Cloning of cDNA for proteinase 3: a serine protease, antibiotic, and autoantigen from human neutrophils.

Abstract

SummaryClosely similar but nonidentical NH2-terminal amino acid sequences have been reported for a protein or proteins in human neutrophils whose bioactivities is/are diverse (as a serine protease, antibiotic, and Wegener's granulomatosis autoantigen) but that share(s) several features: localization in the azurophil granules, a molecular mass of ti29 kD, reactivity with diisopropylfluorophosphate, and the ability to degrade elastin. We previously purified one such entity, termed p29b. Using a monospecific antibody, we have cloned from human bone marrow a cDNA encoding the complete p29b protein in its mature form, along with pre-and pro-sequences . The predicted amino acid sequence agrees closely with the NH2-terminal sequence obtained previously from purified p29b, as well as with sequences newly obtained from CNBr fragments . The primary structure is highly homologous to elastase, cathepsin G, T cell granzymes, and other serine proteases, and shares both the catalytic triad and substrate binding pocket of elastase. Hybridization ofthe full-length cDNA with restriction enzyme digests of human genomic DNA revealed only one fragment. This suggests that the closely related species described previously are the same, and can be subsumed by the term used for the first-described activity, proteinase 3. Proteinase 3 is more abundant in neutrophils than elastase and has a similar proteolytic profile and specific activity. Thus, proteinase 3 may share the role previously attributed to neutrophil elastase in tissue damage, and has the potential to function as an antimicrobial agent .

A proportion of proteinase 3 (PR3)-specific anti-neutrophil cytoplasmic antibodies (ANCA) only react with PR3 after cleavage of its N-terminal activation dipeptide

“…They were prepared by washing the cell monolayers once with serum-free medium, followed by incubation with fresh serum-free medium for 48 h. Saturation of the binding capacity of MCPR3-2 adsorbed to plastic wells, as determined using the rabbit anti-PR3 serum for detection, was achieved with a 1:32 dilution of D-rPR3-S176A and a 1:64 dilution of rPR3-S176A-containing media, respectively. Subsequently, 200 ml of diluted The constructs carrying the mutation S176A have an alanine residue in position 176 of the published amino acid sequence [16] instead of the active site serine. This mutation results in lack of enzyme activity [11] but does not affect recognition by PR3-ANCA [14].…”

“…1. The original cDNA insert of wild-type PR3 spanning from nucleotide positions 9 to 790 of the published sequence [16] as well as the cDNA insert coding for the inactive mutant rPR3-S176A were prepared as described elsewhere [11]. The deletion mutants lacking the codons for the N-terminal propeptide (D-rPR3 and D-rPR3-S176A) were prepared using the splicing by overlap extension method [17] with the primers listed in Table 1.…”

Mapping of Conformational Epitopes on Human Proteinase 3, the Autoantigen of Wegener’s Granulomatosis

“…Amino acid residues at positions 103, 119, and 120 are polymorphic in human populations, accounting for three different protein sequences (V103 or I103 in combination with A119 and T120, and I103 in combination with T119, S120). Evolutionary comparisons, however, suggest that V103, A119, and T120 (20)(21)(22) of hPR3 represent the ancestral allele (Fig. 2).…”

The cell biology of leukocyte-mediated proteolysis

“…Proteinase 3 (PR3). PR3 is a cationic serine proteinase that consists of a 228-residue polypeptide chain with 54% homology to HLE [17]. It is a glycoprotein with two potential amino terminal-linked glycosylation sites.…”