Shedding Light on the Role of Na,K-ATPase as a Phosphatase during Matrix-Vesicle-Mediated Mineralization

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Sebinelli, Heitor Gobbi ; Andrilli, Luiz Henrique Silva ; Favarin, Bruno Zoccaratto ; Cruz, Marcos Aantonio Eufrasio ; Bolean, Mayte ; Fiore, Michele ; Chieffo, Carolina ; Magne, David ; Magrini, Andrea ; Ramos, Ana Paula ; Millan, Jose Luis ; Mebarek, Saida ; Buchet, Rene ; Bottini, Massimo ; Ciancaglini, Pietro

• Document type: Journal article
• Source: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES; v. 23, n. 23, p. 19-pg., 2022-12-01.

Abstract

Matrix vesicles (MVs) contain the whole machinery necessary to initiate apatite formation in their lumen. We suspected that, in addition to tissue-nonspecific alkaline phosphatase (TNAP), Na,K,-ATPase (NKA) could be involved in supplying phopshate (P-i) in the early stages of MV-mediated mineralization. MVs were extracted from the growth plate cartilage of chicken embryos. Their average mean diameters were determined by Dynamic Light Scattering (DLS) (212 +/- 19 nm) and by Atomic Force Microcopy (AFM) (180 +/- 85 nm). The MVs had a specific activity for TNAP of 9.2 +/- 4.6 U center dot mg(-1) confirming that the MVs were mineralization competent. The ability to hydrolyze ATP was assayed by a colorimetric method and by P-31 NMR with and without Levamisole and SBI-425 (two TNAP inhibitors), ouabain (an NKA inhibitor), and ARL-67156 (an NTPDase1, NTPDase3 and Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) competitive inhibitor). The mineralization profile served to monitor the formation of precipitated calcium phosphate complexes, while IR spectroscopy allowed the identification of apatite. Proteoliposomes containing NKA with either dipalmitoylphosphatidylcholine (DPPC) or a mixture of 1:1 of DPPC and dipalmitoylphosphatidylethanolamine (DPPE) served to verify if the proteoliposomes were able to initiate mineral formation. Around 69-72% of the total ATP hydrolysis by MVs was inhibited by 5 mM Levamisole, which indicated that TNAP was the main enzyme hydrolyzing ATP. The addition of 0.1 mM of ARL-67156 inhibited 8-13.7% of the total ATP hydrolysis in MVs, suggesting that NTPDase1, NTPDase3, and/or NPP1 could also participate in ATP hydrolysis. Ouabain (3 mM) inhibited 3-8% of the total ATP hydrolysis by MVs, suggesting that NKA contributed only a small percentage of the total ATP hydrolysis. MVs induced mineralization via ATP hydrolysis that was significantly inhibited by Levamisole and also by cleaving TNAP from MVs, confirming that TNAP is the main enzyme hydrolyzing this substrate, while the addition of either ARL-6715 or ouabain had a lesser effect on mineralization. DPPC:DPPE (1:1)-NKA liposome in the presence of a nucleator (PS-CPLX) was more efficient in mineralizing compared with a DPPC-NKA liposome due to a better orientation of the NKA active site. Both types of proteoliposomes were able to induce apatite formation, as evidenced by the presence of the 1040 cm(-1) band. Taken together, the findings indicated that the hydrolysis of ATP was dominated by TNAP and other phosphatases present in MVs, while only 3-8% of the total hydrolysis of ATP could be attributed to NKA. It was hypothesized that the loss of Na/K asymmetry in MVs could be caused by a complete depletion of ATP inside MVs, impairing the maintenance of symmetry by NKA. Our study carried out on NKA-liposomes confirmed that NKA could contribute to mineral formation inside MVs, which might complement the known action of PHOSPHO1 in the MV lumen. (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: 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
• 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: 22/05026-7 - Training in the isolation of extracellular vesicles (EVs) and MVs modeling biological mineralization
• Grantee: Heitor Gobbi Sebinelli
• Support Opportunities: Scholarships in Brazil - Technical Training Program - Technical Training