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Stefin B, the major low molecular weight inhibitor in ovarian carcinoma
L Kastelic1, B Turk, N Kopitar-Jerala
1Department of Biochemistry, Jozef Stefan Institute, Ljubljana, Slovenia.
Abstract:
Endogenous cysteine proteinase inhibitors (CPIs) presumably regulate lysosomal cysteine endopeptidases (CPs), such as cathepsins B and L, in vivo. An imbalance between CPs and CPIs in carcinomas, possibly due to impaired inhibition of proteinases, was reported. Ovarian carcinoma contain high levels of Stefin B and about twentyfold less Stefin A compared to normal epithelial tissue. Stefin B was isolated and characterized. We used alkaline treatment, affinity chromatography on Cm-papain Sepharose, followed by gel filtration and ion-exchange chromatography and anti-Stefin B-Sepharose 4B to isolate two major isoforms of Stefin B with pI values 5.9 and 6.5. M(r) of ovarian Stefin B was close to 14,000 as judged by SDS-PAGE and had a blocked N-terminus. It strongly inhibited papain (Ki = 0.11 nM) and cathepsin L (Ki = 0.035 nM), but only moderately cathepsin B (Ki = 130 nM). As these properties are similar to Stefin B from human and bovine origin, as well as to Stefin B from human histiosarcoma, we believe that tumor Stefin B does not differ from normal Stefin B.
Insights
Ovarian carcinomas exhibit elevated Stefin B levels, a cysteine proteinase inhibitor (CPI). Isolated ovarian Stefin B strongly inhibits cathepsin L, suggesting tumor CPIs function similarly to normal tissues.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Endogenous cysteine proteinase inhibitors (CPIs) regulate lysosomal cysteine endopeptidases (CPs).
- Imbalances between CPs and CPIs are implicated in carcinomas.
- Ovarian carcinomas show increased Stefin B and decreased Stefin A compared to normal tissue.
Purpose of the Study:
- To isolate and characterize Stefin B from ovarian carcinoma.
- To investigate the inhibitory properties of ovarian Stefin B against specific cathepsins.
Main Methods:
- Isolation of Stefin B using affinity chromatography (Cm-papain Sepharose, anti-Stefin B-Sepharose 4B), gel filtration, and ion-exchange chromatography.
- Characterization by SDS-PAGE and determination of isoelectric points (pI).
- Enzyme inhibition assays to determine inhibition constants (Ki) against papain, cathepsin L, and cathepsin B.
Main Results:
- Two major isoforms of ovarian Stefin B (pI 5.9 and 6.5) were isolated, with an Mr of approximately 14,000 and a blocked N-terminus.
- Ovarian Stefin B strongly inhibited papain (Ki = 0.11 nM) and cathepsin L (Ki = 0.035 nM).
- Moderate inhibition was observed for cathepsin B (Ki = 130 nM).
Conclusions:
- The properties and inhibitory profile of ovarian Stefin B are comparable to Stefin B from other human and bovine sources.
- Tumor-derived Stefin B likely functions similarly to its normal counterpart in regulating cysteine endopeptidases within the tumor microenvironment.