A Purified Aspartic Protease from Akkermansia Muciniphila Plays an Important Role in Degrading Muc2
Abstract
:1. Introduction
2. Results
2.1. Purification of Amuc_1434*
2.2. Amuc_1434* Activity Test and Kinetic Study
2.3. Effect of pH on the Activity of Amuc_1434*
2.4. Effect of Temperature on the Activity and Half-Life of Amuc_1434*
2.5. Effects of Inhibitors on Amuc_1434*
2.6. Association between Amuc_1434* and Muc2
2.7. Amuc_1434* Degradation of Muc2
2.8. Amuc_1434 Localization
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Induction and Expression of Proteins
4.3. Purification and Identification of Proteins
4.4. Western Blot
4.5. Activity Test of Purified Amuc_1434*
4.6. Kinetics of Amuc_1434* Catalyzed Reactions
4.7. Effect of pH and Temperature on Amuc_1434* Activity
4.8. Half-Life Study
4.9. Effect of Inhibitors on Proteolytic Activity
4.10. Cell Attachment Assay
4.11. Muc2 Degradation Experiment
4.12. Sandwich Enzyme-Linked Immunosorbent Assay (ELISA)
4.13. Muc2 Immunofluorescence
4.14. Immunohistochemistry
4.15. Statistical Analysis
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
BCA | bicinchonininc acid |
BSA | bovine serum albumin |
CRC | colorectal cancer |
DAB | diaminobenzidine |
DIO | 3,3′-dioctadecyloxacarbocyanine perchlorate |
DMSO | dimethyl sulfoxide |
ECL | electrochemiluminescence |
EDTA | ethylene Diamine Tetraacetic Acid |
ELISA | enzyme–linked immunosorbent assay |
EP | eppendorf |
HRP | horseradish peroxidase |
IPTG | isopropyl β-D-Thiogalactoside |
MW | molecular weight |
Mucin 2 | Muc2 |
PBS | phosphate buffer saline |
PE | phycoerythrin |
PMP | pseudomyxoma peritonei |
PMSF | phenylmethylsulphonyl fluoride |
PAGE | polyacrylamide gel electrophoresis |
RIPA | radio immunoprecipitation assay |
SDS | sodium dodecylsulphate |
TMB | 3,3′,5,5′-Tetramethylbenzidine |
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Compound | Concentration (mM) | Relative Activity (%) |
---|---|---|
None | - | 100.0 ± 9.1 |
EDTA | 10.0 | 149.9 ± 14.4 |
PMSF | 1.0 | 126.9 ± 2.6 |
Pepstatin A | 0.02 | 40.15 ± 6.7 |
E-64 | 0.01 | 88.5 ± 4.7 |
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Meng, X.; Wang, W.; Lan, T.; Yang, W.; Yu, D.; Fang, X.; Wu, H. A Purified Aspartic Protease from Akkermansia Muciniphila Plays an Important Role in Degrading Muc2. Int. J. Mol. Sci. 2020, 21, 72. https://doi.org/10.3390/ijms21010072
Meng X, Wang W, Lan T, Yang W, Yu D, Fang X, Wu H. A Purified Aspartic Protease from Akkermansia Muciniphila Plays an Important Role in Degrading Muc2. International Journal of Molecular Sciences. 2020; 21(1):72. https://doi.org/10.3390/ijms21010072
Chicago/Turabian StyleMeng, Xin, Wencheng Wang, Tianqi Lan, Wanxin Yang, Dahai Yu, Xuexun Fang, and Hao Wu. 2020. "A Purified Aspartic Protease from Akkermansia Muciniphila Plays an Important Role in Degrading Muc2" International Journal of Molecular Sciences 21, no. 1: 72. https://doi.org/10.3390/ijms21010072