Cathepsin S, a lysosomal cysteine protease, is synthesized as inactive precursor. It is activated in the lysosomes by a proteolytic cleavage of the propeptide. HEK 293-cells which do not express cathepsin S were transfected with cDNA of either wild type human procathepsin S or a mutant procathepsin S in which Asn of the only glycosylation site in the proregion was replaced by Gln. The cells expressed glycosylated and non-glycosylated procathepsin S, respectively. Large amounts of the precursors were secreted into the culture media by both transfectants. Secreted wild type procathepsin S contained Man-6-phosphate in the oligosaccharide chain. Wild type procathepsin S was activated in the cells but no maturation occurred in the culture media. In vitro processing of glycosylated as well as of non-glycosylated procathepsin S gave fully active enzymes thus indicating that the oligosaccharide chain was not necessary for proper folding. A reuptake of the glycosylated and non-glycosylated procathepsin S by HEK 293-cells could be observed. Small amounts of mature cathepsin S were detected in the lysosomes of the mutant transfectants. Subcellular fractionation showed non-glycosylated procathepsin S in the membrane fraction. Non-glycosylated procathepsin S was bound to the plasma membrane at 2 degrees C, suggesting an additional sorting motif in the cathepsin S molecule besides the Man-6-phosphate residue.
Two processes, synthesis and degradation, contribute to the intracellular concentration of a protein. As most malignant tumors or tumor cell lines show elevated levels of proteinases, we studied the half-life of a cysteine proteinase, procathepsin S, in order to determine whether tumor cells can regulate their cathepsin concentration via changing the degradation rate of the enzyme.The following procathepsin S species were examined: wild-type procathepsin S in macrophages, recombinant procathepsin S in human embryonic kidney cells (HEK 293 cells), recombinant nonglycosylated procathepsin S in HEK 293 cells, wild-type procathepsin S in the established nonsmall cell lung carcinoma cell line 97TM1.The half-lives of both wild-type procathepsins S expressed in macrophages and in HEK 293 cells were 1 h, whereas that of procathepsin S in the tumor cell line was 2 h. Nonglycosylated procathepsin S was not processed. The degradation of mature cathepsin S proceeded with a half-life of 16±18 h. All cell lines studied secreted substantial amounts of procathepsin S into the culture medium. No further maturation of secreted procathepsin S has been observed in the culture medium. We suggest a disturbed sorting mechanism in tumor cells.Keywords: procathepsin S; degradation; secretion; processing; tumor cells. Lysosomal proteins show long average half-lives (t12 ) of several days up to 2 weeks [1±3] although the accumulation of proteolytic enzymes in the lysosomes and the acid pH within this organelle create an appropriate environment for rapid protein degradation. The molecular properties rendering the lysosomal proteins resistant in vivo to the proteolytic attack are still unclear. Glycosylation has been reported to stabilize lysosomal proteins against rapid degradation [4], and the inhibition of hydrolytic enzymes by glycosaminoglycans within the lysosomes may also contribute to this stability [5]. In contrast, isolated cathepsins are rapidly inactivated and degraded in vitro under weakly acidic conditions [6] at which these enzymes are most active. It remains to be further elucidated what makes lysosomal proteins, including the cathepsins, relatively long-lived.The maturation of procathepsins is a rapid process compared to the long life of the mature cathepsins, lasting 1 h or less for cathepsin D [1,4,7] and cysteine proteinases [8±12], as well as for other lysosomal proteins [13]. Reports dealing with the t12 -values of individual mature lysosomal cathepsins are sparse [1±3,14]. The biosynthesis and the maturation of inactive precursor molecules contribute to the supply of active lysosomal enzymes in the cell. Loss of active enzymes, either by secretion or by degradation, is the other determinant of lysosomal enzyme concentration. The t1 2 of an enzyme describes the turnover rate and, thus, it allows the calculation of the time scale at which the turnover rate can be regulated.Many malignant tumors and established tumor cell lines are characterized by high levels of proteolytic enzymes, including lysosomal cysteine proteina...
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