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 (Universidad de Zaragoza) ; Vendrell, Marc ; Pardo, Julián (Universidad de Zaragoza) ; Arias, Maykel A. ; Galvez, Eva M. (Universidad de Zaragoza)

Resumen: 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.

Idioma: Inglés

DOI: 10.1021/acsptsci.4c00065

Año: 2024

Publicado en: ACS pharmacology & translational science 7, 5 (2024), 1474-1484
ISSN: 2575-9108

Factor impacto SCIMAGO: 1.23 - Pharmacology (medical) (Q1) - Pharmacology (Q1)

Financiación: info:eu-repo/grantAgreement/ES/AEI/PID2020-113963RB-I00
Financiación: info:eu-repo/grantAgreement/ES/DGA/B29-23R
Financiación: info:eu-repo/grantAgreement/ES/DGA/LMP139-21
Financiación: info:eu-repo/grantAgreement/EC/H2020/859908/EU/Novel Applications in 19F Magnetic Resonance Imaging/NOVA-MRI
Financiación: info:eu-repo/grantAgreement/ES/MCIU-AEI/SAF2017-83120-C2-1-R
Financiación: info:eu-repo/grantAgreement/ES/MINECO/FJCI-2017-31629
Financiación: info:eu-repo/grantAgreement/ES/MINECO/IJC-2019-039192-I

Tipo y forma: Artículo (Versión definitiva)

Área (Departamento): Área Enfermería (Dpto. Fisiatría y Enfermería)
Área (Departamento): Area Medicina (Dpto. Medicina, Psiqu. y Derm.)
Área (Departamento): Área Inmunología (Dpto. Microb.Ped.Radio.Sal.Pú.)