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 PPi 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 PPi 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 PPi 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.
Highlights
Detergent removal, and different reconstitution steps in DPPC liposomes for human TNAP and NPP1 isolated from transfected CHO-K1 and COS-1 culture cells, respectively
When ADP was used as a substrate, the highest rate of catalysis was observed (Fig. 5, ADP) again for TNAP proteoliposomes (ϳ25% of the initial amount), whereas the rate was comparatively lower for NPP1 proteoliposomes
DPPS and DPPC are two of the main lipids found in the MV membranes, and many studies have revealed that they play a crucial role in the biomineralization process, regulating both calcium entry into the MVs and formation of HA crystals (38 – 41)
Summary
TNAP and NPP1 Expression Constructs—The 2.5-kb human TNAP cDNA was cloned into pCMV-Script vector (Stratagene, La Jolla, CA) to express native form of TNAP. Incorporation of GPI-anchored TNAP and NPP1 into Liposomes—Equal volumes of liposomes (100 nmol of Pi/ml) and TNAP (0.02 mg/ml) or NPP1 (0.02 mg/ml) or TNAP (0.01 mg/ml) plus NPP1 (0.01 mg/ml) in 50 mmol/liter Tris-HCl buffer, pH 7.5, containing 2 mmol/liter MgCl2 were mixed and incubated at 25 °C. Enzymatic Release by TNAP and NPP1—Membrane-bound enzymes or reconstituted proteoliposomes were incubated in 50 mmol/liter Tris-HCl buffer, pH 7.4, with specific phosphatidylinositol phospholipase C (0.2 or 1 unit, as indicated, of GPI-specific PLC from Bacillus thuringiensis per ml) for 2 h, under constant rotary shaking, at 37 °C. Measurement of Nucleotide Hydrolysis by HPLC—Hydrolysis of ATP, ADP, and AMP by proteoliposomes was determined at 37 °C in 50 mmol/liter Tris-HCl buffer, pH 7.4, containing 2 mmol/liter MgCl2 and substrate. Images were collected with an L9C cooled CCD, 11.2-megapixel camera (SIA)
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