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Selective Detection of Active Extracellular Granzyme A by Using a Novel Fluorescent Immunoprobe with Application to Inflammatory Diseases.
Senan-Salinas, Ana; Comas, Laura; Esteban, Patricia; Garzón-Tituaña, Marcela; Cheng, Zhiming; Santiago, Llipsy; Domingo, Maria Pilar; Ramírez-Labrada, Ariel; Paño-Pardo, José Ramón; Vendrell, Marc; Pardo, Julián; Arias, Maykel A; Galvez, Eva M.
Afiliação
  • Senan-Salinas A; Instituto de Carboquímica ICB-CSIC, 50018 Zaragoza, Spain.
  • Comas L; Instituto de Carboquímica ICB-CSIC, 50018 Zaragoza, Spain.
  • Esteban P; Fundación Instituto de Investigación Sanitaria Aragón (IIS Aragón), Biomedical Research Centre of Aragón (CIBA), 50009 Zaragoza, Spain.
  • Garzón-Tituaña M; Dept. Microbiology, Preventive Medicine and Public Health, University of Zaragoza, 50009 Zaragoza, Spain.
  • Cheng Z; CIBERINFEC, ISCIII-CIBER de Enfermedades Infecciosas, Instituto de Salud Carlos III, 28029Madrid, Spain.
  • Santiago L; Centre for Inflammation Research, The University of Edinburgh, EH164UU Edinburgh, U.K.
  • Domingo MP; IRR Chemistry Hub, Institute for Regeneration and Repair, The University of Edinburgh, EH16 4UU Edinburgh, U.K.
  • Ramírez-Labrada A; Instituto de Carboquímica ICB-CSIC, 50018 Zaragoza, Spain.
  • Paño-Pardo JR; Instituto de Carboquímica ICB-CSIC, 50018 Zaragoza, Spain.
  • Vendrell M; Fundación Instituto de Investigación Sanitaria Aragón (IIS Aragón), Biomedical Research Centre of Aragón (CIBA), 50009 Zaragoza, Spain.
  • Pardo J; CIBERINFEC, ISCIII-CIBER de Enfermedades Infecciosas, Instituto de Salud Carlos III, 28029Madrid, Spain.
  • Arias MA; Unidad de Nanotoxicología e Inmunotoxicología (UNATI), Centro de Investigación Biomédica de Aragón (CIBA), Aragón Health Research Institute (IIS Aragón), 50009Zaragoza, Spain.
  • Galvez EM; CIBERINFEC, ISCIII-CIBER de Enfermedades Infecciosas, Instituto de Salud Carlos III, 28029Madrid, Spain.
ACS Pharmacol Transl Sci ; 7(5): 1474-1484, 2024 May 10.
Article em En | MEDLINE | ID: mdl-38751645
ABSTRACT
Granzymes (Gzms), a family of serine proteases, expressed by immune and nonimmune cells, present perforin-dependent and independent intracellular and extracellular functions. When released in the extracellular space, GzmA, with trypsin-like activity, is involved in the pathophysiology of different inflammatory diseases. However, there are no validated specific systems to detect active forms of extracellular GzmA, making it difficult to assess its biological relevance and potential use as a biomarker. Here, we have developed fluorescence-energy resonance-transfer (FRET)-based peptide probes (FAM-peptide-DABCYL) to specifically detect GzmA activity in tissue samples and biological fluids in both mouse and human samples during inflammatory diseases. An initial probe was developed and incubated with GzmA and different proteases like GzmB and others with similar cleavage specificity as GzmA like GzmK, thrombin, trypsin, kallikrein, or plasmin. After measuring fluorescence, the probe showed very good specificity and sensitivity for human and mouse GzmA when compared to GzmB, its closest homologue GzmK, and with thrombin. The specificity of this probe was further refined by incubating the samples in a coated plate with a GzmA-specific antibody before adding the probe. The results show a high specific detection of soluble GzmA even when compared with other soluble proteases with very similar cleavage specificity like thrombin, GzmK, trypsin, kallikrein, or plasmin, which shows nearly no fluorescence signal. The high specific detection of GzmA was validated, showing that using pure proteins and serum and tissue samples from GzmA-deficient mice presented a significant reduction in the signal compared with WT mice. The utility of this system in humans was confirmed, showing that GzmA activity was significantly higher in serum samples from septic patients in comparison with healthy donors. Our results present a new immunoprobe with utility to detect extracellular GzmA activity in different biological fluids, confirming the presence of active forms of the soluble protease in vivo during inflammatory and infectious diseases.

Texto completo: 1 Base de dados: MEDLINE Idioma: En Ano de publicação: 2024 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Idioma: En Ano de publicação: 2024 Tipo de documento: Article