title: Characterization of the CD177 interaction with the ANCA antigen proteinase 3.
abstract: Proteinase 3 is a serine protease found in neutrophil granules and on the extracellular neutrophil membrane (mPR3). mPR3 is a major antigen for anti-neutrophil cytoplasmic antibodies (PR3-ANCAs), autoantibodies causing fatal autoimmune diseases. In most individuals, a subpopulation of neutrophils also produce CD177, proposed to present additional PR3 on the surface, resulting in CD177neg/mPR3lowand CD177pos/mPR3highneutrophil subsets. A positive correlation has been shown between mPR3 abundance, disease incidence, and clinical outcome. We present here a detailed investigation of the PR3:CD177 complex, verifying the interaction, demonstrating the effect of binding on PR3 proteolytic activity and explaining the accessibility of major PR3-ANCA epitopes. We observed high affinity PR3:CD177 complex formation by surface plasmon resonance. Using flow cytometry and a PR3-specific FRET assay, we found that CD177 binding reduced the proteolytic activity of PR3 in vitro using purified proteins, in neutrophil degranulation supernatants containing wtPR3 and directly on mPR3highneutrophils and PR3-loaded HEK cells. Finally, CD177pos/mPR3highneutrophils showed no migration advantage in vitro or in vivo when migrating from the blood into the oral cavity. We illuminate details of the PR3:CD177 interaction explaining mPR3 membrane orientation and proteolytic activity with relevance to ANCA activation of the distinct mPR3 neutrophil populations.
authors.affiliation: Experimental and Clinical Research Center, Charité - Universitätsmedizin Berlin, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany.
authors.forename: Uwe
authors.initials: U
authors.lastname: Jerke
authors.affiliation: Experimental and Clinical Research Center, Charité - Universitätsmedizin Berlin, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, MDC, Berlin, Germany.
authors.forename: Stephen F
authors.initials: SF
authors.lastname: Marino
authors.affiliation: Experimental and Clinical Research Center, Charité - Universitätsmedizin Berlin, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, MDC, Berlin, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Daumke
authors.affiliation: Experimental and Clinical Research Center, Charité - Universitätsmedizin Berlin, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany.|Nephrology and Intensive Care Medicine, Charité Campus Virchow, Berlin, Germany.
authors.forename: Ralph
authors.initials: R
authors.lastname: Kettritz
mesh_terms: D019268:Antibodies, Antineutrophil Cytoplasmic / Q000096:biosynthesis*; D015551:Autoimmunity; D015550:Cell Degranulation / Q000276:immunology*; D042783:Endothelial Cells / Q000276:immunology / Q000473:pathology; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D058851:GPI-Linked Proteins / Q000235:genetics / Q000276:immunology; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000276:immunology; D006801:Humans; D007519:Isoantigens / Q000235:genetics / Q000276:immunology*; D008958:Models, Molecular; D053146:Myeloblastin / Q000235:genetics / Q000276:immunology*; D009504:Neutrophils / Q000276:immunology* / Q000473:pathology; D061251:Primary Cell Culture; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000235:genetics / Q000276:immunology*; D015398:Signal Transduction; D020349:Surface Plasmon Resonance
chemical_list: D019268:Antibodies, Antineutrophil Cytoplasmic; C407242:CD177 protein, human; D000939:Epitopes; D058851:GPI-Linked Proteins; D007519:Isoantigens; D011956:Receptors, Cell Surface; D053146:Myeloblastin