Chemistry Department, Faculty of Philosophy, Sciences and Letters at Ribeirao Preto, Department of Chemistry , University of Sao Paulo , Avenida Bandeirantes, 3900, Monte Alegre , Ribeirao Preto , SP Brazil , 14040-901.
Institute of Environmental, Chemical and Pharmaceutical Sciences , Federal University of Sao Paulo , Rua Sao Nicolau, 210, Centro , Diadema , SP Brazil , 09913-030.
Langmuir. 2019 Nov 26;35(47):15232-15241. doi: 10.1021/acs.langmuir.9b02590. Epub 2019 Nov 14.
Matrix vesicles (MVs) are a special class of extracellular vesicles that drive bone and dentin mineralization by providing the essential enzymes and ions for the nucleation and propagation of mineral crystals. Tissue-nonspecific alkaline phosphatase (TNAP) is an integral protein of MV membrane and participates in biomineralization by hydrolyzing extracellular pyrophosphate (PP), a strong mineralization inhibitor, and forming inorganic phosphate (P), necessary for the growth of mineral crystals inside MVs and their propagation once released in the extracellular matrix. MV membrane is enriched in cholesterol (CHOL), which influences the incorporation and activity of integral proteins in biologic membranes; however, how CHOL controls the incorporation and activity of TNAP in MV membrane has not yet been elucidated. In the present study, Langmuir monolayers were used as a MV membrane biomimetic model to assess how CHOL affects TNAP incorporation and activity. Surface pressure-area (π-) isotherms of binary dipalmitoilphosphatidylcholine (DPPC)/CHOL monolayers showed that TNAP incorporation increases with CHOL concentration. Infrared spectroscopy showed that CHOL influences the conformation and orientation of the enzyme. Optical-fluorescence micrographs of the monolayers revealed the tendency of TNAP to incorporate into CHOL-rich microdomains. These data suggest that TNAP penetrates more efficiently and occupies a higher surface area into monolayers with a lower CHOL concentration due to the higher membrane fluidity. However, the quantity of enzyme transferred to solid supports as well as the enzymatic activity were higher using monolayers with a higher CHOL concentration due to increased rigidity that changes the enzyme orientation at the air-solid interface. These data provide new insights regarding the interfacial behavior of TNAP and CHOL in MVs and shed light on the biochemical and biophysical processes occurring in the MV membrane during biomineralization at the molecular level.
基质小泡（MVs）是一类特殊的细胞外囊泡，通过提供核晶和矿化晶体生长所需的必需酶和离子来驱动骨和牙本质矿化。组织非特异性碱性磷酸酶（TNAP）是 MV 膜的整合蛋白，通过水解细胞外焦磷酸（PP），形成无机磷酸盐（P），参与生物矿化，PP 是矿化的强烈抑制剂。在 MV 膜中，TNAP 参与生物矿化，水解细胞外焦磷酸（PP），形成无机磷酸盐（P），为 MV 膜内矿化晶体的生长以及晶体在细胞外基质中释放后的传播提供必要条件。MV 膜富含胆固醇（CHOL），胆固醇影响生物膜中整合蛋白的掺入和活性；然而，CHOL 如何控制 TNAP 在 MV 膜中的掺入和活性尚未阐明。在本研究中，Langmuir 单层被用作 MV 膜仿生模型，以评估 CHOL 如何影响 TNAP 的掺入和活性。二棕榈酰磷脂酰胆碱（DPPC）/CHOL 双层的表面压力-面积（π-A）等温线表明，TNAP 的掺入量随 CHOL 浓度的增加而增加。红外光谱表明，CHOL 影响酶的构象和取向。单层的光学荧光显微镜揭示了 TNAP 倾向于掺入富含 CHOL 的微区。这些数据表明，由于膜流动性较高，TNAP 更有效地渗透并占据低 CHOL 浓度单层的更大表面积。然而，由于刚性增加，改变了酶在气-固界面的取向，因此使用高 CHOL 浓度的单层，转移到固体支持物上的酶的数量以及酶活性更高。这些数据为 MV 中 TNAP 和 CHOL 的界面行为提供了新的见解，并阐明了生物矿化过程中 MV 膜中发生的生化和生物物理过程。
