tailieunhanh - Báo cáo khoa học: Characterization of a monoclonal antibody as the first specific inhibitor of human NTP diphosphohydrolase-3 Partial characterization of the inhibitory epitope and potential applications

The study and therapeutic modulation of purinergic signaling is hindered by a lack of specific inhibitors for NTP diphosphohydrolases (NTPDases), which are the terminating enzymes for these processes. In addition, little is known of the NTPDase protein structural elements that affect enzymatic activity and which could be used as targets for inhibitor design. | Characterization of a monoclonal antibody as the first specific inhibitor of human NTP diphosphohydrolase-3 Partial characterization of the inhibitory epitope and potential applications Mercedes N. Munkonda1 Julie Pelletier1 Vasily V. Ivanenkov2 Michel Fausther1 Alain Tremblay1 Beat Kunzli3 Terence L. Kirley2 and Jean Sevigny1 1 Centre de Recherche en Rhumatologie et Immunologie Centre Hospitalier Universitaire de Quebec Universite Laval Canada 2 Department of Pharmacology and CellBiophysics College of Medicine University of Cincinnati OH USA 3 Department of GeneralSurgery Universitat Munchen Germany Keywords ATP immunologicaltechniques inhibitor monoclonalantibody NTPDase Correspondence J. Sevigny Centre de Recherche en Rhumatologie et Immunologie Centre Hospitalier Universitaire de Quebec CHUQ 2705 Boulevard Laurier local T1-49 Quebec QC G1V 4G2 Canada Fax 1 418 654 2765 Tel 1 418 654 2772 E-mail Received 19 September 2008 revised 11 November 2008 accepted 12 November 2008 doi The study and therapeutic modulation of purinergic signaling is hindered by a lack of specific inhibitors for NTP diphosphohydrolases NTPDases which are the terminating enzymes for these processes. In addition little is known of the NTPDase protein structural elements that affect enzymatic activity and which could be used as targets for inhibitor design. In the present study we report the first inhibitory monoclonal antibodies specific for an NTPDase namely human NTPDase3 EC as assessed by ELISA western blotting flow cytometry immunohistochemistry and inhibition assays. Antibody recognition of NTPDase3 is greatly attenuated by denaturation with SDS and abolished by reducing agents indicating the significance of the native conformation and the disulfide bonds for epitope recognition. Using site-directed chemical cleavage the SDS-resistant parts of the epitope were located in two fragments of the C-terminal lobe of .