Interaction of purified human proteinase 3 (PR3) with reconstituted lipid bilayers

Proteinase 3 (PR3), the major target autoantigen in Wegener's granulomatosi s is a serine proteinase that is normally stored intracellularly in the pri mary granules of quiescent neutrophils and monocytes. Upon cell activation, a significant portion of this antigen is detected on the cell surface memb rane. The nature of the association of PR3 with the membrane and its functi onal significance are unknown. We investigated the interaction of purified human PR3 with mixtures of zwitterionic (dimyristoyl-L-alpha-phosphatidylch oline, DMPC) and anionic (dimyristoyl-L-alpha-phosphatidylglycerol, DMPG) p hospholipids in reconstituted lipid bilayers using differential scanning ca lorimetry and lipid photolabeling, and measured the affinity of this intera ction using spectrophotometry. Two other primary granule constituents, huma n neutrophil elastase (HNE) and myeloperoxidase (MPO) were investigated for comparison. In calorimetric assays, using lipid vesicles of mixed DMPC/DMP G, increasing PR3 concentrations (protein/lipid molar ratio from 0 to 1 : 1 10) induced a significant decrease of the main chain transition enthalpy an d a shift in chain melting temperatures which is indicative of partial inse rtion of PR3 into the hydrophobic region of the lipid membranes. This was c onfirmed by hydrophobic photolabeling using liposomes containing trace amou nts of the photoactivable [I-125]-labeled phosphatidylcholine analog TID-PC /16. The molar affinity of PR3, EWE, and MPO to lipid vesicles of different DMPC/DMPG ratios was then determined by spectrophotometry. At a DMPC/DMPG ratio of 1 : 1, molar affinities of PR3, K-d = 4.5 +/- 0.3 mu M; HNE, 14.5 +/- 1.2 mu M; and MPO, 50 +/- 5 mu M (n = 3) were estimated. The lipid-asso ciated PR3 exhibited two-fold lower V-max and K-m values, and its enzyme ac tivity was slightly more inhibited (K-i) by the natural alpha(1)-proteinase inhibitor (alpha(1)-PI) or an autoantibody to PR3.