Usage of Cell-Free Protein Synthesis in Post-Translational Modification of μ-Conopeptide PIIIA
The post-translational modifications of conopeptides are the most complicated modifications to date and are well-known and closely related to the activity of conopeptides. The hydroxylation of proline in conopeptides affects folding, structure, and biological activity, and prolyl 4 hydroxylase has b...
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| Format: | Article |
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MDPI AG
2023-07-01
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| Series: | Marine Drugs |
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| Online Access: | https://www.mdpi.com/1660-3397/21/8/421 |
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| author | Yanli Liu Zitong Zhao Yunyang Song Yifeng Yin Fanghui Wu Hui Jiang |
| author_facet | Yanli Liu Zitong Zhao Yunyang Song Yifeng Yin Fanghui Wu Hui Jiang |
| author_sort | Yanli Liu |
| collection | DOAJ |
| description | The post-translational modifications of conopeptides are the most complicated modifications to date and are well-known and closely related to the activity of conopeptides. The hydroxylation of proline in conopeptides affects folding, structure, and biological activity, and prolyl 4 hydroxylase has been characterized in <i>Conus literatus</i>. However, the hydroxylation machinery of proline in conopeptides is still unclear. In order to address the hydroxylation mechanism of proline in μ-PIIIA, three recombinant plasmids encoding different hybrid precursors of μ-PIIIA were constructed and crossly combined with protein disulfide isomerase, prolyl 4 hydroxylase, and glutaminyl cyclase in a continuous exchange cell-free protein system. The findings showed that prolyl 4 hydroxylase might recognize the propeptide of μ-PIIIA to achieve the hydroxylation of proline, while the cyclization of glutamate was also formed. Additionally, in <i>Escherichia coli,</i> the co-expression plasmid encoding prolyl 4 hydroxylase and the precursor of μ-PIIIA containing pro and mature regions were used to validate the continuous exchange cell-free protein system. Surprisingly, in addition to the two hydroxyproline residues and one pyroglutamyl residue, three disulfide bridges were formed using Trx as a fusion tag, and the yield of the fusion peptide was approximately 20 mg/L. The results of electrophysiology analysis indicated that the recombinant μ-PIIIA without C-terminal amidate inhibited the current of hNa<sub>V</sub>1.4 with a 939 nM IC<sub>50</sub>. Our work solved the issue that it was challenging to quickly generate post-translationally modified conopeptides in vitro. This is the first study to demonstrate that prolyl 4 hydroxylase catalyzes the proline hydroxylation through recognition in the propeptide of μ-PIIIA, and it will provide a new way for synthesizing multi-modified conopeptides with pharmacological activity. |
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| language | English |
| last_indexed | 2024-03-10T23:47:16Z |
| publishDate | 2023-07-01 |
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| series | Marine Drugs |
| spelling | doaj.art-2f2ec2b090a84eb9a94074f7d16d28d42023-11-19T01:57:46ZengMDPI AGMarine Drugs1660-33972023-07-0121842110.3390/md21080421Usage of Cell-Free Protein Synthesis in Post-Translational Modification of μ-Conopeptide PIIIAYanli Liu0Zitong Zhao1Yunyang Song2Yifeng Yin3Fanghui Wu4Hui Jiang5State Key Laboratory of NBC Protection for Civilian, Beijing 102205, ChinaState Key Laboratory of NBC Protection for Civilian, Beijing 102205, ChinaState Key Laboratory of NBC Protection for Civilian, Beijing 102205, ChinaState Key Laboratory of NBC Protection for Civilian, Beijing 102205, ChinaState Key Laboratory of NBC Protection for Civilian, Beijing 102205, ChinaState Key Laboratory of NBC Protection for Civilian, Beijing 102205, ChinaThe post-translational modifications of conopeptides are the most complicated modifications to date and are well-known and closely related to the activity of conopeptides. The hydroxylation of proline in conopeptides affects folding, structure, and biological activity, and prolyl 4 hydroxylase has been characterized in <i>Conus literatus</i>. However, the hydroxylation machinery of proline in conopeptides is still unclear. In order to address the hydroxylation mechanism of proline in μ-PIIIA, three recombinant plasmids encoding different hybrid precursors of μ-PIIIA were constructed and crossly combined with protein disulfide isomerase, prolyl 4 hydroxylase, and glutaminyl cyclase in a continuous exchange cell-free protein system. The findings showed that prolyl 4 hydroxylase might recognize the propeptide of μ-PIIIA to achieve the hydroxylation of proline, while the cyclization of glutamate was also formed. Additionally, in <i>Escherichia coli,</i> the co-expression plasmid encoding prolyl 4 hydroxylase and the precursor of μ-PIIIA containing pro and mature regions were used to validate the continuous exchange cell-free protein system. Surprisingly, in addition to the two hydroxyproline residues and one pyroglutamyl residue, three disulfide bridges were formed using Trx as a fusion tag, and the yield of the fusion peptide was approximately 20 mg/L. The results of electrophysiology analysis indicated that the recombinant μ-PIIIA without C-terminal amidate inhibited the current of hNa<sub>V</sub>1.4 with a 939 nM IC<sub>50</sub>. Our work solved the issue that it was challenging to quickly generate post-translationally modified conopeptides in vitro. This is the first study to demonstrate that prolyl 4 hydroxylase catalyzes the proline hydroxylation through recognition in the propeptide of μ-PIIIA, and it will provide a new way for synthesizing multi-modified conopeptides with pharmacological activity.https://www.mdpi.com/1660-3397/21/8/421conopeptidepost-translational modificationhydroxylationcell-free protein system |
| spellingShingle | Yanli Liu Zitong Zhao Yunyang Song Yifeng Yin Fanghui Wu Hui Jiang Usage of Cell-Free Protein Synthesis in Post-Translational Modification of μ-Conopeptide PIIIA Marine Drugs conopeptide post-translational modification hydroxylation cell-free protein system |
| title | Usage of Cell-Free Protein Synthesis in Post-Translational Modification of μ-Conopeptide PIIIA |
| title_full | Usage of Cell-Free Protein Synthesis in Post-Translational Modification of μ-Conopeptide PIIIA |
| title_fullStr | Usage of Cell-Free Protein Synthesis in Post-Translational Modification of μ-Conopeptide PIIIA |
| title_full_unstemmed | Usage of Cell-Free Protein Synthesis in Post-Translational Modification of μ-Conopeptide PIIIA |
| title_short | Usage of Cell-Free Protein Synthesis in Post-Translational Modification of μ-Conopeptide PIIIA |
| title_sort | usage of cell free protein synthesis in post translational modification of μ conopeptide piiia |
| topic | conopeptide post-translational modification hydroxylation cell-free protein system |
| url | https://www.mdpi.com/1660-3397/21/8/421 |
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