Fig. 1: hTNAP^18–500 functions as dimeric, pH-dependent phosphatase.

a The phosphatase activity of the purified hTNAP^18–500 protein. The TNAP activities were inhibited by either levamisole (0.9 mM) or EGTA (0.0182 mM) treatment, the latter effect was rescued by the re-supplement of calcium (0.9 mM). optical density (OD). n = 3 biologically independent samples. b The hTNAP activities showed pH-dependence, which increased along with the pH of the reaction solutions. optical density (OD). n = 3 biologically independent samples. c The disruptions of the hTNAP^18–500 activity by the mutations at the dimeric interface. The dimeric mutants of hTNAP^18–500 proteins (TNAP with mutations at the dimeric interface) were overexpressed in HEK293T and the phosphatase activities in cell media were measured and calibrated with the expression level as determined by the Western blotting (see Supplementary Fig. 8). n = 6 biologically independent samples in blank, empty vector, R450C, R71H, N417S, G420S group. n = 4 biologically independent samples in wild-type, G420A group. n = 3 biologically independent samples in R391H group. d The crystal structure of hTNAP^18–500 dimer viewed from “side” (left) and “top” (right). Two TNAP protomers in the dimer were shown as cartoon model and colored in yellow (A) and blue (B). The sugar moieties of N-glycosylation were shown as stick and surface model and colored in pink, with the associated asparagine residues shown as stick model. The metal ions found in TNAP were shown as balls and colored in purple (Ca²⁺), green (Mg²⁺) and gray (Zn²⁺). e The dimeric interface of hTNAP^18–500. The TNAP dimer was shown as cartoon model with the protomer B colored in blue and A in transparency. The residues involved in dimerization were shown as stick model and color by elements (protomer A: gray-blue-red; protomer B: green-blue-red). f The interactions maintaining hTNAP^18–500 dimer. The protomer B was shown as calculated solvent-accessible electrostatic surface-potential maps and the interacting residues of the protomer A were shown as stick model and colored by elements, except that the residues reported with HPP related mutations were shown as stick-ball model and colored in orange-blue-red. Insets at side: the enlarged views showing the interactive sub-regions. All data in this figure are represented as mean ± SD. All experiments were repeated three times independently with similar results. Source data are provided as a Source Data file.