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. 2019 Jul;37(4):607-613.
doi: 10.1007/s00774-018-0962-8. Epub 2018 Oct 15.

Lipid microenvironment affects the ability of proteoliposomes harboring TNAP to induce mineralization without nucleators

Ana Maria Sper Simão #  1 , Maytê Bolean #  1 , Bruno Zoccaratto Favarin  1 , Ekeveliny Amabile Veschi  1 , Camila Bussola Tovani  1 , Ana Paula Ramos  1 , Massimo Bottini  2   3 , Rene Buchet  4   5   6   7   8 , José Luis Millán  3 , Pietro Ciancaglini  9
Affiliations

Lipid microenvironment affects the ability of proteoliposomes harboring TNAP to induce mineralization without nucleators

Ana Maria Sper Simão et al. J Bone Miner Metab. 2019 Jul.

Abstract

Tissue-nonspecific alkaline phosphatase (TNAP), a glycosylphosphatidylinositol-anchored ectoenzyme present on the membrane of matrix vesicles (MVs), hydrolyzes the mineralization inhibitor inorganic pyrophosphate as well as ATP to generate the inorganic phosphate needed for apatite formation. Herein, we used proteoliposomes harboring TNAP as MV biomimetics with or without nucleators of mineral formation (amorphous calcium phosphate and complexes with phosphatidylserine) to assess the role of the MVs' membrane lipid composition on TNAP activity by means of turbidity assay and FTIR analysis. We found that TNAP-proteoliposomes have the ability to induce mineralization even in the absence of mineral nucleators. We also found that the addition of cholesterol or sphingomyelin to TNAP-proteoliposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine reduced the ability of TNAP to induce biomineralization. Our results suggest that the lipid microenvironment is essential for the induction and propagation of minerals mediated by TNAP.

Keywords: Alkaline phosphatase; Biomineralization; Matrix vesicles; Nucleational core; Proteoliposome.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Achievement of Vmax for the hydrolysis of ATP by TNAP-proteoliposomes composed of different lipid compositions: (black) DPPC, (green) DPPC:Chol (9:1), (red) DPPC:SM (9:1) and (blue) DPPC:Chol:SM (8:1:1) (molar ratios). Arrows indicate the saturating ATP concentration for each distinct proteoliposome. Inset: representation to determine Hill coefficient (n). Data were reported as the mean of triplicate measurements of three different enzyme preparations
Fig. 2
Fig. 2
Effect of TNAP-proteoliposomes composed of DPPC, DPPC:Chol (9:1), DPPC:SM (9:1) and DPPC:Chol:SM (8:1:1) (molar ratios), on mineral propagation in the absence of nucleator (blank) and in the presence of ACP (blue) or PS-CPLX-seeded SCL (red), at pH 7.5. The assay was accomplished in SCL buffer with a saturating ATP concentration for TNAP activity for each distinct proteoliposome. ATP concentrations of 6 mM, 10 mM, 5 mM, and 9 mM were used for the proteoliposomes composed of DPPC, DPPC:Chol, DPPC:SM and DPPC:Chol:SM, respectively, as indicated by arrows in Fig. 1. Enzyme-devoid liposomes were used as control and bars show the increment in absorbencies after 48 h of incubation at 37 °C. All results are expressed as mean ± SEM. P < 0.05
Fig. 3
Fig. 3
FTIR spectra of the minerals obtained from the mineralization assays under incubation of TNAP-proteoliposomes composed of (green) DPPC, (black) DPPC:Chol (9:1), (pink) DPPC:SM (9:1) and (blue) DPPC:Chol:SM (8:1:1) (molar ratios), in SCL, at 37 °C, at pH 7.5, in the absence of nucleators. ATP concentrations of 6 mM, 10 mM, 5 mM, and 9 mM were used for the proteoliposomes composed of DPPC, DPPC:Chol, DPPC:SM and DPPC:Chol:SM, respectively, as indicated by arrows in Fig. 1. Mineralization was followed by the differences in the ratio between the areas of the internal reference band of the phospholipid (C=O) at 1740 cm−1 and the band corresponding to the asymmetrical stretching of the PO 43− group at 1032 cm−1
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