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Different N-Glycosylation Sites Reduce the Activity of Recombinant DSPAα2
Bat plasminogen activators α2 (DSPAα2) has extremely high medicinal value as a powerful natural thrombolytic protein. However, wild-type DSPAα2 has two N-glycosylation sites (N185 and N398) and its non-human classes of high-mannose-type N-glycans may cause immune responses in vivo. By mutating the N...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9497888/ https://www.ncbi.nlm.nih.gov/pubmed/36135182 http://dx.doi.org/10.3390/cimb44090270 |
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author | Peng, Huakang Wang, Mengqi Wang, Nan Yang, Caifeng Guo, Wenfang Li, Gangqiang Huang, Sumei Wei, Di Liu, Dehu |
author_facet | Peng, Huakang Wang, Mengqi Wang, Nan Yang, Caifeng Guo, Wenfang Li, Gangqiang Huang, Sumei Wei, Di Liu, Dehu |
author_sort | Peng, Huakang |
collection | PubMed |
description | Bat plasminogen activators α2 (DSPAα2) has extremely high medicinal value as a powerful natural thrombolytic protein. However, wild-type DSPAα2 has two N-glycosylation sites (N185 and N398) and its non-human classes of high-mannose-type N-glycans may cause immune responses in vivo. By mutating the N-glycosylation sites, we aimed to study the effect of its N-glycan chain on plasminogen activation, fibrin sensitivity, and to observe the physicochemical properties of DSPAα2. A logical structure design was performed in this study. Four single mutants and one double mutant were constructed and expressed in Pichia pastoris. When the N398 site was eliminated, the plasminogen activator in the mutants had their activities reduced to ~40%. When the N185 site was inactivated, there was a weak decrease in the plasminogen activation of its mutant, while the fibrin sensitivity significantly decreased by ~10-fold. Neither N-glycosylation nor deglycosylation mutations changed the pH resistance or heat resistance of DSPAα2. This study confirms that N-glycosylation affects the biochemical function of DSPAα2, which provides a reference for subsequent applications of DSPAα2. |
format | Online Article Text |
id | pubmed-9497888 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-94978882022-09-23 Different N-Glycosylation Sites Reduce the Activity of Recombinant DSPAα2 Peng, Huakang Wang, Mengqi Wang, Nan Yang, Caifeng Guo, Wenfang Li, Gangqiang Huang, Sumei Wei, Di Liu, Dehu Curr Issues Mol Biol Article Bat plasminogen activators α2 (DSPAα2) has extremely high medicinal value as a powerful natural thrombolytic protein. However, wild-type DSPAα2 has two N-glycosylation sites (N185 and N398) and its non-human classes of high-mannose-type N-glycans may cause immune responses in vivo. By mutating the N-glycosylation sites, we aimed to study the effect of its N-glycan chain on plasminogen activation, fibrin sensitivity, and to observe the physicochemical properties of DSPAα2. A logical structure design was performed in this study. Four single mutants and one double mutant were constructed and expressed in Pichia pastoris. When the N398 site was eliminated, the plasminogen activator in the mutants had their activities reduced to ~40%. When the N185 site was inactivated, there was a weak decrease in the plasminogen activation of its mutant, while the fibrin sensitivity significantly decreased by ~10-fold. Neither N-glycosylation nor deglycosylation mutations changed the pH resistance or heat resistance of DSPAα2. This study confirms that N-glycosylation affects the biochemical function of DSPAα2, which provides a reference for subsequent applications of DSPAα2. MDPI 2022-08-31 /pmc/articles/PMC9497888/ /pubmed/36135182 http://dx.doi.org/10.3390/cimb44090270 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Peng, Huakang Wang, Mengqi Wang, Nan Yang, Caifeng Guo, Wenfang Li, Gangqiang Huang, Sumei Wei, Di Liu, Dehu Different N-Glycosylation Sites Reduce the Activity of Recombinant DSPAα2 |
title | Different N-Glycosylation Sites Reduce the Activity of Recombinant DSPAα2 |
title_full | Different N-Glycosylation Sites Reduce the Activity of Recombinant DSPAα2 |
title_fullStr | Different N-Glycosylation Sites Reduce the Activity of Recombinant DSPAα2 |
title_full_unstemmed | Different N-Glycosylation Sites Reduce the Activity of Recombinant DSPAα2 |
title_short | Different N-Glycosylation Sites Reduce the Activity of Recombinant DSPAα2 |
title_sort | different n-glycosylation sites reduce the activity of recombinant dspaα2 |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9497888/ https://www.ncbi.nlm.nih.gov/pubmed/36135182 http://dx.doi.org/10.3390/cimb44090270 |
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