The environment influence on NPP1 activity and mineral propagation

Matrix vesicles (MVs) are responsible for nucleating and propagating hydroxyapatite (HAp). This process is regulated by pyrophosphate (PPi) and phosphate (Pi) in the nucleation region. Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1), an integral membrane enzyme present in MVs, produces PPi. NPP1-proteoliposomes were produced using NPP1 at a concentration of 0.2mg/mL and liposomes composed of DPPC, DPPC:SM 9:1, DPPC:Chol 9:1, or DPPC:SM:Chol 8:1:1 (molar ratio). pNP-5`-TMP and ATP were used to investigate the phosphodiesterase and phosphomonohidrolase activities of NPP1 in reaction media with ionic strengths ranging from 0.031 to 0.127 M. The ionic strength of 0.082 M provides the optimal environment for phosphodiesterase activity in all proteoliposomes studied, while the lowest ionic strength yielded the best results for phosphomonohidrolase activity. In vitro mineralization assays were performed in with and without nucleators. DPPC:SM:Chol-proteoliposomes were incapable to propagate mineralization. PS-CPLX nucleators facilitated mineral propagation in DPPC- and DPPC:SM-proteoliposomes, while ACP nucleators enabled mineralization in DPPC:Chol-proteoliposomes. ATR-FTIR spectra of all mineral propagated samples showed the same band profile. RAMAN shifts revealed HAp bands in minerals propagated by DPPC-proteoliposomes without nucleators and DPPC:Chol-proteoliposomes with ACP nucleators. In conclusion, our findings highlight the importance of NPP1 in the propagation of Hap minerals and the influence of liposome composition and ionic strength in this process.
Financial Supports: FAPESP 2019/08568-2; 2022/04885-6 and CNPq 305426/2021-4