Structure–Activity Relationship of the Dimeric and Oligomeric Forms of a Cytotoxic Biotherapeutic Based on Diphtheria Toxin
Protein aggregation is a well-recognized problem in industrial preparation, including biotherapeutics. These low-energy states constantly compete with a native-like conformation, which is more pronounced in the case of macromolecules of low stability in the solution. A better understanding of the st...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-08-01
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| Series: | Biomolecules |
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| Online Access: | https://www.mdpi.com/2218-273X/12/8/1111 |
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| author | Marcin Mielecki Marcin Ziemniak Magdalena Ozga Radosław Borowski Jarosław Antosik Angelika Kaczyńska Beata Pająk |
| author_facet | Marcin Mielecki Marcin Ziemniak Magdalena Ozga Radosław Borowski Jarosław Antosik Angelika Kaczyńska Beata Pająk |
| author_sort | Marcin Mielecki |
| collection | DOAJ |
| description | Protein aggregation is a well-recognized problem in industrial preparation, including biotherapeutics. These low-energy states constantly compete with a native-like conformation, which is more pronounced in the case of macromolecules of low stability in the solution. A better understanding of the structure and function of such aggregates is generally required for the more rational development of therapeutic proteins, including single-chain fusion cytotoxins to target specific receptors on cancer cells. Here, we identified and purified such particles as side products of the renaturation process of the single-chain fusion cytotoxin, composed of two diphtheria toxin (DT) domains and interleukin 13 (IL-13), and applied various experimental techniques to comprehensively understand their molecular architecture and function. Importantly, we distinguished soluble purified dimeric and fractionated oligomeric particles from aggregates. The oligomers are polydisperse and multimodal, with a distribution favoring lower and even stoichiometries, suggesting they are composed of dimeric building units. Importantly, all these oligomeric particles and the monomer are cystine-dependent as their innate disulfide bonds have structural and functional roles. Their reduction triggers aggregation. Presumably the dimer and lower oligomers represent the metastable state, retaining the native disulfide bond. Although significantly reduced in contrast to the monomer, they preserve some fraction of bioactivity, manifested by their IL-13RA2 receptor affinity and selective cytotoxic potency towards the U-251 glioblastoma cell line. These molecular assemblies probably preserve structural integrity and native-like fold, at least to some extent. As our study demonstrated, the dimeric and oligomeric cytotoxin may be an exciting model protein, introducing a new understanding of its monomeric counterpart’s molecular characteristics. |
| first_indexed | 2024-03-09T04:39:41Z |
| format | Article |
| id | doaj.art-16e5a8f46d36429daa0f92672fe79a37 |
| institution | Directory Open Access Journal |
| issn | 2218-273X |
| language | English |
| last_indexed | 2024-03-09T04:39:41Z |
| publishDate | 2022-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomolecules |
| spelling | doaj.art-16e5a8f46d36429daa0f92672fe79a372023-12-03T13:22:50ZengMDPI AGBiomolecules2218-273X2022-08-01128111110.3390/biom12081111Structure–Activity Relationship of the Dimeric and Oligomeric Forms of a Cytotoxic Biotherapeutic Based on Diphtheria ToxinMarcin Mielecki0Marcin Ziemniak1Magdalena Ozga2Radosław Borowski3Jarosław Antosik4Angelika Kaczyńska5Beata Pająk6WPD Pharmaceuticals, Żwirki and Wigury 101, 02-089 Warsaw, PolandWPD Pharmaceuticals, Żwirki and Wigury 101, 02-089 Warsaw, PolandWPD Pharmaceuticals, Żwirki and Wigury 101, 02-089 Warsaw, PolandWPD Pharmaceuticals, Żwirki and Wigury 101, 02-089 Warsaw, PolandWPD Pharmaceuticals, Żwirki and Wigury 101, 02-089 Warsaw, PolandWPD Pharmaceuticals, Żwirki and Wigury 101, 02-089 Warsaw, PolandWPD Pharmaceuticals, Żwirki and Wigury 101, 02-089 Warsaw, PolandProtein aggregation is a well-recognized problem in industrial preparation, including biotherapeutics. These low-energy states constantly compete with a native-like conformation, which is more pronounced in the case of macromolecules of low stability in the solution. A better understanding of the structure and function of such aggregates is generally required for the more rational development of therapeutic proteins, including single-chain fusion cytotoxins to target specific receptors on cancer cells. Here, we identified and purified such particles as side products of the renaturation process of the single-chain fusion cytotoxin, composed of two diphtheria toxin (DT) domains and interleukin 13 (IL-13), and applied various experimental techniques to comprehensively understand their molecular architecture and function. Importantly, we distinguished soluble purified dimeric and fractionated oligomeric particles from aggregates. The oligomers are polydisperse and multimodal, with a distribution favoring lower and even stoichiometries, suggesting they are composed of dimeric building units. Importantly, all these oligomeric particles and the monomer are cystine-dependent as their innate disulfide bonds have structural and functional roles. Their reduction triggers aggregation. Presumably the dimer and lower oligomers represent the metastable state, retaining the native disulfide bond. Although significantly reduced in contrast to the monomer, they preserve some fraction of bioactivity, manifested by their IL-13RA2 receptor affinity and selective cytotoxic potency towards the U-251 glioblastoma cell line. These molecular assemblies probably preserve structural integrity and native-like fold, at least to some extent. As our study demonstrated, the dimeric and oligomeric cytotoxin may be an exciting model protein, introducing a new understanding of its monomeric counterpart’s molecular characteristics.https://www.mdpi.com/2218-273X/12/8/1111biotherapeuticscytotoxindiphtheria toxinIL-13inclusion bodiesrefolding |
| spellingShingle | Marcin Mielecki Marcin Ziemniak Magdalena Ozga Radosław Borowski Jarosław Antosik Angelika Kaczyńska Beata Pająk Structure–Activity Relationship of the Dimeric and Oligomeric Forms of a Cytotoxic Biotherapeutic Based on Diphtheria Toxin Biomolecules biotherapeutics cytotoxin diphtheria toxin IL-13 inclusion bodies refolding |
| title | Structure–Activity Relationship of the Dimeric and Oligomeric Forms of a Cytotoxic Biotherapeutic Based on Diphtheria Toxin |
| title_full | Structure–Activity Relationship of the Dimeric and Oligomeric Forms of a Cytotoxic Biotherapeutic Based on Diphtheria Toxin |
| title_fullStr | Structure–Activity Relationship of the Dimeric and Oligomeric Forms of a Cytotoxic Biotherapeutic Based on Diphtheria Toxin |
| title_full_unstemmed | Structure–Activity Relationship of the Dimeric and Oligomeric Forms of a Cytotoxic Biotherapeutic Based on Diphtheria Toxin |
| title_short | Structure–Activity Relationship of the Dimeric and Oligomeric Forms of a Cytotoxic Biotherapeutic Based on Diphtheria Toxin |
| title_sort | structure activity relationship of the dimeric and oligomeric forms of a cytotoxic biotherapeutic based on diphtheria toxin |
| topic | biotherapeutics cytotoxin diphtheria toxin IL-13 inclusion bodies refolding |
| url | https://www.mdpi.com/2218-273X/12/8/1111 |
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