Human neutrophil proteinases (elastase, proteinase-3, and cathepsin-G) are released at sites of acute inflammation. We hypothesized that these inflammation-associated proteinases can affect cell signaling by targeting proteinase-activated receptor-2 (PAR 2 ). The PAR family of G protein-coupled receptors is triggered by a unique mechanism involving the proteolytic unmasking of an N-terminal self-activating tethered ligand (TL). Proteinases can either activate PAR signaling by unmasking the TL sequence or disarm the receptor for subsequent enzyme activation by cleaving downstream from the TL sequence. We found that none of neutrophil elastase, cathepsin-G, and proteinase-3 can activate G q -coupled PAR 2 calcium signaling; but all of these proteinases can disarm PAR 2 , releasing the N-terminal TL sequence, thereby preventing G q -coupled PAR 2 signaling by trypsin. Interestingly, elastase (but neither cathepsin-G nor proteinase-3) causes a TL-independent PAR 2 -mediated activation of MAPK that, unlike the canonical trypsin activation, does not involve either receptor internalization or recruitment of ␤-arrestin. Cleavage of synthetic peptides derived from the extracellular N terminus of PAR 2 , downstream of the TL sequence, demonstrated distinct proteolytic sites for all three neutrophil-derived enzymes. We conclude that in inflammation, neutrophil proteinases can modulate PAR 2 signaling by preventing/disarming the G q /calcium signal pathway and, via elastase, can selectively activate the p44/42 MAPK pathway. Our data illustrate a new mode of PAR regulation that involves biased PAR 2 signaling by neutrophil elastase and a disarming/silencing effect of cathepsin-G and proteinase-3.

1 Human mast cell tryptase appears to display considerable variation in activating proteinaseactivated receptor 2 (PAR 2 ). We found tryptase to be an ine cient activator of wild-type rat-PAR 2 (wt-rPAR 2 ) and therefore decided to explore the factors that may in¯uence tryptase activation of PAR 2 . 2 Using a 20 mer peptide (P20) corresponding to the cleavage/activation sequence of wt-rPAR 2 , tryptase was as e cient as trypsin in releasing the receptor-activating sequence (SLIGRL...). However, in the presence of either human-PAR 2 or wt-r PAR 2 expressing cells, tryptase could only activate PAR 2 by releasing SLIGRL from the P20 peptide, suggesting that PAR 2 expressed on the cells was protected from tryptase activation. 3 Three approaches were employed to test the hypothesis that PAR 2 receptor glycosylation restricts tryptase activation. (a) pretreatment of wt-rPAR 2 expressing cells or human embryonic kidney cells (HEK293) with vibrio cholerae neuraminidase to remove oligosaccharide sialic acid, unmasked tryptase-mediated PAR 2 activation. (b) Inhibiting receptor glycosylation in HEK293 cells with tunicamycin enabled tryptase-mediated PAR 2 activation. (c) Wt-rPAR 2 devoid of the Nterminal glycosylation sequon (PAR 2 T25 7 ), but not rPAR 2 devoid of the glycosylation sequon located on extracellular loop-2 (PAR 2 T224A), was selectively and substantially (430 fold) more sensitive to tryptase compared with the wt-rPAR 2 . 4 Immunocytochemistry using antisera that speci®cally recognized the N-terminal precleavage sequence of PAR 2 demonstrated that tryptase released the precleavage domain from PAR 2 T25 7 but not from wt-rPAR 2 . 5 Heparin : tryptase molar ratios of greater than 2 : 1 abrogated tryptase activation of PAR 2 T25 7 . 6 Our results indicate that glycosylation of PAR 2 and heparin-inhibition of PAR 2 activation by tryptase could provide novel mechanisms for regulating receptor activation by tryptase and possibly other proteases.

Background: Proteinase-activated receptor-1 (PAR1) is a proteolytically activated G protein-coupled receptor. Neutrophilderived enzymes might regulate PAR1 signaling. Results: Neutrophil elastase and proteinase-3 cleave and activate PAR1 signaling that is distinct from thrombin-triggered responses. Neutrophil elastase and proteinase-3 signaling through PAR1 modulates endothelial cell signaling. Conclusion: Neutrophil enzymes are G␣ i -biased agonists for PAR1. Significance: Biased PAR1-activating compounds may prove of value as therapeutic agents to treat cardiovascular and inflammatory diseases.

The chemistries within phagosomes of APCs mediate microbial destruction as well as generate peptides for presentation on MHC class II. The antimicrobial effector NADPH oxidase (NOX2), which generates superoxide within maturing phagosomes, has also been shown to regulate activities of cysteine cathepsins through modulation of the lumenal redox potential. Using real-time analyses of lumenal microenvironmental parameters, in conjunction with hydrolysis pattern assessment of phagocytosed proteins, we demonstrated that NOX2 activity not only affects levels of phagosomal proteolysis as previously shown, but also the pattern of proteolytic digestion. Additionally, it was found that NOX2 deficiency adversely affected the ability of bone marrow–derived macrophages, but not dendritic cells, to process and present the I-Ab–immunodominant peptide of the autoantigen myelin oligodendrocyte glycoprotein (MOG). Computational and experimental analyses indicated that the I-Ab binding region of the immunodominant peptide of MOG is susceptible to cleavage by the NOX2-controlled cysteine cathepsins L and S in a redox-dependent manner. Consistent with these findings, I-Ab mice that were deficient in the p47phox or gp91phox subunits of NOX2 were partially protected from MOG-induced experimental autoimmune encephalomyelitis and displayed compromised reactivation of MOG-specific CD4+ T cells in the CNS, despite eliciting a normal primary CD4+ T cell response to the inoculated MOG Ag. Taken together, this study demonstrates that the redox microenvironment within the phagosomes of APCs is a determinant in MHC class II repertoire production in a cell-specific and Ag-specific manner, which can ultimately impact susceptibility to CD4+ T cell–driven autoimmune disease processes.

Human protamine P2 was purified to homogeneity by solubilizing whole spermatozoa in guanidinium . HC1 containing 2-mercaptoethanol, alkylating the resulting protamine thiols with vinylpyridine, removing acid-insoluble material by acid dialysis and using CM-cellulose chromatography to remove non-protamine basic proteins and separate protamines P1 and P2. The P2 preparation contained two components, P2a and P2b, which were sequenced completely without being separated. The peptides obtained from thermolysin and endoproteinase Lys-C digestions were purified by reverse-phase high-pressure liquid chromatography and sequenced using a gasphase sequencer. P2a contains 57 amino acids and has a relative molecular mass of 7636 while P2b contains 54 amino acids, which are identical to residues 4-57 of P2a, and has a relative molecular mass of 7242. Protamine P2a is approximately 50% homologous with human protamine PI. The amino acid sequence of P2a is: In a previous paper [I 31 we reported the complete amino acid sequence of human protamine PI whch was found to be quite homologous to the single protamine in bull [I], ram [2] and boar [3] as well as to mouse protamine 1 [14]. In this paper we present the complete amino acid sequences of two forms of the second major human protamine, P2. MATERIALS AND METHODS MaterialsThe materials used have been described previously [13] Methods Two purification methods were used to isolate human protamine P2. The first method was the modified procedure of Kolk and Samuel [9] which was used to obtain pure protamine PI [13]. Whole spermatozoa from 8 -10 fertile donors were washed, dissolved in 6M guanidinium . HC1 containing 2-mercaptoethanol and alkylated with vinylpyridine. The resulting solution was dialyzed against 2% HCl and the dialysate supernatant was fractionated on a Bio-Rex 70 ionexchange column. The second method was the same as the first except that CM-cellulose [I51 was used in place of the Bio-Rex 70 in the ion-exchange step. CM-cellulose (CM-52, Whatman) was equilibrated with 5% guanidinium . HCI in 50 mM lithium acetate buffer at pH 5.0 and packed into a 1 .0-cm-diameter column on top of a 0.5-cm bed of Sephadex G-10 to a height of 8 cm. The acid dialysate supernatant was mixed with 2.5 g guanidinium . HC1, diluted to 50 ml with lithium acetate buffer and run into the column under gravity at room temperature. Only the very basic protamines bound to the CM-cellulose and they were eluted with a linear gradient of 5 -15% guanidinium . HC1 in lithium acetate buffer in a total volume of 200ml. The fractions under each 230-nm absorbance peak were pooled, exhaustively dialysed against 1% acetic acid in Spectrapore 3 tubing, dried down in a vacuum centrifuge, redissolved in 0.2 M acetic acid and stored at -20 "C. Amino acid analysis, automated protein sequencing and reverse-phase peptide HPLC were performed as described previously [13].Protamine P2 was digested with thermolysin by dissolving 18 nmol in 0.2 ml0.5% ammonium bicarbonate, adding 2 pg thermolysin and incubating the solu...