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

Autor(es)

Derradi, R. ^[1] ; Bolean, M. ^[1] ; Simao, A. M. S. ^[1] ; Caseli, L. ^[2] ; Millan, J. L. ^[3] ; Bottini, M. ^{[3, 4]} ; Ciancaglini, P. ^[1] ; Ramos, A. P. ^[1]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Dept Chem, Fac Philosophy Sci & Letters Ribeirao Preto, Dept Chem, Ave Bandeirantes 3900, BR-14040901 Ribeirao Preto, SP - Brazil

^[2] Univ Fed Sao Paulo, Inst Environm Chem & Pharmaceut Sci, Rua Sao Nicolau, 210 Ctr, BR-09913030 Diadema, SP - Brazil

^[3] Sanford Burnham Prebys Med Discovery Inst, La Jolla, CA 92037 - USA

^[4] Univ Roma Tor Vergata, Dept Expt Med, I-00133 Rome - Italy

• Tipo de documento: Artigo Científico
• Fonte: Langmuir; v. 35, n. 47, p. 15232-15241, NOV 26 2019.
• Citações Web of Science: 0

Resumo

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. (AU)

• Processo FAPESP: 17/08892-9 - Superfícies bioativas obtidas a partir de filmes Langmuir-Blodgett e biominerais
• Beneficiário: Ana Paula Ramos
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 15/03594-4 - Estudo da adsorção de fosfatase alcalina em monocamadas de Langmuir contendo microdomínios lipídicos ricos em colesterol
• Beneficiário: Rafael Derradi
• Modalidade de apoio: Bolsas no Brasil - Mestrado
• Processo FAPESP: 14/11941-3 - Colágeno e annexina são responsáveis pelo controle do processo de biomineralização?
• Beneficiário: Pietro Ciancaglini
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 16/21236-0 - Sistemas miméticos de vesículas da matriz extracellular (MVs) para o estudo da regulação do processo de biomineralização: proteolipossomos contendo NPP1 e Anexina V
• Beneficiário: Pietro Ciancaglini
• Modalidade de apoio: Auxílio à Pesquisa - Regular