BIOSYNTHESIS AND PROCESSING OF PROTEINASE-3 IN U937 CELLS - PROCESSING PATHWAYS ARE DISTINCT FROM THOSE OF CATHEPSIN-G

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 t he azurophilic granules, is expressed in progenitor cells of myeloid o rigin. In the present study, the biosynthesis, processing, and intrace llular transport of the enzyme was investigated in the human myelomono cytic 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 us ing a combination of techniques including amino-terminal sequencing, w e identified the 35-kDa form as a zymogen containing an activation dip eptide but lacking the amino-terminal 25 residues, presumably the resu lt of cleavage by a signal peptidase. Tunicamycin treatment and alkali nization of acidic cell compartments with NH4Cl did not prevent the pr ocessing 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 B-phosphate receptor. Brefeldin A inhibited th e zymogen processing, suggesting that the dipeptide cleavage occurred in a post-Golgi organelle. The enzyme responsible for the removal of t he dipeptide is a cysteine proteinase since E-64d, a class-specific in hibitor, prevented processing. However, treatment of cells with a dipe ptidyl peptidase I inhibitor, Gly-Phe-diazomethyl ketone and with the lysosomotropic agents, NH4Cl and chloroquine, did not prevent dipeptid e cleavage, indicating that the processing enzyme for proteinase 3 is not dipeptidyl peptidase I. In contrast, Gly-Phe-diazomethyl ketone in hibited cleavage of the dipeptide from cathepsin G. This indicates tha t processing of proteinase 3 is distinct from that of cathepsin G. Pro teinase 3 is also processed at the COOH-terminal extension. Cleavage t akes place next to Arg-222, suggesting that a trypsin-like proteinase is involved in the COOH-terminal processing.