000135355 001__ 135355
000135355 005__ 20250908131442.0
000135355 0247_ $$2doi$$a10.1021/acsptsci.4c00065
000135355 0248_ $$2sideral$$a138627
000135355 037__ $$aART-2024-138627
000135355 041__ $$aeng
000135355 100__ $$aSenan-Salinas, Ana
000135355 245__ $$aSelective Detection of Active Extracellular Granzyme A by Using a Novel Fluorescent Immunoprobe with Application to Inflammatory Diseases
000135355 260__ $$c2024
000135355 5060_ $$aAccess copy available to the general public$$fUnrestricted
000135355 5203_ $$aGranzymes (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.
000135355 536__ $$9info:eu-repo/grantAgreement/ES/AEI/PID2020-113963RB-I00$$9info:eu-repo/grantAgreement/ES/DGA/B29-23R$$9info:eu-repo/grantAgreement/ES/DGA/LMP139-21$$9info:eu-repo/grantAgreement/EC/H2020/859908/EU/Novel Applications in 19F Magnetic Resonance Imaging/NOVA-MRI$$9This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No H2020 859908-NOVA-MRI$$9info:eu-repo/grantAgreement/ES/MCIU-AEI/SAF2017-83120-C2-1-R$$9info:eu-repo/grantAgreement/ES/MINECO/FJCI-2017-31629$$9info:eu-repo/grantAgreement/ES/MINECO/IJC-2019-039192-I
000135355 540__ $$9info:eu-repo/semantics/openAccess$$aby$$uhttp://creativecommons.org/licenses/by/3.0/es/
000135355 592__ $$a1.23$$b2024
000135355 593__ $$aPharmacology (medical)$$c2024$$dQ1
000135355 593__ $$aPharmacology$$c2024$$dQ1
000135355 655_4 $$ainfo:eu-repo/semantics/article$$vinfo:eu-repo/semantics/publishedVersion
000135355 700__ $$aComas, Laura
000135355 700__ $$aEsteban, Patricia
000135355 700__ $$0(orcid)0000-0001-6778-0636$$aGarzón-Tituaña, Marcela
000135355 700__ $$aCheng, Zhiming
000135355 700__ $$0(orcid)0000-0002-1861-5981$$aSantiago, Llipsy
000135355 700__ $$aDomingo, Maria Pilar
000135355 700__ $$0(orcid)0000-0002-3888-7036$$aRamírez-Labrada, Ariel
000135355 700__ $$0(orcid)0000-0002-9600-8116$$aPaño-Pardo, José Ramón$$uUniversidad de Zaragoza
000135355 700__ $$aVendrell, Marc
000135355 700__ $$0(orcid)0000-0003-0154-0730$$aPardo, Julián$$uUniversidad de Zaragoza
000135355 700__ $$0(orcid)0000-0002-9730-2210$$aArias, Maykel A.
000135355 700__ $$aGalvez, Eva M.$$uUniversidad de Zaragoza
000135355 7102_ $$11006$$2255$$aUniversidad de Zaragoza$$bDpto. Fisiatría y Enfermería$$cÁrea Enfermería
000135355 7102_ $$11007$$2610$$aUniversidad de Zaragoza$$bDpto. Medicina, Psiqu. y Derm.$$cArea Medicina
000135355 7102_ $$11011$$2566$$aUniversidad de Zaragoza$$bDpto. Microb.Ped.Radio.Sal.Pú.$$cÁrea Inmunología
000135355 773__ $$g7, 5 (2024), 1474-1484$$pACS pharmacol. transl. sci.$$tACS pharmacology & translational science$$x2575-9108
000135355 8564_ $$s3369282$$uhttps://zaguan.unizar.es/record/135355/files/texto_completo.pdf$$yVersión publicada
000135355 8564_ $$s3171545$$uhttps://zaguan.unizar.es/record/135355/files/texto_completo.jpg?subformat=icon$$xicon$$yVersión publicada
000135355 909CO $$ooai:zaguan.unizar.es:135355$$particulos$$pdriver
000135355 951__ $$a2025-09-08-12:59:58
000135355 980__ $$aARTICLE