Identification, Biochemical Characterization, and In Vivo Detection of a Zn-Metalloprotease with Collagenase Activity from Mannheimia haemolytica A2
Abstract
:1. Introduction
2. Results
2.1. Zymography of Proteins Precipitated with Ammonium Sulfate (PpAs) from the CS of M. haemolytica A2
2.2. Determination of the Proteolytic Activity of 60% PpAs from M. haemolytica A2 against Several Mammalian Proteins
2.3. The 110-kDa Mh Protease Was Purified through Cationic Exchange Chromatography
2.4. The 110 kDa Protease of M. haemolytica A2 Is a Heat-Resistant Enzyme That Possesses Collagenolytic Activity
2.5. The 110 kDa Protease with Collagenase Activity Secreted by M. haemolytica A2 Belongs to the Zn-Dependent Family of Metalloproteases
2.6. The 110 kDa-Mh Protease Was Confirmed as a Zn-Dependent Metalloprotease through Mass Spectrometry
2.7. Prediction of the 3D Structure and Catalytic Site of the 110-Mh Metalloprotease
2.8. Anti-110-Mh Metalloprotease Antibodies Inhibit Its Proteolytic Activity
2.9. Identification of M. haemolytica A2 from a Suggestive Case of Mannheimiosis in Sheep through Histopathology and PCR
2.10. Detection of the 110-Mh Metalloprotease in Lung Samples from Sheep with Suggestive Pneumonic Damage of M. haemolytica
3. Discussion
4. Materials and Methods
4.1. Bacterial Strains
4.2. Precipitation of Proteins from CS with (NH4)2SO4
4.3. Zymography Assays of the PpAs from CS of M. haemolytica A2
4.4. Purification of the 110 kDa Protease by Ion Exchange Chromatography
4.5. Proteolytic Degradation of Bovine Collagen Type I by the 110 kDa Protease of M. haemolytica A2
4.6. Characterization of the Proteolytic Activity of 110 kDa by Using Protease Inhibitors
4.7. Preparation of Sample for LC-MS/MS Mass Spectrometry
4.8. LC-MS/MS Mass Spectrometry
4.9. Database Search
4.10. Prediction of the 3D Structure and Catalytic Site of the 110-Mh Metalloprotease
4.11. Production of Anti-Protease Serum, Western Blotting, and Inhibition of 110-Mh Metalloprotease Activity by Antibodies
4.12. Determination and Characterization of M. haemolytica A2 Isolates from Lungs
4.13. Histopathology, Immunohistochemistry, and Immunofluorescence
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Ramírez-Rico, G.; Martinez-Castillo, M.; Ruiz-Mazón, L.; Meneses-Romero, E.P.; Palacios, J.A.F.; Díaz-Aparicio, E.; Abascal, E.N.; de la Garza, M. Identification, Biochemical Characterization, and In Vivo Detection of a Zn-Metalloprotease with Collagenase Activity from Mannheimia haemolytica A2. Int. J. Mol. Sci. 2024, 25, 1289. https://doi.org/10.3390/ijms25021289
Ramírez-Rico G, Martinez-Castillo M, Ruiz-Mazón L, Meneses-Romero EP, Palacios JAF, Díaz-Aparicio E, Abascal EN, de la Garza M. Identification, Biochemical Characterization, and In Vivo Detection of a Zn-Metalloprotease with Collagenase Activity from Mannheimia haemolytica A2. International Journal of Molecular Sciences. 2024; 25(2):1289. https://doi.org/10.3390/ijms25021289
Chicago/Turabian StyleRamírez-Rico, Gerardo, Moises Martinez-Castillo, Lucero Ruiz-Mazón, Erika Patricia Meneses-Romero, José Arturo Flores Palacios, Efrén Díaz-Aparicio, Erasmo Negrete Abascal, and Mireya de la Garza. 2024. "Identification, Biochemical Characterization, and In Vivo Detection of a Zn-Metalloprotease with Collagenase Activity from Mannheimia haemolytica A2" International Journal of Molecular Sciences 25, no. 2: 1289. https://doi.org/10.3390/ijms25021289