tag |
| title | The <i>Clostridium botulinum</i> C2 Toxin Subunit C2IIa Delivers Enzymes with Positively Charged N-Termini into the Cytosol of Target Cells |
| title_full | The <i>Clostridium botulinum</i> C2 Toxin Subunit C2IIa Delivers Enzymes with Positively Charged N-Termini into the Cytosol of Target Cells |
| title_fullStr | The <i>Clostridium botulinum</i> C2 Toxin Subunit C2IIa Delivers Enzymes with Positively Charged N-Termini into the Cytosol of Target Cells |
| title_full_unstemmed | The <i>Clostridium botulinum</i> C2 Toxin Subunit C2IIa Delivers Enzymes with Positively Charged N-Termini into the Cytosol of Target Cells |
| title_short | The <i>Clostridium botulinum</i> C2 Toxin Subunit C2IIa Delivers Enzymes with Positively Charged N-Termini into the Cytosol of Target Cells |
| title_sort | i clostridium botulinum i c2 toxin subunit c2iia delivers enzymes with positively charged n termini into the cytosol of target cells |
| topic | bacterial AB-type protein toxins cytosolic drug delivery molecular Trojan horse <i>Clostridium botulinum</i> C2 toxin C2IIa polyhistidine-tag |
| url | https://www.mdpi.com/2072-6651/15/6/390 |
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