NPP1 and TNAP hydrolyze ATP synergistically during biomineralization

Author(s)

Andrilli, Luiz H. S. ; Sebinelli, Heitor G. ; Favarin, Bruno Z. ; Cruz, Marcos A. E. ; Ramos, Ana Paula ; Bolean, Mayte ; Millan, Jose Luis ; Bottini, Massimo ; Ciancaglini, Pietro

• Document type: Journal article
• Source: PURINERGIC SIGNALLING; v. N/A, p. 14-pg., 2022-07-23.

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 (PPi), a calcification inhibitor. The P-i/PPi 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 PPi as well as P-i from ATP and tissue-nonspecific alkaline phosphatase (TNAP) that hydrolyzes both ATP and PPi 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/PPi 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. (AU)

• FAPESP's process: 19/25054-2 - Strontium-containing nanoparticles and their versatility for biomaterials fabrication: implications and applications in biomineralization
• Grantee: Ana Paula Ramos
• Support Opportunities: Regular Research Grants
• FAPESP's process: 19/08568-2 - Investigation of the extracellular vesicles (VEs) role in the initiation, propagation, regeneration, and modeling of biological mineralization
• Grantee: Pietro Ciancaglini
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 21/13140-1 - Study of PHOSPHO1 and nSMase2 interaction with model membranes: a possible correlation in matrix vesicle secretion
• Grantee: Luiz Henrique da Silva Andrilli
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 17/20846-2 - Exploring the role of lipid-protein-mineral matrix on bone biomineralization: a biophysical approach using self-assembled films
• Grantee: Marcos Antonio Eufrásio Cruz
• Support Opportunities: Scholarships in Brazil - Doctorate