The effect of cholesterol on the reconstitution of alkaline phosphatase into liposomes

Tissue-nonspecific alkaline phosphatase (TNAP), present on the surface of chondrocyte- and osteoblast-derived matrix vesicles (MVs), plays key enzymatic functions during endochondral ossification. Many studies have shown that MVs are enriched in TNAP and also in cholesterol compared to the plasma membrane. Here we have studied the influence of cholesterol on the reconstitution of TNAP into dipalmitoylphosphatidylcholine (DPPC)-liposomes, monitoring the changes in lipid critical transition temperature (Tc) and enthalpy variation (∆H) using differential scanning calorimetry (DSC). DPPC-liposomes revealed a Tc of 41.5 °C and ∆H of 7.63 Kcal mol−1. The gradual increase in cholesterol concentration decrease ∆H values, reaching a ∆H of 0.87 Kcal mol−1 for DPPC:cholesterol system with 36 mol% of cholesterol. An increase in Tc, up to 47 °C for the DPPC:cholesterol liposomes (36 mol% of Chol), resulted from the increase in the area per molecule in the gel phase. TNAP (0.02 mg/mL) reconstitution was done with protein:lipid 1:10,000 (molar ratio), resulting in 85% of the added enzyme being incorporated. The presence of cholesterol reduced the incorporation of TNAP to 42% of the added enzyme when a lipid composition of 36 mol% of Chol was used. Furthermore, the presence of TNAP in proteoliposomes resulted in a reduction in ∆H. The gradual proportional increase of cholesterol in liposomes results in broadening of the phase transition peak and eventually eliminates the cooperative gel-to-liquid-crystalline phase transition of phospholipids bilayers. Thus, the formation of microdomains may facilitate the clustering of enzymes and transporters known to be functional in MVs during endochondral ossification.

Graphical AbstractDifferential scanning calorimetry (DSC) thermograms were registered using a Nano-DSC II calorimeter, and processed in Excess heat capacity, Cp (kcal/K mol) as function of temperature (°C) of liposomes or proteoliposomes constituted by DPPC and cholesterol in different mol% proportion. The exothermic flow values are normalized and are multiplied by a factor in order for small exothermic peaks to be visible. Inset: phase diagram representing the initial (Ti) and final (Tf) temperature of phase transition. With increasing cholesterol concentration there is a greater range where both phases coexist: gel phase (Lβ′) and liquid-crystalline phase (Lα).Research Highlights►A pronounced decrease in ∆H when DPPC-liposomes are compared with the proteoliposomes. ►DPPC-liposome with 36 mol% of cholesterol impairs efficient protein incorporation. ►Cholesterol in the lipid vesicles results in broadening of the phase transition peak. ►Enzyme incorporation influences cooperativity, induces changes in Tc and ∆H values. ►Cholesterol increase provides decrease in ∆H and increases Tc of the phase transition.