Characterization, protein modeling, and molecular docking of factor C from Indonesian horseshoe crab (Tachypleus gigas)
Abstract Background Horseshoe crab (Tachypleus gigas) amebocytes are useful biomedical components for endotoxin detection, and their growing needs for biomedical purposes cause the horseshoe crab population to decline. Factor C synthesis via genetic engineering offers a solution to replace natural h...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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SpringerOpen
2023-04-01
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| Series: | Journal of Genetic Engineering and Biotechnology |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s43141-023-00496-8 |
| _version_ | 1797845793119404032 |
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| author | Apon Zaenal Mustopa Ayu Fitri Izaki Suharsono Suharsono Fatimah Fatimah Fauziyah Fauziyah Rahmi Damarani Arwansyah Arwansyah Setyanto Tri Wahyudi Siswi Sekar Sari Rozirwan Rozirwan Zubaidi Bachtiar |
| author_facet | Apon Zaenal Mustopa Ayu Fitri Izaki Suharsono Suharsono Fatimah Fatimah Fauziyah Fauziyah Rahmi Damarani Arwansyah Arwansyah Setyanto Tri Wahyudi Siswi Sekar Sari Rozirwan Rozirwan Zubaidi Bachtiar |
| author_sort | Apon Zaenal Mustopa |
| collection | DOAJ |
| description | Abstract Background Horseshoe crab (Tachypleus gigas) amebocytes are useful biomedical components for endotoxin detection, and their growing needs for biomedical purposes cause the horseshoe crab population to decline. Factor C synthesis via genetic engineering offers a solution to replace natural horseshoe crab’s factor C and prevent its excessive harvest from nature. In response to these concerns, this study aimed to characterize the amebocyte lysates and factor C protein modeling of T. gigas originated from Banyuasin South Sumatra Estuary. Methods and results Sampling of T. gigas was carried out in Banyuasin South Sumatra Estuary, Indonesia. The endotoxin test or TAL (Tachypleus amebocyte lysates) assay was performed using gel coagulation method. Protein characterization of protease enzyme was conducted by protease activity, SDS-PAGE, and zymogram analysis. The cDNA of mitochondrial COI gene was amplified for molecular identification followed by cDNA cloning of factor C. Protein modeling was investigated by molecular docking and molecular dynamic (MD) simulation. Endotoxin test results showed that TAL-35 had endotoxin sensitivity in a range of 0.0156–1 EU/ml, while TAL 36 had a sensitivity between 00,625 and 1 EU/ml. T. gigas amebocytes have protease activity in molecular mass sizes less than 60 kDa, with 367 U/ml for TAL 35 and 430 U/ml for TAL 36. The molecular identification revealed 98.68% identity similarity to T. gigas. The docking results suggested three ligands; i.e., diphosphoryl lipid A, core lipid A, and Kdo2 lipid A can be activators of the factor C protein by binding to the region of the receptor to form a ligand-receptor complex. Conclusions Endotoxins can be detected using horseshoe crab amebocytes. The presence of proteases is considered responsible for this ability, as evidenced by casein zymogram results. According to docking and MD analysis, we found that lipopolysaccharides (LPS) participate to the binding site of factor C. |
| first_indexed | 2024-04-09T17:44:40Z |
| format | Article |
| id | doaj.art-19d5f5fa33584499b9fe218645f7d43c |
| institution | Directory Open Access Journal |
| issn | 2090-5920 |
| language | English |
| last_indexed | 2024-04-09T17:44:40Z |
| publishDate | 2023-04-01 |
| publisher | SpringerOpen |
| record_format | Article |
| series | Journal of Genetic Engineering and Biotechnology |
| spelling | doaj.art-19d5f5fa33584499b9fe218645f7d43c2023-04-16T11:22:00ZengSpringerOpenJournal of Genetic Engineering and Biotechnology2090-59202023-04-0121111510.1186/s43141-023-00496-8Characterization, protein modeling, and molecular docking of factor C from Indonesian horseshoe crab (Tachypleus gigas)Apon Zaenal Mustopa0Ayu Fitri Izaki1Suharsono Suharsono2Fatimah Fatimah3Fauziyah Fauziyah4Rahmi Damarani5Arwansyah Arwansyah6Setyanto Tri Wahyudi7Siswi Sekar Sari8Rozirwan Rozirwan9Zubaidi Bachtiar10Research Center for Genetic Engineering, National Research and Innovation Agency (BRIN)Research Center for Genetic Engineering, National Research and Innovation Agency (BRIN)School of Biotechnology, IPB UniversityResearch Center for Genetic Engineering, National Research and Innovation Agency (BRIN)Marine Science Study Program, Faculty of Mathematics and Natural Science, University of SriwijayaMarine Science Study Program, Faculty of Mathematics and Natural Science, University of SriwijayaDepartment of Chemistry Education, Faculty of Teacher Training and Education, Tadulako UniversityDepartment of Physics, Faculty of Mathematics and Natural Sciences, IPB UniversityResearch Center for Genetic Engineering, National Research and Innovation Agency (BRIN)Marine Science Study Program, Faculty of Mathematics and Natural Science, University of SriwijayaResearch Center for Genetic Engineering, National Research and Innovation Agency (BRIN)Abstract Background Horseshoe crab (Tachypleus gigas) amebocytes are useful biomedical components for endotoxin detection, and their growing needs for biomedical purposes cause the horseshoe crab population to decline. Factor C synthesis via genetic engineering offers a solution to replace natural horseshoe crab’s factor C and prevent its excessive harvest from nature. In response to these concerns, this study aimed to characterize the amebocyte lysates and factor C protein modeling of T. gigas originated from Banyuasin South Sumatra Estuary. Methods and results Sampling of T. gigas was carried out in Banyuasin South Sumatra Estuary, Indonesia. The endotoxin test or TAL (Tachypleus amebocyte lysates) assay was performed using gel coagulation method. Protein characterization of protease enzyme was conducted by protease activity, SDS-PAGE, and zymogram analysis. The cDNA of mitochondrial COI gene was amplified for molecular identification followed by cDNA cloning of factor C. Protein modeling was investigated by molecular docking and molecular dynamic (MD) simulation. Endotoxin test results showed that TAL-35 had endotoxin sensitivity in a range of 0.0156–1 EU/ml, while TAL 36 had a sensitivity between 00,625 and 1 EU/ml. T. gigas amebocytes have protease activity in molecular mass sizes less than 60 kDa, with 367 U/ml for TAL 35 and 430 U/ml for TAL 36. The molecular identification revealed 98.68% identity similarity to T. gigas. The docking results suggested three ligands; i.e., diphosphoryl lipid A, core lipid A, and Kdo2 lipid A can be activators of the factor C protein by binding to the region of the receptor to form a ligand-receptor complex. Conclusions Endotoxins can be detected using horseshoe crab amebocytes. The presence of proteases is considered responsible for this ability, as evidenced by casein zymogram results. According to docking and MD analysis, we found that lipopolysaccharides (LPS) participate to the binding site of factor C.https://doi.org/10.1186/s43141-023-00496-8Amebocyte lysateEndotoxinFactor CTachypleus gigasMolecular dynamic |
| spellingShingle | Apon Zaenal Mustopa Ayu Fitri Izaki Suharsono Suharsono Fatimah Fatimah Fauziyah Fauziyah Rahmi Damarani Arwansyah Arwansyah Setyanto Tri Wahyudi Siswi Sekar Sari Rozirwan Rozirwan Zubaidi Bachtiar Characterization, protein modeling, and molecular docking of factor C from Indonesian horseshoe crab (Tachypleus gigas) Journal of Genetic Engineering and Biotechnology Amebocyte lysate Endotoxin Factor C Tachypleus gigas Molecular dynamic |
| title | Characterization, protein modeling, and molecular docking of factor C from Indonesian horseshoe crab (Tachypleus gigas) |
| title_full | Characterization, protein modeling, and molecular docking of factor C from Indonesian horseshoe crab (Tachypleus gigas) |
| title_fullStr | Characterization, protein modeling, and molecular docking of factor C from Indonesian horseshoe crab (Tachypleus gigas) |
| title_full_unstemmed | Characterization, protein modeling, and molecular docking of factor C from Indonesian horseshoe crab (Tachypleus gigas) |
| title_short | Characterization, protein modeling, and molecular docking of factor C from Indonesian horseshoe crab (Tachypleus gigas) |
| title_sort | characterization protein modeling and molecular docking of factor c from indonesian horseshoe crab tachypleus gigas |
| topic | Amebocyte lysate Endotoxin Factor C Tachypleus gigas Molecular dynamic |
| url | https://doi.org/10.1186/s43141-023-00496-8 |
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