Isolation and characterization of chicken bile matrix metalloproteinase.

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.

Tibial dyschondroplasia-associated proteomic changes in chicken growth plate cartilage.

Tibial dyschondroplasia (TD) is a poultry leg problem that affects the proximal growth plate of the tibia, preventing its transition to bone. To understand the disease-induced proteomic changes, we compared the protein extracts of cartilage from normal and TD-affected growth plates. TD was induced by feeding thiram to chickens 2 wk before tissue harvest. Proteins were extracted from whole tissues and from conditioned media (CM) prepared by incubating appropriate growth plate tissues in serum-free culture medium for 48 hr. The extracts were prefractionated to contain proteins ranging between 10 and 100 kD. Equal amounts of proteins were subjected to 2D gel electrophoresis with three individual samples per group. The gels were silver stained, and digital images were compared and analyzed with Melanie software to determine differentially expressed protein spots. On comparison of two sets of gels, 47 matching spots were detected in tissue extracts and 27 in CM extracts. Among the matching spots, 12 were determined to be down-regulated in tissue extracts (P < or = 0.05) and two in CM extracts (P < or = 0.05) of TD-affected growth plates. Altogether, 32 protein spots could be identified in both tissue and CM extracts by in-gel trypsin digestion, followed by peptide mass fingerprinting and mass spectrometry (MS)/MS fragmentation. The down-regulated proteins included alpha-enolase, G protein, origin recognition complex, peptidyl prolyl isomerase, calumenin, type II collagen precursor, and the expressed sequence tag pgm2n.pk014.f20, a protein with homology to human reticulocalbin-3 (RCN3). Most of the downregulated proteins are associated with signal transduction, energy metabolism, and secretory functions that are integral to cell viability. Consistent with our earlier findings that the TD chondrocytes are nonviable, the current results suggest that thiram very likely interferes with basic metabolic functions of chondrocytes, leading to their death and, consequently, to the pathogenesis of TD.