Inhibitory activity of alpha-1 antitrypsin genetic variants towards neutrophil serine proteinases
A1AT is an endogenous inhibitor of the three cognate neutrophil serine proteases (NSPs): human neutrophil elastase (HNE), proteinase 3 (PR3) and cathepsin G (CG). Korkmaz et al. has recently suggested that A1AT preferentially inhibits HNE, therefore PR3 and CG might exert their destructive effect in the lungs much longer and play more important role in pathogenesis of COPD than previously thought. Accordingly, significantly increased levels of neutrophil gene transcripts encoding CG, HNE and PR3 proteins have been documented by Almansa et al. in COPD patients with acute respiratory failure in comparison to COPD subjects with no critical disease exacerbation. Moreover, Sinden et al. has recently showed that PR3 activity was greater than HNE activity (largely undetectable) in sputum from subjects with AATD (PI*ZZ) and COPD (PI*MM) as well as in non-deficient COPD patients during exacerbations. The role of HNE in pathogenesis of COPD has been studied extensively and is quite well understood.
Meanwhile, the role of PR3 and CG in lung tissue destruction is not clear and much underestimated. F and Z A1AT variants are considered to be less functional against HNE and trypsin but there are no data on their activity against PR3 and CG. We intend to apply the innovative highly specific fluorescence resonance energy transfer (FRET) substrates for HNE, PR3 and CG which enable measurement of sub-nanomolar concentrations in biological fluids.
Therefore, this project is aimed at evaluation of genetic A1AT variants inhibitory activity towards NSPs and trypsin (as a control). We hypothesize that both mutated A1AT alleles might present diminished inhibitory activity against PR3 and CG in addition to the defective anti-HNE and anti-trypsin protection. We hope to provide insight into the individual role of each serine proteinase in tissue lung destruction and thus to better characterize the role of qualitative A1AT deficiency in the pathogenesis of COPD.
To verify this hypothesis following problems will be researched:
- Are distorted A1AT protein conformations, dysfunctional (PI*F) and deficiency (PI*Z), responsible for diminished inhibitory activity against PR3 and CG?
- If so, then to what extent the anti-PR3 and CG protection deficit is comparable to that seen for HNE and trypsin?
The specific aims of the study:
- To investigate the in vitro inhibitory activity of serum-derived mutated A1AT proteins towards: HNE, PR3, CG and trypsin in comparison to normal MM variant by FRET and colorimetric methods. Z, M and F variants of AAT will be purified from peripheral blood of subjects with FM, ZZ and MM genotypes by the exchange chromatography.
- To analyze systemic concentration of NSPs: HNE, PR3 and CG and NSPs inhibitors (AAT, alpha(1)-antichymotrypsin, antithrombin) in sera of respiratory patients with FM, ZZ and MM genotypes as well as healthy MM controls by ELISA method.
- To investigate systemic in vivo inhibitory activity of serum-derived mutated FM and ZZ AAT protein towards HNE, PR3, CG and trypsin in comparison to normal MM variant by FRET and colorimetric methods.
- To semi-quantitatively evaluate the presence of cleaved AAT form in urine samples from COPD subjects with FM, ZZ, MM genotypes as well as healthy MM controls collected under stable conditions and repeatedly during exacerbation by Western-blot method followed by mass spectrometry.
Curriculum Vitae of Beata Poplawska:
Beata has been working since February 2011 as a PhD student at the National Institute of Tuberculosis and Lung Diseases, Department of Genetics and Clinical Immunology in Warsaw under the supervision of Prof. Joanna Chorostowska-Wynimko. Her PhD thesis and current interest are focused on the biological activity of A1AT and its mutated forms, in particular inhibitory activities in the context of lung pathology in alpha-1 antitrypsin deficient subjects (A1ATD).
Beata is actively involved in the nation-wide AATD diagnostic program for respiratory patients in Poland organized and managed by their Department. Likewise, she is involved with the international scientific program for Central Eastern European Alpha-1 Antitrypsin Network and is part of the team responsible for the on-going large scale population screening in newborns from Central Poland.
Her eALTA project "Inhibitory activity of alpha-1 antitrypsin genetic variants towards neutrophil serine proteinases" focuses on the inhibitory activity of AAT genetic variants towards three related neutrophil serine proteinases (NSPs): human neutrophil elastase (HNE), proteinase 3 (PR3) and cathepsin G (CG). The role of HNE has been studied extensively in COPD and is generally well understood, but the role of PR3 and CG in lung tissue destruction is not clear and perhaps underestimated. Beata hopes to elucidate the effect that two common gene mutations, PI*F and PI*Z A1AT, have on inhibiting these NSPs compared to the ‘normal' gene variant PI*M in in vitro and in vivo conditions. Furthermore she will also evaluate the serum concentration of both the NSPs and their inhibitors as well as the presence of cleaved AAT form in urine samples.
National Institute of Tuberculosis and Lung Diseases
Department of Genetics and Clinical Immunology