Isolation and characterization of chicken bile matrix metalloproteinase
B Packialakshmi1, R Liyanage2, K S Rasaputra3
1Cell and Molecular Biology Program, University of Arkansas, Fayetteville 72701 Department of Poultry Science, University of Arkansas, Fayetteville 72701.
Abstract:
Avian bile is rich in matrix metalloproteinases (MMP), the enzymes that cleave extracellular matrix proteins such as collagens and proteoglycans. Changes in bile MMP expression have been correlated with hepatic and gall bladder pathologies, but the significance of their expression in normal, healthy bile is not understood. We hypothesized that the MMP in bile may aid the digestion of native collagens that are resistant to conventional gastric proteases. Hence, the objective of this study was to characterize the bile MMP and check its regulation in association with dietary factors. We used substrate zymography, azocoll protease assay, and gelatin affinity chromatography to identify and purify the MMP from chicken bile. Using zymography and SDS PAGE, 5 bands at 70, 64, 58, 50, and 42 kDa were detected. The bands corresponding to 64, 50, and 42 kDa were identified as MMP2 using trypsin in-gel digestion and matrix-assisted laser desorption time-of-flight mass spectrometry and peptide mass fingerprinting. Chickens fed diets containing gelatin supplements showed higher levels of MMP expression in the bile by both azocoll assay and zymography. We conclude that the bile MMP may be associated with the digestion of collagens and other extracellular matrix proteins in avian diets.
Insights
Avian bile contains matrix metalloproteinases (MMPs) that may help digest dietary collagens. Supplementing chicken diets with gelatin increased bile MMP levels, suggesting a role in breaking down resistant proteins.
Area of Science:
- Biochemistry
- Animal Science
- Digestive Physiology
Background:
- Avian bile contains matrix metalloproteinases (MMPs), enzymes crucial for extracellular matrix breakdown.
- The role of bile MMPs in healthy avian digestion, particularly for dietary collagens, remains unclear.
- Previous studies linked altered MMP expression to avian liver and gallbladder diseases.
Purpose of the Study:
- To characterize matrix metalloproteinases (MMPs) present in avian bile.
- To investigate the regulation of bile MMPs in response to dietary factors.
- To explore the potential role of bile MMPs in digesting resistant dietary collagens.
Main Methods:
- Substrate zymography and azocoll protease assays were employed to detect and quantify MMP activity.
- Gelatin affinity chromatography was used for MMP purification from chicken bile.
- SDS-PAGE, in-gel digestion, and mass spectrometry identified specific MMPs, including MMP2.
Main Results:
- Five distinct MMP bands were detected in chicken bile via zymography and SDS-PAGE.
- MMP2 was identified as a significant component of avian bile MMPs through mass spectrometry.
- Chickens fed diets supplemented with gelatin exhibited elevated bile MMP levels.
Conclusions:
- Avian bile contains functional matrix metalloproteinases (MMPs), notably MMP2.
- Bile MMPs likely play a role in the digestion of collagens and other extracellular matrix proteins in avian diets.
- Dietary factors, such as gelatin, can modulate the expression of bile MMPs in chickens.
