title: Biosynthesis and processing of proteinase 3 in U937 cells. Processing pathways are distinct from those of cathepsin G.
abstract: Proteinase 3 is a human polymorphonuclear leukocyte serine proteinase that degrades elastin in vitro and causes emphysema when administered by intratracheal insufflation into hamsters. Proteinase 3, stored in the azurophilic granules, is expressed in progenitor cells of myeloid origin. In the present study, the biosynthesis, processing, and intracellular transport of the enzyme was investigated in the human myelomonocytic cell line U937. Proteinase 3 is initially identified as a 35-kDa precursor and converted into the 29-kDa mature form within 3 h. By using a combination of techniques including amino-terminal sequencing, we identified the 35-kDa form as a zymogen containing an activation dipeptide but lacking the amino-terminal 25 residues, presumably the result of cleavage by a signal peptidase. Tunicamycin treatment and alkalinization of acidic cell compartments with NH4Cl did not prevent the processing of the proteinase 3 zymogen into the mature form, suggesting that the enzyme is targeted to the cytoplasmic granules by a mechanism other than the mannose 6-phosphate receptor. Brefeldin A inhibited the zymogen processing, suggesting that the dipeptide cleavage occurred in a post-Golgi organelle. The enzyme responsible for the removal of the dipeptide is a cysteine proteinase since E-64d, a class-specific inhibitor, prevented processing. However, treatment of cells with a dipeptidyl peptidase I inhibitor, Gly-Phe-diazomethyl ketone and with the lysosomotropic agents, NH4Cl and chloroquine, did not prevent dipeptide cleavage, indicating that the processing enzyme for proteinase 3 is not dipeptidyl peptidase I. In contrast, Gly-Phe-diazomethyl ketone inhibited cleavage of the dipeptide from cathepsin G. This indicates that processing of proteinase 3 is distinct from that of cathepsin G. Proteinase 3 is also processed at the COOH-terminal extension. Cleavage takes place next to Arg-222, suggesting that a trypsin-like proteinase is involved in the COOH-terminal processing.
authors.affiliation: Department of Internal Medicine, Division of Respiratory, Critical Care, and Occupational Medicine, University of Utah Health Sciences Center, Salt Lake City, Utah 84132, USA.
authors.forename: N V
authors.initials: NV
authors.lastname: Rao
authors.forename: G V
authors.initials: GV
authors.lastname: Rao
authors.forename: B C
authors.initials: BC
authors.lastname: Marshall
authors.forename: J R
authors.initials: JR
authors.lastname: Hoidal
mesh_terms: D000595:Amino Acid Sequence; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001345:Autoradiography; D020126:Brefeldin A; D020789:Cathepsin C; D056649:Cathepsin G; D002403:Cathepsins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D002738:Chloroquine / Q000494:pharmacology; D006224:Cricetinae; D003517:Cyclopentanes / Q000494:pharmacology; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000378:metabolism; D006801:Humans; D007532:Isoleucine / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D053146:Myeloblastin; D009504:Neutrophils / Q000201:enzymology; D010446:Peptide Fragments / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D014316:Tritium
chemical_list: D003517:Cyclopentanes; D004792:Enzyme Precursors; D010446:Peptide Fragments; D011500:Protein Synthesis Inhibitors; D013462:Sulfur Radioisotopes; D000643:Ammonium Chloride; D007532:Isoleucine; D014316:Tritium; D020126:Brefeldin A; D002738:Chloroquine; D008715:Methionine; D002403:Cathepsins; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D020789:Cathepsin C; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D053146:Myeloblastin