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    Cholesterol Regulates the Incorporation and Catalytic Activity of Tissue-Nonspecific Alkaline Phosphatase in DPPC Monolayers

    Year: 2019

    Journal: Langmuir, Volume 35, NOV 26, page 15232–15241

    Authors: Derradi, R.; Bolean, M.; Simao, A. M. S.; Caseli, L.; Millan, J. L.; Bottini, M.; Ciancaglini, P.; Ramos, A. P.

    Organizations: Sao Paulo Research Foundation (FAPESP grants)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2015/03594-4, 2014/11941-3, 2016/21236-0, 2017/08892-9]; Coordination for the Improvement of Higher Education Personnel (CAPES)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES) [001]; Brazilian National Council of Technological and Scientific Development (CNPq)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPQ) [442834/2014-4, 304021/2017-2, 167497/2017-0]; project NanOArt from the University of Rome Tor Vergata; NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of Dental & Craniofacial Research (NIDCR) [R01DE012889] Funding Source: NIH RePORTER

    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 (PPi), a strong mineralization inhibitor, and forming inorganic phosphate (P-i), 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 (pi-A) 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.
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