Fig. 2. Mapping of the polypeptide fragment that determines the extracellular release of NPP2.(A) The domain structure of NPP1 and NPP2. The hydrophobic fragment in the N-terminal domain is represented by a thick black vertical line. (B) Secretion by HEK293 cells of NPP1, NPP2 and the indicated chimaeras of the N-terminal, catalytic and nuclease-like domains of NPP1 and NPP2, as defined in panel A. NPP1-1-2 refers to a fusion of the N-terminal and catalytic domains of NPP1 and the nuclease-like domain of NPP2. The other chimaeras are annotated in the same manner. All proteins were expressed with an N-terminal HA-tag and a C-terminal Myc-tag, and were visualized after 72 hours by immunoblotting with anti-Myc antibodies. (C) Extracellular release of NPP1, NPP2 and the indicated chimaeras of the N-terminal subdomains, all expressed for 72 hours with an N-terminal HA-tag and a C-terminal Myc-tag and visualized by immunoblotting with anti-Myc antibodies. NPP1-2_12-30-1 refers to NPP1 with its N-terminal hydrophobic subdomain (residues 59-79) swapped for the corresponding subdomain (residues 12-30) of NPP2. The other fusions are annotated in the same manner. (D) Effect of mutation of the N-terminal hydrophobic domain on the secretion of HA-NPP2-Myc. The residues of the hydrophobic domain of NPP2 were replaced four by four by the corresponding residues of NPP1. The secretion of wild-type NPP1, NPP2 and the NPP2 mutants was visualized by immunoblotting with anti-Myc antibodies. All data shown in panels B-D are representative for at least three independent experiments.