Characterization of a monoclonal antibody as the first specific inhibitor of human NTP diphosphohydrolase‐3

Abstract

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 3.6.1.5), 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 NTPDase3 (i.e. Leu220–Cys347 and Cys347–Pro485), which are both required for antibody binding. Additional site-directed mutagenesis revealed the importance of Ser297 and the fifth disulfide bond (Cys399–Cys422) for antibody binding, indicating that the discontinuous inhibitory epitope is located on the extracellular C-terminal lobe of NTPDase3. These antibodies inhibit recombinant NTPDase3 by 60–90%, depending on the conditions. More importantly, they also efficiently inhibit the NTPDase3 expressed in insulin secreting human pancreatic islet cells in situ. Because insulin secretion is modulated by extracellular ATP and purinergic receptors, this finding suggests the potential application of these inhibitory antibodies for the study and control of insulin secretion.

Cystic fibrosis remodels the regulation of purinergic signaling by NTPDase1 (CD39) and NTPDase3

“…The NTPDase1 and NTPDase3 activities were measured on both surfaces of airway epithelial cultures in Krebs buffer (pH 7.4) at 37°C (5% CO 2/95% O2) (56). They were preincubated either with the vehicle, an inhibitor of both NTPDases (1-20 mM azide), or 1-100 ng/ml monoclonal antibodies demonstrated to selectively inhibit the activity of human NTPDase3 (hN3-H10 s) (49). All reactions were initiated with the substrate and stopped by periodically collecting aliquots to be processed for HPLC analysis (56).…”

“…The tissue distribution of NTPDase1 and NTPDase3 was established in human airways as previously described (49). In brief, 6-m-thick sections of snap-frozen airway tissue from healthy donors or CF patients were fixed in an ice-cold solution of 95% acetone and 5% of 10% formalin (Fisher Scientific, Pittsburgh, PA) and then blocked in 0.1% PBS containing 7% goat serum.…”

“…In brief, 6-m-thick sections of snap-frozen airway tissue from healthy donors or CF patients were fixed in an ice-cold solution of 95% acetone and 5% of 10% formalin (Fisher Scientific, Pittsburgh, PA) and then blocked in 0.1% PBS containing 7% goat serum. The sections were incubated with anti-NTPDase1 (BU61) or anti-NTPDase3 antibodies (hN3-H10 s) (49) overnight, treated with 0.15% H2O2 in PBS to inactivate endogenous peroxidase, followed by avidin/biotin (Vector Labs, Burlington, Ontario, Canada) to prevent nonspecific staining. The sections were incubated with biotin-labeled goat antimouse secondary IgG (Jackson Immuno Research Laboratories, West Grove, PA) in the presence of the avidin-biotinylated HRP complex (VectaStain Elite ABC kit, Vector Laboratories) to optimize the reaction.…”

“…Human NTPDase1 has been extensively investigated and assigned critical roles in the prevention of ischemia-reperfusion injury (31) and transplant rejection (2,67), in thrombus formation, and inflammatory responses (6,20). In contrast, few studies addressed human NTPDase3 (49,73). In the respiratory system, NTPDase1 was detected at the mRNA level in mouse lung extract (33,61) and immunolocalized on murine alveolar cells (21).…”

“…In the respiratory system, NTPDase1 was detected at the mRNA level in mouse lung extract (33,61) and immunolocalized on murine alveolar cells (21). The recent development of a selective inhibitor of human NTPDase3 (49) provided the necessary tool to distinguish the contributions of NTPDase1 and NTPDase3 to the regulation of the P2Y 2 R agonist in human airways.…”