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Wegener's GranulomatosisHuman Proteinase 3, the Autoantigen of Mapping of Conformational Epitopes on

JenneKniepert, Ulf Schönermarck, Ulrich Specks and Dieter E. Angelika Kuhl, Brice Korkmaz, Bert Utecht, Andrea

http://www.jimmunol.org/content/185/1/387doi: 10.4049/jimmunol.09038872010;

2010; 185:387-399; Prepublished online 7 JuneJ Immunol 

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The Journal of Immunology

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

Angelika Kuhl,*,1,2 Brice Korkmaz,*,1,3 Bert Utecht,† Andrea Kniepert,*,‡

Ulf Schonermarck,x Ulrich Specks,{ and Dieter E. Jenne*

Anti-neutrophil cytoplasmic Abs (cANCAs) against conformational epitopes of proteinase 3 (PR3) are regarded as an important

pathogenic marker in Wegener’s granulomatosis (WG). Although the three-dimensional structure of PR3 is known, binding sites

of mAbs and cANCAs have not been mapped to date. Competitive binding and biosensor experiments suggested the existence of

four nonoverlapping areas on the PR3 surface. In this paper, we present an approach to identify these discontinuous surface

regions that cannot be mimicked by linear peptides. The very few surface substitutions found in closely related PR3 homologs

from primates, which were further varied by the construction of functional human-gibbon hybrids, resulted in the differential loss

of three Ab binding sites, two of which were mapped to the N-terminal b-barrel and one to the linker segment connecting the N-

and C-terminal barrels of PR3. The sera from WG patients differed in their binding to gibbon PR3 and the gibbon-human PR3

hybrid, and could be divided into two groups with similar or significantly reduced binding to gibbon PR3. Binding of almost all

sera to PR3–a1-protease inhibitor (a1–PI) complexes was even more reduced and often absent, indicating that major antigenic

determinants overlap with the active site surface on PR3 that associates with a1-PI. Similarly, the mouse mAbs CLB12.8 and 6A6

also did not react with gibbon PR3 and PR3–a1-PI complexes. Our data strongly suggest that cANCAs from WG patients at least

in part recognize similar surface structures as do mouse mAbs and compete with the binding of a1-PI to PR3. The Journal of

Immunology, 2010, 185: 387–399.

Wegener’s granulomatosis (WG) is an autoimmunesmall vessel vasculitis with granulomas affecting theupper and lower respiratory tract and kidneys in its

generalized form (1). Anti-neutrophil cytoplasmic autoantibodies(cANCAs) recognizing conformational epitopes on proteinase 3(PR3) are a disease-specific diagnostic marker for WG and areable to activate cytokine-primed neutrophils in vitro by binding tomembrane-bound PR3 (2–4). Although the vast majority of PR3 isstored intracellularly in primary granules together with other pro-teases, small amounts of PR3 prestored in as yet poorly charac-terized small vesicles are rapidly mobilized during priming. Thissmall fraction of PR3 is externalized together with NB1 (CD177),a glycolipid-anchored membrane protein (5, 6), and is presented

on the neutrophil surface as a complex with NB1 (7). Simulta-neous interaction of cANCAs with membrane-bound PR3 andFcgR is believed to trigger full activation and premature degran-ulation of extravasating neutrophils around small vessels inpatients with active disease (3).Although the cause and autoimmune pathogenesis ofWG are not

yet known, interactions of certain PR3–cANCA subpopulationswith PR3 on the surface of neutrophils are believed to contributeto clinical progression and to the propagation of small vessel de-struction. This hypothesis, however, has been challenged in view ofpatients with WG who are constantly cANCA negative, by obser-vations that cANCAs are often absent in early or localized stages ofthe disease, and by whole-blood analyses indicating that circulatingneutrophils are not carrying ANCAs on their surfaces even in activedisease (8). As purified PR3-cANCA IgG preparations, but notcANCAs in whole blood, were able to bind to PR3 on the neutrophilsurface, conformational alterations or steric inaccessibility ofsurface-bound PR3 was inferred to explain the low-affinity inter-action between PR3 and cANCA. Although cANCAmeasurementsare generally accepted as a valuable diagnostic tool and disease-specific biomarker, their utility for the monitoring of WG patientsappears to be unreliable (9). Titer decreases and increases werefound to be weakly correlated with remissions and relapses, respec-tively, and disease activity in general. In addition, absence ofcANCA-PR3 deposits in inflammatory lesions and coexistence ofnormal neutrophil numbers and PR3 autoantibodies in peripheralblood favored the view that cANCAs are rather a consequence thana cause of the disease.Several attempts were made to determine cANCA binding spe-

cificities using PR3-derived linear peptides (10–12) or chimeric mol-ecules composed of human PR3 (hPR3) and human neutrophilelastase or mouse PR3 (13). The differential binding patterns ofmAbs and cANCA sera, however, did not lead to the identificationof distinct surface regions that were targeted by the Abs. Nonspecific

*Department of Neuroimmunology, Max-Planck-Institute of Neurobiology, Planegg/Martinsried; †Utecht and Ludemann, Klausdorf/Schwentine; ‡Department of Biology,University of Konstanz, Konstanz; xUniversity Hospital Munich-Großhadern, Munich,Germany; and {Thoracic Diseases Research Unit, Division of Pulmonary and CriticalCare Medicine, Mayo Clinic and Foundation, Rochester, MN 55905

1A.K. and B.K. contributed equally to this work.

2Current address: Roche Diagnostics, Penzberg, Germany.

3Current address: Institut National de la Sante et de la Recherche Medicale U-618,Proteases et Vectorisation Pulmonaires, Universite Francois Rabelais, Tours, France.

Received for publication December 3, 2009. Accepted for publication April 20, 2010.

This work was supported by the Deutsche Forschunsgemeinschaft (SFB571). Fund-ing for B.K. was provided by the Alexander von Humboldt Foundation.

Address correspondence and reprint requests to Dr. Dieter E. Jenne, Department ofNeuroimmunology, Max-Planck-Institute of Neurobiology, D-82152, Planegg/Mar-tinsried, Germany. E-mail address: djenne@neuro.mpg.de

Abbreviations used in this paper: a1-PI, a1-protease inhibitor; cANCA, anti-neutrophil cytoplasmic Ab; gPR3, gibbon proteinase 3; gPR3v1, gibbon proteinase 3variant 1; HNE, human neutrophil elastase; hPR3, human proteinase 3; PBS-T,PBS plus 0.05% Tween-20; PDB, Brookhaven Protein Data Bank; pNPP, 4-nitrophenyl phosphate disodium salt hexahydrate; PR3, proteinase 3; WG, Wegener’sgranulomatosis.

Copyright� 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00

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binding of unrelated autoimmune sera to PR3 peptides turned outto be an insurmountable obstacle for meaningful conclusions (14).As these mapping results were of dubious value, biosensor-basedcompetition analyses between different mAbs, on one hand, andcANCA sera from different patients, on the other hand, were per-formed. With biosensor technology, 13 mAbs to hPR3 were charac-terized and grouped into four major subsets of Abs that recognizeddifferent surface regions of hPR3 (15, 16). mAbs within the samegroup inhibited binding of each other and hence were assumed totarget similar epitope areas on the PR3 surface. mAb 4A5, for exam-ple, representing group 3, partially or completely interfered withthe binding of 50% of PR3-cANCA sera (15), suggesting that a frac-tion of PR3-cANCAs and murine mAbs interact with similar sur-face patches.In a clinical study with a small number of patient sera, cANCA

heterogeneity between patients and changes of binding propertiesin the same patient have been observed (3). PR3-cANCAs wereshown to recognize a surface area on PR3 that is targeted by most,if not all, cANCA sera at the time of active disease presentation(3). At subsequent stages of the disease, PR3-cANCAs changedtheir epitope specificity and interacted with larger or smaller sur-face areas on PR3. Such differences in cANCA properties may belinked to their variable pathogenic potential but cannot be recog-nized by the currently available diagnostic techniques.To distinguish cANCA subpopulations and Ab titers to individ-

ual nonoverlapping epitopes, we aimed at the construction of cata-lytically active PR3 Ag variants that differ minimally from the hu-man Ag, yet lack one or more major epitopes. To this end, weinvestigated the natural surface diversity of PR3 homologs fromclosely related primates. As PR3-cANCAs from WG patientsappeared to show little to no cross-reactivity with murine PR3(17), we assumed that PR3 epitopes are conserved only in homo-logs of more closely related primate species. Natural residue sub-stitutions in PR3 homologs of other primates should thereforereduce or abolish the binding affinity of PR3-cANCA sera, whereascleavage and inhibitor specificity as well as overall surface proper-ties should be very similar and most likely identical to hPR3.To locate a distinct immunogenic subregion within the two sub-

domains of PR3, we adopted a very conservative approach for thedesign of unnatural PR3 variants and recombined only the entireN-terminal and C-terminal b-barrels from the human and gibbonspecies. These two subdomains of chymotrypsin-type serine pro-teases are homologous to each other and can be reassembled intocatalytically active hybrid molecules that retain the functional prop-erties of the parental subdomains (18). To narrow down the surfacelocation of distinct epitopes further, we substituted gibbon-specificresidues with those of hPR3 to rebuild human epitopes, and wesubstituted other residues with those from the more distantly relatedopossum homolog (Ensembl ID ENSMODP00000008115) to elim-inate a third epitope that was common to both gibbon PR3 (gPR3)and hPR3.The overall aim of this study was to define the structural position

of individual Ab epitopes and to construct PR3 templates that permitthe measurement of titers of major cANCA subsets independently.Three nonoverlapping Ab binding areas have been found, and one ofthese binding areas clearly overlaps with the contact area buried bya1-protease inhibitor (a1-PI) after complexation.

Materials and MethodsSera from healthy volunteers and WG patients

Sera from 34 patients with diagnosed WG were collected. Sera from eighthealthy blood donors were used as negative controls. This study was ap-proved by the ethics committee of the Ludwig-Maximilians University(Munich, Germany).

Construction of the proPR3 expression plasmids

The expression vector used in this study was based on pcDNA5/FRT(Invitrogen, Carlsbad, CA). An Igk-chain secretion signal was integratedinto pcDNA5/FRT, using the oligoduplex 59-Pho-CTA GCCACCATGGA-GACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTACCAG-GTTCCAC-39 and 59-Pho-GTGGAACCTGGTACCCAGAGCAGCAGT-ACCCATAGCAGGAGTGTGTCTGTCTCCATGGTGG-39 (Metabion, Mar-tinsried, Germany) to allow the secretion of the expressed protein into thecell culture medium followed by a S-peptide tag. Full-length cDNA forhPR3 was amplified with 59-TGGACACGTGGATGACGACGACAAAAT-CGTGGGCGGTCACGAGGC-39 and 59-CCCCACCGGTTTTCAGGGT-AGAACGGATCCA-39 (Metabion). In this experiment, an enterokinasecleavage site (DDDDK) is introduced at the N terminus of PR3. Theresulting PCR product was digested with PmlI/AgeI and cloned into thereading frame of the S-tag. The construct was named pcDNA5/FRT/hPR3-H6. Gibbon cDNA was amplified from granulocyte cDNA, using the pri-mers 59-TGCGGAGATCGTGGGCGG-39 and 59-GGAACGGATCCAGT-CCACGTA-39 (Metabion). The PCR fragment was reamplified with 59-TGGACACGTGGATGACGACGACAAAATCGTGGGCGGTCACGAG-GC-39 and 59-CCCCACCGGTTTT CAGGGTAGAACGGATCCA-39 andcloned into pcDNA5/FRT as described for hPR3 and named pcDNA5/FRT/gPR3-H6. The construction of two human/gibbon chimeras wasperformed by digestion of pcDNA5/FRT/PR3-H6 with BlpI and AgeI(Fig. 1) and exchange of the respective cDNA segments. A chemicallysynthesized 390-bp hPR3 fragment was digestedwith PmlI/BlpI and clonedinto the same sites of the pcDNA5/FRT/gPR3-H6 plasmid to reconstruct thehuman CLB12.8 epitope in gPR3 (gibbon proteinase 3 variant 1 [gPR3v1]).A further modification of this gPR3v1 was done by replacing the A116 toQ119 tetrapeptide with the respective opossum residues Q-V-A-S. To thisend, a DNA fragment was chemically synthesized, digested with BlpI andBstEII, and subcloned into the same sites of the gPR3v1 plasmid. In addi-tion, the two glycosylation sites Asn113 andAsn159weremutated to lysineresidues in the gPR3v2 construct. Each construct was verified by sequenceanalysis. Fig. 1 shows the schematic description of the proPR3 expressionconstructs with N- and C-terminal tags.

Cell culturing and transfection

Flp-in HEK293 cells (Invitrogen) were cultured in FreeStyle 293 expressionmedium or DMEM (Life Technologies, Rockville, MD) supplemented with10% FCS. For each construct, 1.43 106 flp-in HEK293 cells were plated in35-mm cell culture dishes 1 d prior to transfection. Cells incubating in 3 mlDMEM containing 10% FCS were transfected by the addition of 1.8 mgpOG44 (encoding the flp-in recombinase; Invitrogen) and 0.2 mgpcDNA5/FRT recombinant plasmids in 100 ml serum-free OptiMEM me-dium and 7 ml FuGENE HD transfection reagent (Roche, Penzberg, Ger-many). At 48 h after transfection, the medium was replaced with DMEMplus 10% FCS containing 75 mg/ml hygromycin B (Invitrogen) every 2 to3 d. Two weeks posttransfection, visible circular hygromycin B-resistantcolonies were present. Cells were pooled and cultured until a desirednumber of plates had reached confluence. Cell culture supernatant wastested for recombinant PR3 expression via Western blot, using S-proteinconjugated to HRP (Novagen, Madison, WI). For protein expression, cellswere cultured in DMEM supplemented with 5% FCS and 75 mg/mlhygromycin B for 8–10 d before supernatants were harvested.

Purification of recombinant protein

A total of 500 ml of harvested cell culture supernatant was filtered througha 0.22-mm membrane (Millipore, Schwalbach, Germany), concentrated to100 ml, and dialyzed against starting buffer (20 mM Na2HPO4; 500 mMNaCl; 50 mM imidazole, pH 7.5) at 4˚C. 6xHis-tagged proPR3 was puri-fied using affinity chromatography. The protein solution was applied toa HisTrap HP column (Amersham Biosciences, Munich, Germany) pre-viously equilibrated in starting buffer. The column was washed with start-ing buffer, and bound proteins were eluted with a linear imidazole gradientfrom 50 mM to 1 M imidazole in 20 mM Na2HPO4, 500 mM NaCl, pH7.5. Fractions were collected and analyzed for proPR3 by SDS-PAGE andCoomassie staining. The expected size is 32 kDa. Nearly pure 6xHis-tagged proPR3-containing fractions (.85%) were pooled and concen-trated, and protein concentration was determined by measuring the absor-bance at 280 nm (spectrophotometer; Eppendorf, Hamburg, Germany) andby the bicinchoninic acid assay.

Processing of proPR3 by enterokinase and activity assay

Purified 6xHis-tagged proPR3 was dialyzed into 20 mM Tris-HCl, 50 mMNaCl, and 2 mM CaCl2, pH 7.4, at 4˚C. The N-terminal S-peptide tagwas cleaved off by calf enterokinase (Roche) to generate native active

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6xHis-tagged PR3. Enterokinase cleavage was carried out at an enzyme/substrate ratio of 1/20 for 20 h at room temperature. After S-peptide tagcleavage, PR3 was again analyzed using Coomassie-stained SDS-PAGE(reduction in molecular size), and the enzymatic activity was determinedusing a PR3 synthetic substrate to ascertain that the S-peptide tag wascleaved off. Activity was assessed using 1 mM Boc-Ala-Pro-Nva-4-chloro-SBzl (Bachem, Switzerland) and 0.5 mM 59,59-dithio-bis(2-nitrobenzoicacid) in 100 mM Tris-HCl, pH 8.1; 700 mM NaCl; and 1% Igepal (Sigma-Aldrich, Munich, Germany). Substrate hydrolysis was measured in a spec-trofluorometer (Fluostar Optima, BMG Labtech, Offenburg, Germany).Inhibition of recombinant PR3 was performed by incubation of a1-PI(Sigma-Aldrich) at 10-fold M concentrations for 30 min at 37˚C.

SDS-PAGE and immunoblotting

Proteins from the supernatants of degranulated neutrophils were separatedby 15% NaDodSO4-polyacrylamide gel electrophoresis (SDS-PAGE) un-der native conditions and transferred to a Hybond-ECL membrane (GEHealthcare, Munich, Germany), using a semidry electroblotter (Biometra,Gottingen, Germany). The membrane was blocked in PBS plus 0.05%Tween-20 (PBS-T) containing 5% milk for 1 h at room temperature. Themembranewas thenwashedwith PBS-T. S-protein-HRP (Merck,Darmstadt,Germany) was diluted 1 in 5000 and incubated overnight at 4˚C. The mem-brane was washed again and developed with Super Signal West Chemilumi-nescence Signal (Thermo Fisher Scientific, Bonn, Germany).

Isolation of granulocyte proteins from human andmonkey blood samples,as well as native SDS-PAGE followed by immunoblotting, was performed byUtecht&Ludemann (Klausdorf/Schwentine,Germany).Natural granulocyteproteins from human and primates were separated by nonreducing SDS-PAGE (10% T, 2.5% C) of whole-cell extracts from isolated granulocytesusing one wide slot per sample. After semidry transfer, nitrocellulose mem-branes were cut into 2.5-mm-wide strips, each representing 0.253 106 gran-ulocytes. Immunodetection with mAbs or patient sera was done by blockingthe strips with milk proteins followed by 2 h of incubation with primary Absand 1 h of incubation with species-specific alkaline phosphatase-conjugatedsecondary Abs. 5-Bromo-4-chloro-3-indolyl phosphate and nitro-blue tetra-zolium were used for color development.

ELISA

The 6xHis-tagged PR3 was used in a capture ELISAwith precoated nickel-chelate microtiter 96-well plates (Thermo Fisher Scientific). Plates were in-cubated with purified 6xHis-tagged PR3 at a concentration of 1 mg/ml in

PBS-T at room temperature overnight. The plates were washed three timeswith washing-buffer (Utecht & Ludemann) and incubated for 1 h withmAbs against PR3 or human sera diluted in diluent buffer (Utecht &Ludemann). mAbs to PR3 CLB12.8 (Sanquin, Amsterdam, The Nether-lands); MCPR3-2 (19), 4A5, 6A6 (Wieslab AB, Malmo, Sweden), 1B10,1F11, 1F10, and 2E1 (HyTest, Turku, Finland) were diluted 1 in 1000.Monoclonal anti-PR3 WGM2, WGM3, provided by Dr. W. Gross, and WGpatient’s sera were diluted 1 to 50. Again the plate was washed three timeswith washing buffer and then incubated with a secondary anti–mouse AP(1:5000; Sigma-Aldrich) or anti–human AP Ab (1:50; Sigma-Aldrich),respectively. Finally, color was developed using 4-nitrophenyl phosphatedisodium salt hexahydrate (pNPP) as a substrate. The OD values weremeasured at 405 nm. As a negative control, an irrelevant IgG1 mAb wasused (control IgG1 Ab was purchased from BD Biosciences, Frankfurt,Germany). All tests were performed in duplicates.

ELISA assays with PR3 complexed to a1-PI was performed as describedabove. Briefly, PR3 (100 mg/ml) was incubated with a 10-fold M excess ofa1-PI (1 mg/ml) for 1 h at 37˚C. Completeness of PR3 inhibition by a1-PIwas checked via activity measurements. In competition experiments, con-stant amounts of hPR3 and mAb CLB12.8 were incubated with variousconcentrations of a1-PI for 30 min at 37˚C prior to immobilization of His-tagged PR3 to Ni-NTA plates. Bound CLB12.8 Ab was detected by ELISAusing a secondary anti–mouse AP labeled Ab, followed by substrate de-velopment with pNPP, as described above.

ResultsStrategy for mapping conformational epitopes

As conformation-dependent epitopes of hPR3 were not sharedwith murine PR3 (17), we used more closely related mammalianhomologs for hPR3 (Fig. 1) to evaluate the extent of Ab cross-reactivities. Natural residue substitutions in PR3 homologs of pri-mates should lead to a partial loss in binding affinity of cANCAsera, but should not interfere with the biological function andfolding pathway of PR3. Fig. 2 shows the sequence alignment ofseveral homologs of mature PR3 from humans, other primates, andmice. We assumed C-terminal trimming after R243 in all speciesand hence aligned the mammalian homologs only up to this struc-turally fixed position. With phylogenetic distance, the number of

FIGURE 1. Schematic diagram of cDNA constructs used for the recombinant expression of hPR3, gPR3, h/gPR3, g/hPR3, gPR3v1, and gPR3v2 in flp-in

293 cells. The N-terminal S-tag followed by an enterokinase cleavage site (DDDDK), the native enzyme, and the C-terminal 6xHis-tag are indicated.

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surface substitutions increases: chimpanzee, gibbon, rhesus mon-key, and mouse PR3 carry 7, 16, 35, and 68 substitutions, respec-tively, compared with hPR3. Amino acid residues at positions 103,119, and 120 are polymorphic in human populations, accountingfor three different protein sequences (V103 or I103 in combinationwith A119 and T120, and I103 in combination with T119, S120).Evolutionary comparisons, however, suggest that V103, A119, andT120 (20–22) of hPR3 represent the ancestral allele (Fig. 2).Using SDS-PAGE under nonreducing conditions followed by

Western blotting, we were able to demonstrate the binding of mu-rine mAbs and cANCAs from several WG patients to the structur-ally preserved hPR3 Ag on a nitrocellulose membrane (Fig. 3). Todetermine the influence of multiple natural amino acid variationson Ab recognition, we purified neutrophils from the blood ofthree primate species: chimpanzee (Pan troglodytes verus), gibbon(Hylobates pileatus), and macaque (Macaca mulatta), and extr-acted the granule proteins. PR3 from these supernatants, togetherwith other granule proteins, was separated by native SDS-PAGEand blotted onto membranes. Detection of human myeloperoxi-dase, neutrophil elastase, azurocidin, lactoferrin, and lysozymeserved as a control for granule proteins in the supernatants. All11 WG sera and all mouse anti-PR3 mAbs tested by immuno-blotting recognized hPR3 (Fig. 3). The very closely related chim-panzee PR3 was detected by 9 of 11 WG sera and by all anti-PR3mAbs except for 1F11 (Fig. 3). Furthermore, the cANCA bindingsignal intensity compared with hPR3 was identical. In contrast,macaque PR3 showed nearly no reactivity to cANCAs. Onlytwo WG sera and two mAbs, WGM1 and 4A5, gave weak signals

(Fig. 3). gPR3 showed an intermediate cANCA binding pattern,whereas some anti-PR3 mAbs (e.g., 6A6) and some patient serarecognized the Ag, but the signal intensity was weaker comparedwith that of hPR3. A negative signal was observed for four WGsera and four anti-PR3 mAbs, namely CLB12.8, 1F10, 1F11, andMCPR3-2 (Fig. 3). Using the same approach, we were able toshow an almost complete absence of cANCA binding to rhesusmonkey PR3. We suggested that cross-reactivity of PR3-cANCAswith other PR3 homologs declined with increasing evolutionarydistance between primates. The most useful mammalian PR3 ho-molog for epitope discrimination appeared to be gPR3, because itwas recognized by some mAbs and most patient sera with signif-icantly diminished signal intensity as compared with the humanhomolog. This favorable starting point prompted us to determinethe complete cDNA and cDNA-derived protein sequence for ma-ture gPR3 by PCR cloning. The natural substitutions found ingPR3 most likely accounted for the difference in cANCA reactiv-ity. gPR3 appeared to be most suited for epitope mapping andfurther distinction of cANCA subpopulations.

Structural characteristics of gPR3

gPR3 was cloned from PMNs of a freshly isolated gibbon (H.pileatus) blood sample and sequenced. It differs from its humanhomolog by only 16 residues (Fig. 2). Comparative structuralanalysis of hPR3 and gPR3 revealed the location of amino acidvariations, which almost exclusively mapped to and modified themolecular surface. Fig. 4A shows the solvent-accessible surface ofgPR3 based on the atom coordinates for hPR3 atoms (1FUJ) (23)

FIGURE 2. Amino acid sequence alignment of hPR3

with chimpanzee (P. troglodytes, chimpPR3), gibbon

(H. pileatus, gibPR3), macaque (M. mulatta, mulPR3),

and mouse (mPR3) homologs. Amino acid variations

compared with hPR3 are indicated in red. The catalytic

triad consisting of H57, D102, and S195 is shown in

bold letters. The amino acid polymorphism V103I of

hPR3 is marked by a gray background. Amino acids are

numbered according to the chymotrypsinogen num-

bering. The BlpI restriction enzyme cleavage site for

generation of the h/gPR3 and g/hPR3 chimeras is in-

dicated (arrow). ChimpPR3 and mulPR3 sequences

were obtained from genomic analysis and translation of

the coding exons into amino acid sequences. The

gibPR3 was amplified from granulocyte cDNA. The

resulting PCR product was sequenced and translated

into amino acids.

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(views from the front and the rear) after replacing the humanresidues by the respective gPR3 amino acids. The surface substi-tutions found on gPR3 (dyed green) are not equally distributed butare clustered on one side of the surface of the two b-barrels at

some distance from the extended substrate binding cleft (Fig. 4A).The only exception is the very conservative Ile-208 substitution onthe back side of the hPR3 molecule by a Val, which is one methylgroup shorter. Active site residues K99, D61, and R143 implicatedin the substrate binding of hPR3 and conferring its specificity are notsubstituted on gPR3 (24–26). Fig. 4A also illustrates the interfacebetween the two densely packed subdomains, which runs as thesubstrate binding cleft from northwest to southeast. The homo-logous N-terminal subdomain carries the amino acid substitutionsV35M, S38AN, L39BP, R60Q, Q63AH, Q74R, and L90Q; the C-terminal subdomain substitutions are localized at positions 146,166, 187, 208, 218, 219 and 223 (Figs. 2, 4A, residue numberingaccording to Brookhaven Protein Data Bank (PDB) entry 1FUJ).

Recombinant production and purification of gPR3 and hPR3variants

Recombinant gibbon proPR3 and human proPR3 were expressedin HEK293 cells. Single stable integration of the cDNA constructsinto the nucleus was achieved by homologous recombinationusing the flp-in technology. The flip-in system was faster and moreefficient than the conventional transfection protocol (27), and ex-pression from the same nuclear integration site resulted in repro-ducible stable levels of expression. Constitutively secreted recom-binant human and gibbon proPR3 containing an N-terminal S-tagand a C-terminal 6xHis-tag was detected by Western blotting inthe culture supernatant using S-protein-HRP (Fig. 5A). Subse-quently, proPR3 precursors were purified by single-step chroma-tography on Ni-NTA Sepharose and activated by cleavage ofthe N-terminal S-tag propeptide with bovine enterokinase intoa highly active mature enzyme, resulting in almost complete con-version. Purified proPR3 and active PR3 were then analyzed bySDS-PAGE. The glycosylated proPR3 variants had the expectedrelative molecular mass of ∼32 kDa, whereas N-terminallytrimmed activated PR3 ran faster with a relative molecular massof ∼29 kDa (Fig. 5B). The gPR3 variant 2, gPR3v2 (V35M, S38A

N, I38BP, Q74R, N113K, N159K, 119-122QVAS) (Fig. 4D), lacksboth glycosylation sites at positions 113 and 159 and thereforeruns faster at a different position before and after conversion. Mostimportantly, all N-terminally processed PR3 variants showed enzy-matic activity, thereby confirming correct formation of disulfidebridges and folding of the molecules. The highly specific FRETsubstrate previously developed for hPR3 [Abz-VADCADQ-EDDnp(24, 28)] was cleaved by both gPR3 and hPR3 with similar effi-cacy. Purified human a1-PI inhibited gPR3 and formed a cova-lently linked complex with it, as shown by SDS-PAGE. Likewise,gPR3 was inhibited by purified human elafin, a canonical low-m.w. inhibitor of hPR3 (not shown) confirming the strong conser-vation of its functional properties.

Ab binding to recombinant gPR3 and hPR3

A capture ELISA was developed using the C-terminal 6xHis-tagfor immobilization of PR3 to nickel-coated microtiter plates. Thistechnique avoids the drawbacks of direct immobilization or captur-ing of the autoantigen with an immobilized murine Ab. The naturalsubstitutions found on gPR3 prevented the interaction of mousemAbsCLB12.8andMCPR3-2belonging, respectively, toAbgroups1 and 4, as previously defined (16). The ELISA data with purifiedrecombinant Ags agreed with Western blot results showing thatCLB12.8 and MCPR3-2 could not bind to gPR3. mAb 6A6, how-ever, gave a signal with gPR3, although clearly weaker than withhPR3 in Western blots, but did not bind to gPR3 on ELISA plates.In all ELISA experiments, recombinant hPR3 served as a positivecontrol (Fig. 6A).

FIGURE 3. Western blotting of granule proteins from human and pri-

mate neutrophils after separation by SDS-PAGE under nonreducing con-

ditions. Granulocytes of total blood from human (upper row), chimpanzee

(second row), gibbon (third row), and macaque (fourth row) were isolated

and separated using nonreducing SDS-PAGE. Proteins were blotted onto

nitrocellulose membranes, and each membrane was cut into 28 strips. PR3

was detected using sera from WG patients (lanes 2–12) and mAbs (lanes

13, 16–26) to PR3, respectively. As a control, five other granulocyte pro-

teins were tested using human autoantibodies to myeloperoxidase (1) and

mAbs to human neutrophil elastase (14), human azurocidin (15), human

lactoferrin (27) and human lysozyme (28). Monoclonal mouse anti-hPR3

Abs used are indicated as follows: CLB12.8 (13), WGM1 (16), WGM2

(17), 1B10 (18), 1F11 (19), 1F10 (20), 2E1 (21), PR3G-4 (22), PR3G-6

(23), 4A5 (24), 6A6 (25), MCPR3-2 (26).

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As the residue substitutions in gPR3 are distributed over thesurfaces of bothbarrels,weconstructedchimericmolecules betweenhPR3 and gPR3 by recombining the N- and C-terminal six-strandedb-barrels of both homologs. These compact subdomains representthe smallest foldingmodule of a serine protease domain. As a result,we obtained four sequence variants of the autoantigen, the human,the gibbon, the g/h (N-terminal b-barrel of gibbon origin) andthe h/g (N-terminal b-barrel of human origin, Fig 4B) variant.The mAbs CLB12.8 and MCPR3-2 bound to h/gPR3, but not tog/hPR3. A murine isotype control Ab did not show any reactivity toeither hPR3 and gPR3 nor gibbon-human chimeras (Fig. 6A). Theseresults clearly indicated that amino acid residues in the N-terminalsubdomain of hPR3 were essential for the binding of CLB12.8 andMCPR3-2 to hPR3. The four residues, M35, N38A, P38B, and R74,are substituted in gPR3 and are locally clustered on the hPR3 sur-face, whereas R60, Q63A, and L90 form a second nonoverlappingcluster of surface exposed residues.

Our next goal was to map the location of the two nonoverlappingepitopes recognized by CLB12.8 and MCPR3-2, respectively. Inagreement with Rooney et al. (29), the His-tagged hPR3 in a com-plex with a1-PI was not recognized by CLB12.8 in the Ni-NTAcapture ELISA (Fig. 6B). Structural examination and inspection ofthe related covalent a1-PI–pancreatic elastase complex (PDB2D26) (30) showed that residues of the 143–149 loop andresidues from the 186–190 stretch of pancreatic elastase werelocated within the contact area between porcine pancreaticelastase and a1-PI. On the basis of the known porcine pancreaticelastase a1-PI structure, we, moreover, suggested that the residuesof the loop at positions 35, 38A, 38B, and 74 (PDB 1FUJ) weresterically shielded or distorted at least in part by a1-PI complex-ation. These four residues appeared to directly represent importantstructural determinants of the CLB12.8 epitope, but distortionsand loss of structural order in the complexed hPR3 at more distantsites could also explain the loss of CLB12.8 binding. To determine

FIGURE 4. Accessible surface structures of

gPR3 (A), h/gPR3 (B), gPR3v1 (C), and gPR3v2

(D). Based on the atom coordinates for hPR3

(1FUJ) the amino acids were changed to the re-

spective gPR3 sequence (DeepView/Swiss-Pdb

Viewer, v3.7). The molecular surface is colored

according to the electrostatic potential (blue, pos-

itive region; red, negative region). Amino acid

differences from hPR3 are indicated in green.

The reactive serine residue S195 is yellow. The

standard orientation of PR3 with the active site

pointing to the spectator (left image) and the back

side of the molecule after rotating it 180˚ around

the vertical axis are depicted (right image).

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which of the two hypotheses was correct, we replaced the residuesat these four positions in gPR3 by the respective human residuesV35M, S38AN, L39BP, and Q74R, resulting in the gPR3 variant 1(gPR3v1) (Fig. 4C). By changing only these four amino acidresidues in gPR3, the binding site for mAb CLB12.8, but not thatfor MCPR3-2, was reconstituted (Fig. 6C). These data clearlyindicate that two distinct epitopes are targeted by the two mAbs.Moreover, our experiments prove that the CLB12.8 epitope iscritically shaped by the surface residues M35, N38A, P38B, andR74, whereas R60, Q63A, and L90 are the major determinants ofthe MCPR3-2 epitope. Another mAb 6A6 that was previouslyshown to compete with the binding of CLB12.8 also displayedthe same binding properties. First, 6A6 weakly interacted withuncomplexed gPR3 and hPR3–a1-PI complexes in the ELISA

(Fig. 6B, 6C). Second, 6A6 bound to the gPR3v1 (Fig. 6D). Theseexperiments clearly demonstrate that 6A6 recognizes similarstructural determinants on hPR3 as CLB12.8. We also evaluatedthe Ab WGM3, which mutually inhibited the binding of MCPR3-2but was previously not assigned to a single epitope group. LikeMCPR3-2, WGM3 bound neither to gPR3 nor to the humanizedgPR3v1 variant, demonstrating that these two mAbs recognizehighly overlapping surface structures on hPR3 (not shown). Asthe MCPR3-2 and 12.8 epitopes were different, we examinedwhether the binding region of MCPR3-2 also overlapped withthe interface of the hPR3–a1-PI complex and was, therefore, notaccessible after complexation to a1-PI. Capture ELISA assays,however, showed that MCPR3-2 binding was not impaired or abol-ished by a1-PI complexation (Fig. 6B). These data indicate that

FIGURE 5. Production of recombinant proPR3 variants in flp-in HEK293 cells and conversions to active forms. A, Western blot of cell culture

supernatant of stably transfected flp-in 293 cells using S-protein-HRP shows proPR3 expression and secretion into the supernatant. B, Coomassie-

stained SDS-PAGE after purification via nickel-affinity chromatography shows proPR3 (arrow) and after enterokinase treatment successful removal of

the N-terminal propeptide, resulting in a molecular mass reduction of 3 kDa, representing active PR3 (dotted arrow). Note: gPR3v2 and its proform run

somewhat faster because of the absent sugar moieties.

FIGURE 6. Interaction of mouse monoclonal anti-hPR3 Abs (CLB12.8, MCPR3-2, 4A5, and WGM2) with hPR3 and gPR3, hPR3–a1-PI complexes,

and two gPR3 variants. A, Recombinant PR3 was captured via the C-terminal 6xHis-tag to nickel-coated microtiter plates. Primary Ab binding was detected

using a secondary anti-mouse AP-labeled Ab followed by substrate development with pNPP. The two monoclonal mouse anti-hPR3 Abs CLB12.8 (0.2 mg/

ml) and MCPR3-2 (0.2 mg/ml) bind to hPR3, but not to gPR3. The epitope can be reconstituted using the chimera h/gPR3, but not g/hPR3. B, hPR3 was

incubated with a 10-fold M excess of a1-PI prior to coating, and binding of CLB12.8, 6A6, and MCPR3-2 to the complex was determined via ELISA, as

described above. As previously shown, binding of CLB12.8 to hPR3 is reduced in the presence of a1-PI. In contrast, recognition of MCPR3-2 to the

hPR3–a1-PI complex is not affected. C, To narrow these epitopes, Ab binding to a minimally modified gPR3 variant, called gPR3v1, was tested. Here,

amino acids in gPR3 at positions 35, 38, 39, and 74 (chymotrypsinogen numbering) were changed to the respective human sequence. This mutant was

tested in capture ELISA, too. CLB12.8 and 6A6, but not MCPR3-2, were able to bind gPR3v1, indicating similar target specificity. D, Antigenicity of the

mutants gPR3v1 and gPR3v2. The mAb 4A5 and WGM2 belonging to group 3 cannot interact with gPR3v2 when four gibbon residues were substituted

with opossum residues.

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MCPR3-2 is targeting a surface region of hPR3 that does not un-dergo conformational changes after complexation with a1-PI.Regarding the epitope recognized by 4A5 and other Abs with

similar target specificity, we suspected its location on the back sideof PR3 around the linker connecting the two b-barrels. Naturallyoccurring striking deviations from the human linker sequence werenot observed in primates, but in more distantly related mammals, inparticular in the oppossum homolog for PR3. The starting templatefor further modifications was gPR3v1, which carried the recon-structed CLB12.8 epitope and differed from hPR3 at 12 positionsin total. In this template, we modified the gibbon ATVQ linkersequence at four positions (119–122) to match exactly the opossumsequence QVAS (Fig. 4D). Moreover, the two N-linked glycosyl-ation sites at positions 113 and 159 have been removed by convert-ing the asparagines to lysines. This gibbon-based gPR3v2 variantwas no longer able to interact with the mAbs 4A5 and WGM2(Fig. 6D), but retained its binding to CLB12.8. As a complementto these findings, we observed tight binding of 4A5 and WGM2to the hPR3–a1-PI complex (not shown). In summary, we identi-fied the precise structural determinants of three groups of mAbs(represented by the mAbs MCPR3-2, CLB12.8, and 4A5, res-pectively), as defined by van der Geld and coworkers (16). In thisway we also developed the tools to measure epitope-specificcANCA titers.

Epitopes recognized by sera of WG patients

Sera from 34 patients with WG and sera from 6 healthy blooddonors were analyzed by a 6xHis-capture ELISAwith recombinantPR3 Ags. Results are shown in Fig. 7. All WG sera recognized thehPR3 Ag, whereas sera from healthy controls did not bind to anyof the tested recombinant proteins (not shown). Reactivity ofcANCAs with gPR3, human-gibbon chimeras, and gPR3 variantswas determined as the percentage of the signal obtained withthe recombinant hPR3 Ag. In this way we divided our set ofWG ANCA sera into two groups. WG1 sera (n = 15) containedautoantibodies that bound to gPR3 and h/gPR3 with similar effi-cacy and showed at least 70% of the reactivity obtained with hPR3(Fig. 7A). The second group, WG2 (n = 19), showed ,70% bind-ing to gPR3 (Fig. 7B). Binding of cANCAs to the h/gPR3 chime-ras, however, was about the same as observed with hPR3 in

the WG2 subgroup. To narrow the major target specificity ofWG2 ANCAs further, we tested several sera from the WG2 sub-group (n = 10) for gPR3v1 reactivity (Fig. 7C). Again, PR3 bind-ing to this PR3 mutant was reconstituted close to those levelsobtained with the human Ag. These data indicate that majorsubpopulations of cANCAs in the WG2 subgroup are directedtoward the N-terminal hPR3 subdomain and interact with similarconformational determinants as a major group of murine Abs(CLB12.8, 6A6) that were previously defined by their pattern ofmutual binding competition.To map the approximate location of additional cANCA binding

sites, we examined the influence of a1-PI complexation on cANCAbinding, using the Ni-NTA capture ELISA. With the exception oftwo serum samples, cANCA titers from both patient groups WG1and WG2 were strongly reduced when the a1-PI–PR3 complexeswere used as the target Ag (Fig. 8A). These results indicate thata1-PI obscures more binding sites than just the CLB12.8 epitope(Fig. 9). As the 4A5/WGM2 andMCPR3-2/WGM3 regions are stillaccessible in the a1-PI–hPR3 complex, we anticipate that theseadditional a1-PI–sensitive cANCA binding sites are locatedsomewhere else, most likely in proximity with the substrateinteracting region.Autoantibody-induced neutrophil activation via membrane-

bound Ag and IgG receptors is widely regarded as an importantpathogenetic mechanism in small-vessel vasculitis (3). Under tem-porary conditions of low a1-PI levels and cANCAs with highaffinity and fast association rates, cANCAs may well outcompetethe initial substrate-like interaction between a1-PI and hPR3 (Fig.10, red arrow, situation 2). To simulate this situation with purifiedcomponents, we used the mAb CLB12.8, which did not bind toa1-PI–inhibited hPR3 complexes. We varied the a1-PI concentra-tion and examined the extent of CLB12.8 binding to the His-tagged hPR3 at physiological and subphysiological plasma inhib-itor concentrations (Fig. 8B). At physiological concentrations ofa1-PI, but not at low concentrations, the serpin was able to forma complex with hPR3. As shown previously, hPR3–a1-PI com-plexes are no longer retained by CD177 or the lipid bilayer oncellular membranes. Hence our experiments illustrate why theneutrophil-activating potential of cANCAs can vary in patients,depending on the association rates, target specificities, and titers ofAbs, as well as on local concentrations of hPR3 inhibitors.

FIGURE 7. The epitope for monoclonal anti-hPR3 Ab CLB12.8 overlaps with the cANCA binding region from WG patients. hPR3, gPR3, h/gPR3, and

g/hPR3 were coupled to nickel-coated microtiter plates via the C-terminal 6xHis-tag. WG sera (n = 34) and healthy control sera (n = 8) were diluted 1:50,

and cANCA binding was detected using a secondary anti-human AP-labeled Ab, followed by substrate development with pNPP. For each sample, PR3

recognition was calculated in percent, and binding to hPR3 was normalized to 100%. The signals for gPR3, h/gPR3, g/hPR3, and gPR3v1, are given as

a percentage with respect to hPR3. Each dot represents the result from one WG patient. Healthy controls did not show reactivity to each of the tested

recombinant proteins. A, One group of WG patient sera (n = 15) shows similar binding to hPR3, gPR3, and the two chimeras. This group is named WG1. B,

The second group (n = 19), WG2, shows diminished binding to gPR3. The cANCA binding signal can be reconstituted using the PR3 chimera h/gPR3. g/

hPR3 has no effect on an enhanced cANCA signal. C, Ten WG-2 patients were again tested for binding to the mutant gPR3v1. Here, the ANCA binding

signal increases compared with that of gPR3.

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DiscussioncANCAs fromWG patients are known to be directed against three-dimensional features of hPR3, though linear peptides were initiallyused to distinguish different antigenic regions with conflicting andambiguous results (14). Peptide-coated pins display a high densityof various peptide conformations and are, therefore, prone to cap-ture irrelevant Abs from complex patients’ sera. By definition, so-called continuous, linear, or sequential epitopes can be mimickedby synthetic soluble peptides that adopt a huge number of differentconformations in solutions or occasionally a partially ordered sec-ondary structure. Long linear peptides have the advantage of mim-icking secondary structures and can also form an epitope thatconsists of two discontinuous determinants on a long peptide.Hence longer 15-mer peptides derived from hPR3 have also beenemployed to screen the sera of WG patients (11). Four peptideareas consisting of several 15-mer peptides were better recognizedby WG sera than by control sera, but not by mouse mAbs.The value of peptide arrays in mapping the epitopes of native

proteins, however, is limited. It is known that as many as 90% of allB cell epitopes on native proteins are conformational rather thanlinear. During an autoimmune response, B cells most likely interactwith the folded stable autoantigen, not with partially degraded andunfolded peptide fragments. Such fragments would be removed anddegraded very rapidly in biological fluids. The solvent-accessiblesurface area that is typically buried by Ab binding amounts to 700–1000 A2 (31), whereas the entire surface area of hPR3 is only9800 A2. As the typical contact area between a native proteinAg and an Ab is much larger than between a peptide or a haptenand an Ab, a conformational epitope of hPR3 is expected to com-prise a number of residues from at least two different noncontin-uous peptide segments that have close surface proximity.Crystallographic studies of Ag–Ab complexes revealed somestructural characteristics of Ab binding sites on protein surfaces(32). Five to seven core residues (hot spot residues) of the proteinAg are clustered in the center of the interface and dominate themolecular interaction with the Ab (32). Up to 10 additional resi-dues at the periphery of the interface shield this core from the bulksolvent, but do not contribute so much to the binding free energyof the complex (32). Core interacting residues are often located in

highly flexible turn or surface loops that are not under strong evo-lutionary constraints. Binding site selection by B cells is, con-versely, an evolutionary process that operates in an organism’sown body and exploits a highly diversified repertoire of Ab tem-plates in the body. We hypothesized that favorable shape comple-mentarities and optimized interactions are more easily obtained forflexible regions of an Ag, which are also known to be reshapedmore often during phylogenetic evolution. PR3 homologs of theprimate lineage were, therefore, considered as useful initial probesto evaluate the involvement of nonconserved residues in Ab bind-ing. By further variations of the PR3 surface, we were able todistinguish three nonoverlapping epitope areas that are targetedby murine mAbs.The first group of Abs, represented by CLB12.8, 6A6, and

PR3G-2 (16), binds to a region on hPR3 that differs from gPR3 intwo closely spaced surface loops, the so-called 37-loop and 70-loop. M35, N38A, and P38B are substituted by V35, S38A, andI38B, respectively, whereas R74 of the 70-loop is replaced by Q74in gPR3 (Fig. 9) (33). Reversing these four gibbon substitutionswith the respective human residues reconstitutes the CLB12.8epitope, proving that these four residues are located within thecontact area of CLB12.8. In addition, complex formation betweenhPR3 and a1-PI impairs the accessibility of both loops as pre-dicted from a model of the pancreatic elastase a1-PI. In line withthis prediction, binding of CLB12.8 to the hPR3–a1-PI complexwas lost. By contrast to all other epitopes we identified, theCLB12.8 epitope is unique in that it is occluded or altered bya1-PI binding and is very close to the active site region. Elafin,another physiological peptide inhibitor of PR3, binds noncova-lently in a canonical manner to PR3 (34). As it consists only of57 residues, the anticipated substrate-like contacts between elafinand PR3 are fewer and are restricted to the S5 to S29 pockets.According to the crystal structure of the homologous porcineelastase-elafin complex (PDB 1FLE) (35), the 37- and the 70-loops of porcine elastase are not rearranged after elafin binding,and hence the CLB12.8 epitope in the PR3-elafin complex is fullyaccessible, as demonstrated experimentally.The second Ab binding region of the N-terminal subdomain is

located to the north of the active site binding cleft and has been

FIGURE 8. Influence of a1-PI on Ab binding to hPR3. A, hPR3 was inhibited with a 10-fold M excess of a1-PI and coupled to nickel-coated microtiter

plates via the C-terminal His6-tag. WG sera (n = 11) and healthy control sera (n = 3) were diluted 1:50, and cANCA binding was detected using a secondary

anti-human AP-labeled Ab followed by substrate development with pNPP. cANCA binding of WG-1, except for two, and all WG-2 patient sera were

affected by a1-PI, and binding to the hPR3–a1-PI complex could not be observed. Healthy controls did not react with hPR3. B, mAb CLB12.8 was used in

a competition experiment with a1-PI. In this experiment, 200 ng of hPR3 and 100 ng of CLB12.8 were incubated at different concentrations of a1-PI,

ranging from 0 to 2 mg/ml, for 30 min at 37˚C in a volume of 20 ml. The hPR3 was diluted with PBS-T and 350 mM NaCl to a final concentration of 1 mg/

ml. A 100-ml volume of these dilutions was used in duplicates to incubate Ni-NTA plates. Bound CLB12.8 Ab was detected using a secondary anti-mouse

AP-labeled Ab, followed by substrate development with pNPP, as described above.

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identified with the help of three natural variations of the gibbon

homolog. The 60-loop of gPR3 displays two differences. The di-vergent residues in gPR3 are Q60 and H63A, instead of R60 andQ63A in hPR3. The third variation concerns the hydrophobic L90,which is Q in gPR3. This residue is at the beginning of the long99-loop and in close proximity to the R60 and Q63A residues (Fig.9). We found that MCPR3-2 and WGM3, a mAb of the IgM class,bind to this surface area, previously designated as epitope region 4(16). Both Abs can bind to PR3–a1-PI complexes and evidentlydo not extend into the a1-PI contact area. These findings areconsistent with previous competition experiments showing thatMCPR3-2 and WGM3 mutually inhibit their binding to immobi-lized PR3. Our attempts to map the mAb PR3G-4 were unsuccess-ful because of its low and unspecific binding. Hence no Ab couldbe assigned to the hypothetical epitope area 2 (16).Another group of Abs belonging to the epitope group 3 (16)

showed binding to both hPR3 and gPR3. The only larger surfacecompletely shared between both species is found on the back sideof PR3, opposite the active site region. The linker (residues S115to H132) that connects the N- and C-terminal b-barrel runs acrossthe back side of PR3 and has been reported as one of several linear

regions that bind Abs from WG sera (10, 11). We have, therefore,replaced the residues ATVQ between positions 119 and 122 by thenaturally occurring sequence QVAS, which is present in the opos-sum homolog of PR3 (Figs. 4D, 9). Indeed, these minor sequencechanges led to the loss of the binding site for two mAbs we tested:4A5 and WGM2.The binding of PR3-specific ANCAs to neutrophil cell surfaces

presenting either constitutively expressed or degranulation-inducedPR3 is the most attractive explanation for the contribution of theseautoantibodies to the pathogenesis of WG. Locally primed neutro-phils expose the autoantigen on their surface either in a complexwith CD177 or in direct association with plasma membrane lipidstransiently. Peripheral blood neutrophils from WG patients withANCAs, however, do not carry surface-bound autoantibodies(8). Specific IgG could not be detected on neutrophils after theirexposure to ANCA sera. Various explanations for the absence ofPR3-ANCAs on neutrophils suspended in whole blood or plasmadilutions have been put forward, but the role of the a1-PI fromplasma has so far been neglected. As shown previously, this abun-dant plasma inhibitor interacts with membrane-bound active PR3and rapidly removes it as a covalent complex from neutrophil

FIGURE 9. Nonoverlapping epitopes recognized by three groups of murine mAbs with similar target specificity and membrane interacting region on

hPR3. A, Main chain ribbon plot of hPR3 based on its crystal structure (1FUJ) and complemented by ball-and-stick models for those residues that are

critical for recognition of mAbs and neutrophil membrane receptors. Epitope 1 (green) involves M35, N38A, P38B, and R74; epitope 2 (pink) involves R60,

Q63A, and L90; epitope 3 (blue) encompasses the consecutive residues A119, T120, Q121, and V122 on the back side of the molecule (right panel) in

Bode’s standard orientation. Numbering of epitopes corresponds to the groups of Abs with overlapping specificities as defined by van der Geld et al. (16).

Hydrophobic residues that are implicated in membrane binding and CD177 interactions on the C-terminal b-barrel (33) are depicted in orange. B, Solid

surface representation of the hPR3 monomer. The orientations are identical as in A; the colors indicate negative (red) and positive (blue) electrostatic

potential at the solvent-accessible molecular surface. The image was generated with DeepView (Swiss-Pdb Viewer, v3.7, Swiss Institute of Bioinformatics,

Lausanne, Switzerland).

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membranes (36). Certain Abs (e.g., those from rabbits or murinemAbs with fast association kinetics, high affinity, and the appro-priate target specificity) may well be able to anticipate the actionof a1-PI and to stabilize the membrane-bound PR3 by secondaryinteractions with FcgRs. When such PR3-autoantibodies areformed in WG patients and a1-PI levels are low, newly exposedmembrane-associated PR3 could readily mediate neutrophil acti-vation via FcgRs upon autoantibody binding (Fig. 10).In support of this view, we mapped the target specificity for

a group of mAbs (CLB12.8, 6A6, PR3G-2) that bind in close prox-imity to the active site region. One of these, CLB12.8, was previ-ously shown to induce a respiratory burst, and this pathogeniceffect was indeed suppressed by a1-PI at higher concentrations(29). Although these authors did not observe binding to PR3-inhibitor complexes, another group (37) reported successful quan-tification of PR3–a1-PI complexes in inflammatory fluids using thesame CLB12.8 monoclonal as a capture Ab. We re-examined theseconflicting findings and confirmed that the epitope of CLB12.8 wasinaccessible in PR3–a1-PI complexes in agreement with the loca-tion of amino acid substitutions that abrogated CLB12.8 binding.To evaluate the functional significance of the CLB12.8 epitope in

WG, we compared the reactivity of WG sera with hPR3, gPR3, andvariants thereof lacking the CLB12.8 epitope. In this way, we

identified a major group of sera whose ANCAs were directedtoward a similar conformational epitope as recognized by group 1mAbs (CLB12.8, 6A6, PR3G-2). According to our observations,a significant number of cANCA sera appear to contain IgG popu-lations that share the binding specificity with CLB12.8 and couldthus stimulate neutrophils under certain conditions like CLB12.8(“the bad ANCAs”).As shownbyseveral previous studies, purifiedcANCAsare able to

interferewith the enzymatic activity of soluble PR3 (38–40), but notin all studies (10, 41–43). In view of our results with PR3 mutantsand PR3-a1-PI complexes, some ANCA populations most likelyrecognize structural determinants around the active site region andcould, therefore, prevent or delay the interaction between mem-brane-bound PR3 anda1-PI. TheseAbswould therefore have a highpathogenic potential, as these Ab-PR3 complexes cannot be clearedand stripped off from membranes by a1-PI (“the ugly ANCAs”). Athird, so far theoretical, category of PR3 autoantibodies (“harmlessANCAs”) consists of those whose epitope is buried by CD177 orlipid interactions. Such Abs would not be able to activate neutro-phils and hence would have the lowest pathogenic potential. So far,no Ab that prevents membrane binding of secreted PR3 has beendescribed. Abs with this target specificity may be beneficial, as theyprevent the association of PR3 with cellular membranes. As

FIGURE 10. Schematic illustrating situative conditions and epitope dependence of PR3-cANCA pathogenicity. Locally primed neutrophils, such as those

exposed to TNF-a, express membrane- and CD177 receptor-bound PR3 (CD177, green U-shaped symbols; PR3, red circles) on their surface. Autoanti-

bodies to PR3 (ANCAs, Y-shaped symbols) differing in epitope specificities, affinities, association rates, and plasma levels are present in the patients’

plasma and biological fluids, along with a1-proteinase inhibitor (a1-PI, yellow pentagons). In many instances (blue arrow, situation 1), externalized

membrane-associated PR3 is inactivated by a1-PI before ANCAs interact with PR3 and FcgRs on neutrophils via their Ag binding sites and Fcg domains.

The autoantigen is rapidly removed from the membranes and scavenged by complexation. Thus, ANCAs can persist without triggering premature

neutrophil activation. Under certain conditions (red arrow, situation 2), ANCAs are faster than a1-PI and interact with the Ags on neutrophil membranes

and FcgRs. Neutrophils are activated and generate reactive oxygen species. These ANCAs probably bind to structural determinants that are also important

for the initial encounter between a1-PI and PR3. Binding of ANCA is stabilized by FcgR interactions. Oxygen radicals inactivate a1-PI (yellow pentagons

with stars) and create a local ground for further activation of bystander neutrophils. Genetic factors, such as increased density of CD177 membrane

receptors, dysfunctional a1-PI variants (PiZ alleles), or aberrant de novo synthesis of PR3 or proPR3 by circulating neutrophils, increase the risk of

inappropriate neutrophil activation by PR3-ANCA.

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circulating PR3-cANCAs are not always pathogenic and can evenpersist during remission, target specificity, plasma levels, and asso-ciation kinetics of autoantibodies with PR3 may be critical for theirpathogenicity and should be taken into account. According to ourconcept, the most harmful PR3 autoantibodies react with PR3 onneutrophil surfaces faster thana1-PI and activate neutrophils beforea1-PI can interfere (Fig. 10). Alternatively, a catalytically inactiveproform of PR3 may be released by neutrophils in WG patients thatcannot be trapped and removed by a1-PI. Besides that, a1-PI var-iants in the plasma of WG patients that poorly react with catalyti-cally active PR3 may favor neutrophil activation by PR3-ANCAs.Certaina1-PI alleles are indeed correlated with an increased risk forWG (44). Although a number of observations underscore the poten-tial relevance of epitope-specific ANCA discrimination, the predic-tive value and clinical impact of epitope-specific ANCAmeasurements remain to be determined. Larger studies with statis-tical power for deeper subgroup analyses are needed to develop newprognostic parameters forWG relapses and appropriateness of long-term therapeutic interventions.

AcknowledgmentsWe thank Dr. E. Csernok, Dr. W. Gross (both from the Department of

Rheumatology, University of Lubeck, Lubeck, Germany), and Dr. Heer-

inga (Department of Pathology and Medical Biology, University Medical

Center Groningen, Groningen, The Netherlands) for providing mouse

monoclonal anti-human proteinase 3 Abs (WGM-2, WGM-3, and

PR3G-4) and H. Kittel and E. Stegmann (both from the Max-Planck-

Institute of Neurobiology, Martinsried, Germany) for skillful technical

assistance.

DisclosuresThe authors have no financial conflicts of interest.

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