Publication: Caracterización de anticuerpos monoclonales funcionales específicos de ADAM-17
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2018-09-25
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Universidad Complutense de Madrid
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Abstract
ADAM-17 es una proteína transmembrana que forma parte de la superfamilia de lasmetaloproteasas dependientes de zinc. Presenta una organización compleja en dominiosfuncionales y participa en gran variedad de procesos celulares. Por ello, es muy importanteprofundizar en el estudio de los mecanismos que regulan la actividad de ADAM-17. El trabajorealizado en esta tesis se ha centrado en obtener herramientas que permitan regular algunosde los procesos controlados por ADAM-17: i) adhesión celular, mediada por la interacción de laintegrina α5β1 con ADAM-17; ii) procesamiento proteolítico, determinado por el corte y laliberación de dos de los sustratos de ADAM-17 (ALCAM y TNF-α). Hemos obtenido ycaracterizado dos anticuerpos monoclonales (denominados 2A10 y YES-2) que reconocenespecíficamente el dominio desintegrina de ADAM-17 humano. La especificidad de estosanticuerpos monoclonales frente a ADAM17 se determinó mediante ensayos deinmunoabsorción ligada a enzimas (ELISA), citometría de flujo y western-blot. Estos anticuerposmonoclonales ejercen un efecto inhibitorio en la interacción celular de la integrina α5β1 conADAM-17 y con fibronectina en líneas celulares tumorales y leucocitarias y, en consecuencia,inhiben la adhesión mediada por la integrina α5β1. Además, los anticuerpos monoclonales 2A10y YES-2 aumentan la actividad sheddasa de ADAM-17 en linfocitos B y monocitos. Por lo tanto,estos anticuerpos podrían utilizarse como herramientas para modular la capacidad adhesiva yproteolítica de ADAM-17.
ADAM-17 is a transmembrane protein belonging to the superfamily of zinc-dependentmetalloproteinases. This protein possesses a complex organization in functional domains andparticipates in a wide variety of cellular processes. Therefore, it is very important advance in thestudy of the mechanisms that regulate ADAM-17 activity. In this Doctoral Thesis, we havefocused on obtaining tools to regulate some of the processes controlled by ADAM-17: i) celladhesion, mediated by the interaction between α5β1 integrin with ADAM-17; ii) proteolyticectodomain release, determined by the cleavage and release of two ADAM-17 substrates(ALCAM and TNF-α). We have generated and characterized two monoclonal antibodies (named2A10 and YES-2), against the desintegrin domain of human ADAM-17. The specificity of thesemonoclonal antibodies against ADAM-17 was tested by enzyme-linked immunoadsorbent assay(ELISA), flow cytometry and western blotting. These monoclonal antibodies inhibit the cellularinteraction between the α5β1 integrin and ADAM-17 or with fibronectin in tumor and leukocytecell lines and consequently, inhibit cell adhesion mediated by the α5β1 integrin. In addition,monoclonal antibodies 2A10 and YES-2 increase the sheddase activity of ADAM-17 inB lymphocytic and monocytic cell lines. Taken together, these results suggest that theseantibodies could be used as tools to modulate the adhesive and sheddase activities of ADAM-17.
ADAM-17 is a transmembrane protein belonging to the superfamily of zinc-dependentmetalloproteinases. This protein possesses a complex organization in functional domains andparticipates in a wide variety of cellular processes. Therefore, it is very important advance in thestudy of the mechanisms that regulate ADAM-17 activity. In this Doctoral Thesis, we havefocused on obtaining tools to regulate some of the processes controlled by ADAM-17: i) celladhesion, mediated by the interaction between α5β1 integrin with ADAM-17; ii) proteolyticectodomain release, determined by the cleavage and release of two ADAM-17 substrates(ALCAM and TNF-α). We have generated and characterized two monoclonal antibodies (named2A10 and YES-2), against the desintegrin domain of human ADAM-17. The specificity of thesemonoclonal antibodies against ADAM-17 was tested by enzyme-linked immunoadsorbent assay(ELISA), flow cytometry and western blotting. These monoclonal antibodies inhibit the cellularinteraction between the α5β1 integrin and ADAM-17 or with fibronectin in tumor and leukocytecell lines and consequently, inhibit cell adhesion mediated by the α5β1 integrin. In addition,monoclonal antibodies 2A10 and YES-2 increase the sheddase activity of ADAM-17 inB lymphocytic and monocytic cell lines. Taken together, these results suggest that theseantibodies could be used as tools to modulate the adhesive and sheddase activities of ADAM-17.
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Tesis de la Universidad Complutense de Madrid, Facultad de Ciencias Biológicas, leída el 24-11-2017