NPP1 and TNAP hydrolyze ATP synergistically during biomineralization

Abstract

Matrix vesicles (MVs) are a special class of extracellular vesicles released by mineralizing cells during bone and tooth mineralization that initiate the precipitation of apatitic minerals by regulating the extracellular ratio between inorganic phosphate (P_i), a calcification promoter, and pyrophosphate (PP_i), a calcification inhibitor. The P_i/PP_i ratio is thought to be controlled by two ecto-phosphatases present on the outer leaflet of the MVs' membrane: ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) that produces PP_i as well as P_i from ATP and tissue-nonspecific alkaline phosphatase (TNAP) that hydrolyzes both ATP and PP_i to generate P_i. However, if and how these enzymes act in concert in MVs are still unclear. Herein, we investigated the role of NPP1 and TNAP in ATP hydrolysis during MV-mediated biomineralization using proteoliposomes as a biomimetic model for MVs. Proteoliposomes composed by 1,2-dipalmitoylphosphatidylcholine (DPPC) and harboring NPP1 alone, TNAP alone, or both together at different molar ratios (1:1, 10:1, and 1:10) were fabricated. After 48 h of incubation with ATP, TNAP-containing proteoliposomes consumed more ATP than NPP1-containing vesicles (270 and 210 nmol, respectively). Both types of vesicles comparatively formed ADP (205 and 201 nmol, respectively), while NPP1-containing vesicles hydrolyzed AMP less efficiently than TNAP-containing proteoliposomes (10 and 25 nmol, respectively). In vitro mineralization assays showed that in the presence of ATP, TNAP-harboring proteoliposomes mineralized through a sigmoidal single-step process, while NPP1-harboring vesicles displayed a two-step mineralization process. ATR-FTIR analyses showed that the minerals produced by TNAP-harboring proteoliposomes were structurally more similar to hydroxyapatite than those produced by NPP1-harboring vesicles. Our results with proteoliposomes indicate that the pyrophosphohydrolase function of NPP1 and the phosphohydrolase activity of TNAP act synergistically to produce a P_i/PP_i ratio conducive to mineralization and the synergism is maximal when the two enzymes are present at equimolar concentrations. The significance of these findings for hypophosphatasia is discussed.

Keywords: NPP1; Phosphohydrolase; Proteoliposomes; Pyrophosphohydrolase; TNAP.

Fig. 1

Hydrolysis of ATP and formation of ADP and AMP by proteoliposomes harboring TNAP and/or NPP1 in the absence (A, B, and C) and in the presence (D, E, and F) of 0.5 mM of suramin. Changes with time of the concentration of ATP (A, D), ADP (B, E), and AMP (C, F), in dispersions of proteoliposomes harboring NPP1 (red symbols), TNAP (black symbols), NPP1:TNAP at a 1:1 molar ratio (blue symbols), NPP1:TNAP at a 1:10 molar ratio (green symbols), and NPP1:TNAP at a 10:1 molar ratio (magenta symbols). The reaction was initiated by adding 2.0 mM (1000 nmol) of ATP to the vesicle solutions. The insets showed the concentrations of ATP, ADP, and AMP in the proteoliposome dispersions, and the statistical differences among them, after 48 h of reaction, in the absence and in the presence of 0.5 mM of suramin. Data are reported as the mean ± SD of triplicate measurement of three independent enzyme/proteoliposome preparations. One-way ANOVA test was used to determine the statistically significant differences among the groups at a specific reaction time, while two-way ANOVA was used to determine the statistically significant differences among the reaction times for each group. *0.01 ≤ p < 0.05, **0.001 ≤ p < 0.01, ***0.0001 ≤ p < 0.001, and ****p < 0.0001

Fig. 2

Percentage of inhibition of the activity of NPP1 and/or TNAP reconstituted into DPPC-liposomes by suramin. Percentage of inhibition of ATP hydrolysis (blue bars), ADP production (dark purple bars), and AMP production (light purple bars) by proteoliposomes, after 48 h of reaction. The reaction was initiated by adding 2.0 mM of ATP to the vesicle solutions, either in the absence and in the presence of 0.5 mM of suramin. Data are reported as the mean ± SD of triplicate measurement of three independent enzyme/proteoliposome preparations. One-way ANOVA test was used to determine the statistically significant differences in the percentage of suramin inhibition on ATP hydrolysis, ADP production, and AMP production among the groups of vesicles. **0.001 ≤ p < 0.01, ***0.0001 ≤ p < 0.001, and ****p < 0.0001

Fig. 3

Mineralization curves of proteoliposomes harboring NPP1 and/or TNAP in the presence of ATP. Time-dependent changes in the value of absorbance at 340 nm of dispersions of DPPC-liposomes (control, gold symbols), and proteoliposomes harboring NPP1 (red symbols), TNAP (black symbols), NPP1:TNAP at a 1:1 molar ratio (blue symbols), NPP1:TNAP at a 1:10 molar ratio (green symbols), and NPP1:TNAP at a 10:1 molar ratio (magenta symbols) in the presence of 2.0 mM (1000 nmol) of ATP, during the first 20 h of reaction. Bar graphs (insets) showed the statistical differences in the values of turbidity after 20 h of reaction. Data are reported as the mean ± SD of triplicate measurement of three independent in vitro mineralization assays. One-way ANOVA test was used to determine the statistically significant differences among the groups after 20 h of reaction (inset), while two-way ANOVA was used to determine the statistically significant differences among the reaction times for each group during the first 8 h of reaction. **0.001 ≤ p < 0.01, ***0.0001 ≤ p < 0.001, and **** p < 0.0001

Fig. 4

ATR-FITR spectra of the minerals produced by proteoliposomes harboring NPP1 and/or TNAP after 20 h of incubation with ATP. ATR-FTIR spectra of the minerals produced by proteoliposomes harboring NPP1 (red), TNAP (black), NPP1:TNAP at a 1:1 molar ratio (blue), NPP1:TNAP at a 1:10 molar ratio (green), and NPP1:TNAP at a 10:1 molar ratio (magenta) in the presence of 2.0 mM of ATP, after 20 h of propagation. The dashed lines indicate the bands used for the analysis of the mineral phase. Data are reported as the mean of triplicate measurement of three independent ATR-FTIR mineral spectra

Fig. 5

Correlation between enzymatic pathways catalyzed by proteoliposomes harboring NPP1 and/or TNAP and mineral propagation. The rate-determining pathways are indicated in boldface