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

Abstract

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 i)-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 (π-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 the light on the biochemical and biophysical processes occurring in MV membrane during biomineralization at the molecular level.

The Past and the Future of Langmuir and Langmuir–Blodgett Films

“…Adsorption of membrane proteins has also been studied using Langmuir monolayers. This is the case of alkaline phosphatase, a protein whose polypeptide moiety is exposed outside the outer leaflet of the biomembrane but linked to the membrane layer through a glycophosphatidylinositol (GPI) anchor. − As expected, the hydrophobic anchor plays an essential role in the amphiphilicity of the enzyme. Even if solubilized with detergents but with micelles protecting the anchor, the enzyme may be incorporated into a lipid Langmuir monolayer .…”

“…205 The incorporation of alkaline phosphatase was favored in more fluid mixed monolayers of DPPC and cholesterol (CHOL), as indicated in the cartoon in Figure 14. 206 Table 3 summarizes recent, selected papers, especially from the last 3 years, dedicated to the interaction of soluble bioactive compounds in lipid monolayers representing biomembranes.…”

“…Enzyme activity was found to occur at optimized surface pressure, decreasing when the LC state for DMPA was reached . The incorporation of alkaline phosphatase was favored in more fluid mixed monolayers of DPPC and cholesterol (CHOL), as indicated in the cartoon in Figure …”

Matrix Vesicles: Role in Bone Mineralization and Potential Use as Therapeutics

“…It was found that the lipid composition could influence the formation of minerals and the activity of the enzymes or proteins [ 206 , 207 ]. For instance, the addition of cholesterol to the lipid membrane of the vesicles had an influence on the incorporated ALP [ 208 ]. A higher concentration of cholesterol in the membrane resulted in the formation of a more rigid monolayer which hampered ALP incorporation.…”

“…A higher concentration of cholesterol in the membrane resulted in the formation of a more rigid monolayer which hampered ALP incorporation. Nevertheless, the enzymatic activity of incorporated ALP was enhanced in membranes with higher concentrations of cholesterol [ 208 , 209 ]. Moreover, a recent study has shown that the presence of cholesterol and sphingomyelin in the membrane of ALP-harboring proteoliposomes promoted amorphous calcium phosphate precipitation in vitro [ 210 ].…”