SORTING OF NONGLYCOSYLATED HUMAN PROCATHEPSIN-S IN MAMMALIAN-CELLS

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 wer e transfected with cDNA of either wild type human procathepsin S or a mutant procathepsin S in which Asn of the only glycosylation site in t he proregion was replaced by Gin. The cells expressed glycosylated and non-glycosylated procathepsin S, respectively. Large amounts of the p recursors were secreted into the culture media by both transfectants. Secreted wild type procathepsin S contained Man-6-phosphate in the oli gosaccharide chain. Wild type procathepsin S was activated in the cell s but no maturation occurred in the culture media. In vitro processing of glycosylated as well as of non-glycosylated procathepsin S gave fu lly active enzymes thus indicating that the oligosaccharide chain was not necessary for proper folding. A reuptake of the glycosylated and n on-glycosylated procathepsin S by HEK 293-cells could be observed. Sma ll amounts of mature cathepsin S were detected in the lysosomes of the mutant transfectants. Subcellular fractionation showed non-glycosylat ed procathepsin S in the membrane fraction. Non-glycosylated procathep sin S was bound to the plasma membrane at 2 degrees C, suggesting an a dditional sorting motif in the cathepsin S molecule besides the Man-6- phosphate residue.