Processing and transport of the precursor of cathepsin C during its transfer into lysosomes.
The biosynthesis and processing of a lysosomal cysteine proteinase, cathepsin C (dipeptidylaminopeptidase I), was investigated by pulse-chase experiments in cultured rat macrophages. Cathepsin C is first synthesized as procathepsin C with a molecular mass of 55 kDa. Procathepsin C is then cleaved and modified within 1 h into mature cathepsin C with two chains of 25 and 7.8 kDa. A combination of pulse-chase experiments and the subcellular fractionation analysis showed that procathepsin C and cathepsin C are located in low-buoyant-density organelles and lysosomes, respectively. The reactivity of endoglycosidase H and N-glycanase and analysis of phosphorylation indicated that both precursor and mature cathepsin C are phosphorylated and N-glycosylated to give a high-mannose-type. The addition of 300-kDa mannose 6-phosphate receptor antiserum to the chase medium caused extensive release of procathepsin C into the medium, whereas the addition of control serum did not. The membrane association of procathepsin C was tested by successive extraction of cells pulse labeled for 75 min with hypotonic buffer, alkaline solution, and Triton X-100. Procathepsin C was totally extracted by hypotonic solution, whereas procathepsin D was a membrane-associated form requiring Triton X-100 for its extraction. Gel-filtration chromatography analysis of the pulse-labeled products revealed that the precursor product exists as an oligomeric form. It is suggested that the oligomerization of cathepsin C occurs before its entry into lysosomes.
Muno
D
D
Department of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan.
Ishidoh
K
K
Ueno
T
T
Kominami
E
E
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001345:Autoradiography; D001692:Biological Transport; D020789:Cathepsin C; D002402:Cathepsin D / Q000096:biosynthesis / Q000302:isolation & purification; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000096:biosynthesis* / Q000302:isolation & purification; D004792:Enzyme Precursors / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008247:Lysosomes / Q000378:metabolism*; D017737:Macrophages, Peritoneal / Q000201:enzymology*; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D051381:Rats; D017208:Rats, Wistar; D013462:Sulfur Radioisotopes
D000906:Antibodies; D004792:Enzyme Precursors; D013462:Sulfur Radioisotopes; D008715:Methionine; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D020789:Cathepsin C; D002402:Cathepsin D