Related Experiment Video
Updated: Mar 7, 2026
Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation
Published on: February 28, 2019
Characterization of the CD177 interaction with the ANCA antigen proteinase 3
Uwe Jerke1, Stephen F Marino2, Oliver Daumke2
1Experimental and Clinical Research Center, Charité - Universitätsmedizin Berlin, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany.
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/mPR3low and CD177pos/mPR3high neutrophil 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 mPR3high neutrophils and PR3-loaded HEK cells. Finally, CD177pos/mPR3high neutrophils 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.
Insights
The CD177 protein binds to Proteinase 3 (PR3) on neutrophils, reducing its activity. This interaction is key to understanding autoimmune diseases like ANCA vasculitis.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Proteinase 3 (PR3) is a neutrophil serine protease and a major autoantigen in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis.
- Neutrophil subsets expressing CD177 and high levels of membrane-bound PR3 (mPR3) are associated with disease incidence and severity.
- The precise role of CD177 in modulating PR3 function and its implications in ANCA pathogenesis remain unclear.
Purpose of the Study:
- To investigate the molecular interaction between PR3 and CD177.
- To determine the effect of CD177 binding on PR3 proteolytic activity.
- To elucidate the accessibility of PR3 epitopes to PR3-ANCAs in the context of the PR3:CD177 complex.
Main Methods:
- Surface plasmon resonance (SPR) to assess PR3:CD177 binding affinity.
- Flow cytometry and Förster resonance energy transfer (FRET) assays to measure PR3 activity.
- In vitro assays using purified proteins, neutrophil supernatants, and engineered cells (HEK cells).
- In vitro and in vivo migration assays.
Main Results:
- High-affinity complex formation between PR3 and CD177 was confirmed.
- CD177 binding significantly reduced PR3 proteolytic activity.
- The interaction did not confer a migration advantage to CD177-positive neutrophils.
- Epitope accessibility for PR3-ANCAs was modulated by CD177 binding.
Conclusions:
- CD177 binding to mPR3 regulates PR3 activity and may influence ANCA binding.
- Understanding the PR3:CD177 interaction provides insights into ANCA pathogenesis and neutrophil subset heterogeneity.
- This interaction is relevant for the activation of distinct mPR3 neutrophil populations in autoimmune diseases.
