Figure 6.

Translation of PR3 and PRTN3–002 mRNA in neutrophils from patients with ANCA disease. (A) RT-PCR using transcript-specific primers indicated that both PR3 and PRTN3–002 mRNAs were the expected full length. (B) Predicted protein sequences for PR3 versus p24^PR3/MBN. The star indicates the first methinonine of p24^PR3/MBN, which is in frame with PR3. (C) Available anti-PR3 antibodies were screened for crossreactivity against p24^PR3/MBN by Western blot (Supplemental Figure 6). PMN, polymorphonuclear leukocytes; R&D, R&D Systems. (D) Anti-PR3 antibody reacted with a 24-kD band in THP-1 protein lysates. (E) Metabolically labeled protein lysates from neutrophils of patients with AAV (n=2; patients 56 and 79 listed in Supplemental Table 1) and one disease control (Ctl) were processed for detection of newly synthesized proteins. Anti-PR3 immunoblot showed reactivity with three bands in lysates from patient 1. The position corresponding to prepro-PR3 (upper band in E) and processed PR3 (C–E) is marked by solid arrows. The position corresponding to p24^PR3/MBN in C–E is marked by open arrows. (F) Neutrophils from two patients (Pts; 6 and 26 in Supplemental Table 1) were metabolically labeled, and extracts were immunoprecipitated (IP) with anti-PR3 antibody. Unbound (U) and bound (B) fractions were analyzed by Western blot. Bands testing positive for both streptavidin probe (left panel) and anti-PR3 antibody (right panel) were excised, digested enzymatically, and analyzed by tandem MS. (G) The MS/MS spectrum acquired from a parent ion of 567.9 (+2) confirmed that PR3 was present in the bands. The most abundant peptide fragment ions extending from the N and C termini are denoted b and y, respectively. Neutrophils from patients 6, 26, 56, and 79 listed in Supplemental Table 1 and one disease control was used for metabolic labeling to detect synthesis of PR3. M, protein molecular weight marker. Molecular mass in kilodaltons is shown to the left of the images.