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 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.

Proteoliposomes in nanobiotechnology

“…Experiments confirmed that the interaction between detergentsolubilized TNAP, free of detergent excess, and the lipid bilayer of liposomes occurs via the GPI-anchor of the enzyme and our method of incorporation via direct insertion guarantees that the enzyme is associated with the external leaflet of the liposome membrane (Camolezi et al 2002). Enzyme incorporation is time-dependent and the incorporation yields depend of the lipid composition used to form the vesicles (Simão et al 2010a). An important point to be observed is that the enzyme bound to liposomes retains the ability to hydrolyze the different substrates, such as ATP, ADP, AMP, PP i , and p-nitrophenylphosphate (pNPP) Simão et al 2010a, b;Bolean et al 2010).…”

“…1). By changing the type and proportion of the lipids, incubation time, method used for detergent removal, or even the velocity by which the detergent is removed, different vesicular systems can be obtained, varying in the type and quantity of proteins that can be reconstituted (Camolezi et al 2002;Ierardi et al 2002;Daghastanli et al 2004;Santos et al 2005Santos et al , 2006bRigos et al 2008Rigos et al , 2010Simão et al 2010a;Bolean et al 2010Bolean et al , 2011.…”

“…Na,K-ATPase. For simpler proteins, the preferred source is the expression and purification of recombinant proteins (Simão et al 2007(Simão et al , 2010a.…”

Cellular function and molecular structure of ecto-nucleotidases

“…Of relevance in the present context, APs can hydrolyze extracellular ATP via ADP and AMP to adenosine [362,363,514] (Fig. 4).…”

“…Recently, it has been demonstrated that soluble heterologously expressed murine NPP1 [362,363] or NPP3 [7] hydrolyzes (at pH 7.4) PP i in addition to ATP. This result corroborates earlier observations that NPP2 cleaves PP i into P i [364].…”