Mutations of the C-terminal end of cathepsin K affect proenzyme secretion and intracellular maturation.
Transfection of the human cathepsin K cDNA into CHO cells results in the expression of mature catalytically active 27-kDa protein and in cells secreting the 39-kDa proenzyme form. Monensin, which neutralizes the pH of acidic organelles, was found to inhibit intracellular processing of the proenzyme and to stimulate its secretion into the culture medium. Brefeldin A caused alterations in immunofluorescence staining consistent with interference of lysosomal targeting and inhibited both intracellular processing and secretion of cathepsin K. Inhibition of glycosylation by tunicamycin also abolished cathepsin K maturation. Furthermore, the processing of the proenzyme to the mature form was abolished by a single mutation of the terminal Met(329) to Ala. The triple mutation of Ser(325), Pro(327), and Met(329) (all to Ala) inhibited both maturation and secretion, using either transient or stable expression systems. The results indicate that intracellular maturation and secretion of cathepsin K can be affected differentially by various treatments and by mutations of the C-terminal end of the protein. These results are consistent with the involvement of both the secreted proenzyme and the intracellularly processed enzyme in cathepsin K-mediated processes.
Claveau
D
D
Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Kirkland, Quebec, Canada H9H 3L1.
Riendeau
D
D
D000143:Acids / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D016466:CHO Cells; D056657:Cathepsin K; D002403:Cathepsins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D004274:DNA, Recombinant; D004792:Enzyme Precursors / Q000187:drug effects / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008360:Mannosephosphates / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D008985:Monensin / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
D000143:Acids; D004274:DNA, Recombinant; D004792:Enzyme Precursors; D008360:Mannosephosphates; D014415:Tunicamycin; D020126:Brefeldin A; C027693:mannose-6-phosphate; D008985:Monensin; D002403:Cathepsins; C534130:CTSK protein, human; D056657:Cathepsin K