Subsequently, plates were washed three times with TBS to remove both bound and free AMPPCP, as well as free hN3-B3S, and ATPase activity was assayed. a non-hydrolyzable substrate analog AMPPCP, as well as location of the epitope adjacent to the active site, suggest a noncompetitive mechanism of inhibition by steric hindrance. The described technique should be useful for systematic epitope mapping in cell membrane proteins for which either a 3D structure is available, or a sufficiently accurate 3D model can be obtained by homology modeling. Keywords:alanine scanning mutagenesis, ecto-nucleotidase, epitope mapping, NTPDase, site-directed masking == Introduction == Identification of discontinuous B-cell epitopes remains NS-2028 a challenging task with no common solution despite a variety of available experimental techniques and epitope prediction methods (Gershoniet al., 2007;Ponomarenkoet al., 2008;Yang and Yu, 2009). The recently invented technique of site-directed masking offers a systematic means of epitope mapping, if a three-dimensional structure of a protein antigen is available (Paus and Winter, 2006). In this technique, a panel of single cysteine mutations distributed evenly on a protein antigen surface are constructed, and then used to chemically tether the antigen via a cysteine residue and a linker to a solid phase, NS-2028 hence masking an area around the cysteine and preventing access of antibody to this area. The mutations which block antibody binding define the location of the antibody epitope, which can be further refined by alanine scanning mutagenesis. We adapted this technique, which was originally described for purified soluble antigens, to map epitopes in unpurified proteins expressed on the cell surface. We used this method to define the binding site of an inhibitory monoclonal antibody to human ecto-nucleotidase NTPDase3, and to gain insight into the mechanism of inhibition. Plasma membrane nucleoside triphosphate diphosphohydrolases (NTPDase1, 2, 3 and 8) are integral membrane proteins with two membrane-spanning domains and a catalytic site facing the extracellular space (Robsonet al., 2006). These enzymes hydrolyze the and phosphates of extracellular nucleotides, and function as the major regulators and terminators of purinergic signaling (Kukulskiet al., 2005). They are viewed as potential therapeutic targets, and efforts were made to develop subtype-selective NTPDase inhibitors (Gendronet al., 2002). Modulation of nucleotide signaling by specific NTPDase inhibitors is thought to have a profound impact on different physiological systems under normal and pathological conditions, and thus could find applications as both experimental and pharmacological tools. Many synthetic compounds have been reported to inhibit NTPDases, including nucleotide analogs and mimetics (Brunschweigeret al., 2008), sulfonated dyes (Iqbalet al., 2005;Munkondaet al., 2007) and polyoxometalates (Mulleret al., 2006), but all of them have relatively low affinity to the enzymes, and none can clearly discriminate between different isoenzymes [discussed in (Munkondaet al., 2009)]. We have recently described a monoclonal antibody hN3-B3Sas a novel and specific inhibitor of human NTPDase3 that decreased enzymatic activity by 6090% depending on the assay conditions (Munkondaet al., 2009). Considering the location of NTPDases on the plasma membrane, which makes them readily accessible, as well as high affinity and selectivity characteristic of antibodies, the antibodies could function as potent and highly specific inhibitors of NTPDases bothin vitroandin vivo. In order to better understand the mechanism of inhibition and to define the means for improvement of inhibitory activity of hN3-B3S, Rabbit polyclonal to LAMB2 we report here the mapping of NS-2028 the antibody epitope in human NTPDase3 using a modification of the site-directed masking technique and a 3D structure of NTPDase3 generated by homology modeling. Our data suggest that hN3-B3Sdoes not compete with substrate, but rather binds NTPDase3 in close proximity to the active site and inhibits the enzymatic activity by steric hindrance. The described method is likely to be useful for systematic epitope mapping in unpurified cell surface NS-2028 NS-2028 proteins for which a 3D structure is available, or can be obtained with adequate accuracy by homology modeling. == Materials and methods == == Monoclonal antibody == Production and characterization of monoclonal antibodies hN3-B3Sand hN3-H10Shas been described (Munkondaet al., 2009). These antibodies are specific to human NTPDase3 and are unique in their ability to inhibit enzymatic activity of NTPDase3 by 6090% depending on conditions. hN3-B3Sand hN3-H10Santibodies are produced by two hybridoma clones, but they performed similarly in all assays, and therefore are likely.