Differential Localization of Cysteine Protease Inhibitors and a Target Cysteine Protease, Cathepsin B, by Immuno-Confocal Microscopy

Abstract

SUMMARYThe cystatin superfamily of cysteine protease inhibitors and target cysteine proteases such as cathepsin B have been implicated in malignant progression. The respective cellular/extracellular localization of cystatins and cysteine proteases in tumors may be critical in regulating activity of the enzymes. Confocal microscopy has enabled us to demonstrate the differential localization of cystatins and cathepsin B in an embryonic liver cell line and an invasive hepatoma cell line. In both, stefins A and B were distributed diffusely throughout the cytoplasm, whereas cystatin C was distributed in juxtanuclear vesicles. Stefin A and cystatin C, but not stefin B, were present on the cell surface. Cystatin C was found on the top surfaces of both cell lines, whereas stefin A was found only on the top surface of the embryonic liver cells. Cathepsin B staining was concentrated in perinuclear vesicles in the embryonic liver cells. In the hepatoma cells, staining for cathepsin B was also present in vesicles adjacent to the cell membrane and on localized regions of the bottom surface. Such a disparate distribution of cathepsin B and its endogenous inhibitors may facilitate proteolysis by the hepatoma cells and thereby contribute to their invasive phenotype.

Cystatin C Rescues Degenerating Neurons in a Cystatin B-Knockout Mouse Model of Progressive Myoclonus Epilepsy

“…CysB immunostaining in brains of WT mice in this study shows cytoplasmic and lysosomal localization, supporting earlier findings that CysB is distributed diffusely throughout the cytoplasm 42 and localized mainly within lysosomal compartments. 43 We confirm and extend the reports that CysB has a role in lysosomal functioning and that loss-of-function in EPM1 and subsequent lysosomal dysfunction are the major pathogenic factors in the disease.…”

Modulation of cystatin C expression impairs the invasive and tumorigenic potential of human glioblastoma cells

“…Quantitatively different combinations of cystatins are the major constituents of the inhibition of cathepsin B in squamous cell lung cancer and normal lung tissue (Krepela et al, 1998). Cathepsin B and its endogenous inhibitors may facilitate proteolysis by hepatoma cells and thereby contribute to the invasive phenotype of this type of cancer (Calkins et al, 1998). An inverse correlation between amounts of cathepsin B and cystatin C has been found in a study of human colorectal cancer (Hirai et al, 1999).…”

Proteomic analysis of pancreatic ductal carcinoma cells treated with 5‐aza‐2'‐deoxycytidine

“…Cystatin B (also called stefin B) belongs to the cystatin superfamily of cysteine protease inhibitors and target cysteine proteases, such as cathepsin B. Cystatin B has been implicated in malignant progression [33] and alterations in its expression, processing, and localization have been observed at various levels in malignant human tumors [34]. Progressively higher levels of cystatin B have been associated, e.g., with short survival in patients Table 1 for their classification).…”