Proteoliposomes harboring alkaline phosphatase and nucleotide pyrophosphatase as matrix vesicle biomimetics

Abstract

We have established a proteoliposome system as an osteoblast-derived matrix vesicle (MV) biomimetic to facilitate the study of the interplay of tissue-nonspecific alkaline phosphatase (TNAP) and NPP1 (nucleotide pyrophosphatase/phosphodiesterase-1) during catalysis of biomineralization substrates. First, we studied the incorporation of TNAP into liposomes of various lipid compositions (i.e. in pure dipalmitoyl phosphatidylcholine (DPPC), DPPC/dipalmitoyl phosphatidylserine (9:1 and 8:2), and DPPC/dioctadecyl-dimethylammonium bromide (9:1 and 8:2) mixtures. TNAP reconstitution proved virtually complete in DPPC liposomes. Next, proteoliposomes containing either recombinant TNAP, recombinant NPP1, or both together were reconstituted in DPPC, and the hydrolysis of ATP, ADP, AMP, pyridoxal-5'-phosphate (PLP), p-nitrophenyl phosphate, p-nitrophenylthymidine 5'-monophosphate, and PP(i) by these proteoliposomes was studied at physiological pH. p-Nitrophenylthymidine 5'-monophosphate and PLP were exclusively hydrolyzed by NPP1-containing and TNAP-containing proteoliposomes, respectively. In contrast, ATP, ADP, AMP, PLP, p-nitrophenyl phosphate, and PP(i) were hydrolyzed by TNAP-, NPP1-, and TNAP plus NPP1-containing proteoliposomes. NPP1 plus TNAP additively hydrolyzed ATP, but TNAP appeared more active in AMP formation than NPP1. Hydrolysis of PP(i) by TNAP-, and TNAP plus NPP1-containing proteoliposomes occurred with catalytic efficiencies and mild cooperativity, effects comparable with those manifested by murine osteoblast-derived MVs. The reconstitution of TNAP and NPP1 into proteoliposome membranes generates a phospholipid microenvironment that allows the kinetic study of phosphosubstrate catabolism in a manner that recapitulates the native MV microenvironment.

FIGURE 1.

Incorporation of polidocanol-solubilized detergent-free osteoblast-derived TNAP into DPPC liposomes (A) and SDS-PAGE of osteoblast-derived TNAP-containing DPPC liposomes (B). Lane 1, M_r standards; lane 2, TNAP-DPPC proteoliposomes, stained with silver nitrate, of ∼60 kDa, corresponding to the monomers of TNAP; lane 3, phosphohydrolytic activity of non-denaturated TNAP-DPPC proteoliposomes of ∼120 kDa, corresponding to the dimer of TNAP. The lower bands in lanes 1 and 2 represent the leading edge of the gel.

FIGURE 2.

Kinetic activity of liposome-associated osteoblast-derived TNAP. Shown is hydrolysis of pNPP (A), ATP (B), and PP_i (C) for TNAP reconstituted in DPPC (●), DPPC/DPPS (8:2) (○), and DPPC/DODAB (9:1) (▵) liposomes (A); DPPC (●), DPPC/DPPS (8:2) (○), and DPPC/DODAB (9:1) (▵) liposomes (B); and DPPC (●), DPPC/DPPS (8:2) (○), and DPPC/DODAB (8:2) (▵) liposomes (C). D, hydrolysis of pNPP (●), ATP (○), and PP_i (■). Assays were determined at 37 °C in 50 mmol/liter AMPOL, containing 2 mmol/liter MgCl₂, pH 10.0 (pNPP), pH 9.5 (ATP), and pH 9.0 (PP_i). Inset, Hill plot of the interaction of the substrate with the enzyme.

FIGURE 3.

Western blot analysis of proteoliposomes containing recombinant TNAP derived from CHO-K1 cells, recombinant NPP1 derived from COS-1 cells, and TNAP plus NPP1. Each lane was loaded with titered amounts of proteoliposomes. Positive controls are 15 ng of recombinant human TNAP and 50 ng of recombinant mouse NPP1.

FIGURE 4.

Progression with time of the disappearance of 1,000 nmol of ATP (2 mmol/liter) (A) and formation of ADP (B) and AMP (C), during hydrolysis of ATP by recombinant CHO-K1 cell-derived TNAP-DPPC proteoliposomes (3. 5 μg of total protein) (●), recombinant COS-1 cell-derived NPP1-DPPC proteoliposomes (4.1 μg of total protein) (■), and TNAP + NPP1 proteoliposomes (4.8 μg of total protein) (▴). The nucleotide concentrations were monitored by HPLC analysis.

FIGURE 5.

Progression with time of the disappearance of 1,000 nmol of ADP (2 mmol/liter) (A) and formation of AMP (B) during hydrolysis of ADP by recombinant CHO-K1 cell-derived TNAP-DPPC proteoliposomes (3.5 μg of total protein) (●), recombinant COS-1 cell-derived NPP1-DPPC proteoliposomes (4.1 μg of total protein) (■), and TNAP plus NPP1 proteoliposomes (4.8 μg of total protein) (▴). The nucleotide concentrations were monitored by HPLC analysis.

FIGURE 6.

Representation of all the possible simultaneous enzymatic reactions during catalysis of ATP by TNAP and NPP1. Indicated in boldface type are those pathways that are rate-determining in proteoliposomes, simultaneously harboring TNAP and NPP1.