Immunolocalization of cathepsin B in equine dyschondroplastic articular cartilage.
Abstract: A polyclonal antiserum raised in sheep against human cathepsin B was tested for specificity and cross-reactivity with the horse homologue by SDS-PAGE and Western blotting, prior to being used for immunolocalization of the enzyme in equine articular cartilage. In Western blots, the antiserum recognized the 30 kDa single chain and 25 kDa heavy chain of the mature enzyme in purified bovine cathepsin B, and corresponding bands at 32 and 27 kDa in equine chondrocyte and fibroblast lysates. This antiserum was then used to compare the expression and distribution of cathepsin B in normal and dyschondroplastic cartilage of young horses. In normal articular cartilage (n = 6 animals), significant amounts of enzyme were detected only in hypertrophic chondrocytes in the deep zone. The enzyme was intracellular, located in the lysosomal granules. No extracellular matrix staining was observed. Levels of cathepsin B were increased slightly above normal in the deep zone in age-matched dyschondroplastic cartilage (n = 5 animals). The most striking finding, however, was the abundance of the enzyme in chondrocyte clonal clusters associated with the lesions. Cathepsin B levels were low in chondrocytes isolated from normal cartilage (n = 6), but increased progressively during serial subculture, reaching a maximum at passage 5-6. In contrast, primary cultures of dyschondroplastic chondrocytes (n = 3) expressed abundant cathepsin B.
Publication Date: 1999-01-12 PubMed ID: 9883087DOI: 10.1016/s1090-0233(98)80122-6Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research is about the location and presence of an enzyme called cathepsin B in the articular cartilage of horses, specifically comparing the levels in normal cartilage and in dyschondroplastic cartilage, which is a disease affecting the cartilage.
Methodology
- The researchers used a polyclonal antiserum made in sheep against human cathepsin B.
- They tested the antiserum for specificity and cross-reactivity with the horse version of the enzyme using SDS-PAGE and Western blotting. SDS-PAGE is a method used in biochemistry and molecular biology to separate proteins according to their electrophoretic mobility. Western blotting is an analytic technique used to detect specific proteins in a sample.
- In Western blots, the antiserum recognized the 30kDa single chain and 25kDa heavy chain of mature cathepsin enzymes in bovine (cow) cathepsin B, and corresponding bands at 32 and 27 kDa in horse chondrocyte (cartilage cells) and fibroblast (connective tissue cells) lysates. Lysate is a fluid containing the contents of lysed cells.
Findings
- The antiserum was then used to compare the expression and distribution of cathepsin B in normal and dyschondroplastic cartilage in young horses.
- In normal cartilage, significant amounts of the enzyme were only found in hypertrophic chondrocytes in the deep zone, and it was located inside the cells, in the lysosomal granules. Lysosomes are small organelles that contain enzymes.
- In dyschondroplastic cartilage, the levels of cathepsin B were slightly higher in the deep zone than in normal cartilage. The enzyme was abundant in the chondrocyte clonal clusters associated with the lesions.
- The researchers also found that the levels of cathepsin B were low in chondrocytes isolated from normal cartilage, but those levels increased during serial subculture, peaking at passage 5-6. In contrast, the primary cultures of dyschondroplastic chondrocytes expressed abundant cathepsin B.
Significance
- The findings suggest that cathepsin B plays a role in the disease process of dyschondroplasia in horses.
- More research would be needed to fully understand this role and how it could potentially be targeted for disease treatment.
Cite This Article
APA
Hernández-Vidal G, Jeffcott LB, Davies ME.
(1999).
Immunolocalization of cathepsin B in equine dyschondroplastic articular cartilage.
Vet J, 156(3), 193-201.
https://doi.org/10.1016/s1090-0233(98)80122-6 Publication
Researcher Affiliations
- Strangeways Research Laboratory, Cambridge, UK.
MeSH Terms
- Animals
- Blotting, Western / veterinary
- Cartilage, Articular / enzymology
- Cartilage, Articular / pathology
- Cathepsin B / analysis
- Cathepsin B / immunology
- Cattle
- Cells, Cultured
- Chondrocytes / enzymology
- Cross Reactions
- Electrophoresis, Polyacrylamide Gel / veterinary
- Horse Diseases / enzymology
- Horse Diseases / etiology
- Horse Diseases / pathology
- Horses
- Humans
- Immunohistochemistry
- Molecular Weight
- Osteochondritis / enzymology
- Osteochondritis / etiology
- Osteochondritis / pathology
- Osteochondritis / veterinary
- Sheep
Citations
This article has been cited 2 times.- Bourebaba L, Röcken M, Marycz K. Osteochondritis dissecans (OCD) in Horses - Molecular Background of its Pathogenesis and Perspectives for Progenitor Stem Cell Therapy. Stem Cell Rev Rep 2019 Jun;15(3):374-390.
- Lotz MK, Otsuki S, Grogan SP, Sah R, Terkeltaub R, D'Lima D. Cartilage cell clusters. Arthritis Rheum 2010 Aug;62(8):2206-18.
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