NPP1 and TNAP hydrolyze ATP synergistically during biomineralization

Autor(es)

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

• Tipo de documento: Artigo Científico
• Fonte: PURINERGIC SIGNALLING; v. N/A, p. 14-pg., 2022-07-23.

Resumo

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)

• Processo FAPESP: 19/25054-2 - Nanopartículas contendo estrôncio e sua versatilidade na fabricação de biomateriais: implicações e aplicações em biomineralização
• Beneficiário: Ana Paula Ramos
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 19/08568-2 - Investigação do papel de vesículas extracelulares (VEs) na iniciação, propagação, regeneração e modelação da mineralização biológica
• Beneficiário: Pietro Ciancaglini
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 21/13140-1 - Estudo da interação de PHOSPHO1 e nSMase2 com membranas modelo: uma possível correlação na secreção de vesículas da matriz
• Beneficiário: Luiz Henrique da Silva Andrilli
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 17/20846-2 - Explorando o papel da matriz lipídico-proteica-mineral na biomineralização óssea: uma abordagem biofísica empregando-se filmes automontados
• Beneficiário: Marcos Antonio Eufrásio Cruz
• Modalidade de apoio: Bolsas no Brasil - Doutorado