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. 2015 Oct 15:584:79-89.
doi: 10.1016/j.abb.2015.08.018. Epub 2015 Aug 29.

Proteoliposomes with the ability to transport Ca(2+) into the vesicles and hydrolyze phosphosubstrates on their surface

Maytê Bolean  1 , Ana Maria S Simão  1 , Tina Kiffer-Moreira  2 , Marc F Hoylaerts  3 , José Luis Millán  2 , Rosangela Itri  4 , Pietro Ciancaglini  5
Affiliations

Proteoliposomes with the ability to transport Ca(2+) into the vesicles and hydrolyze phosphosubstrates on their surface

Maytê Bolean et al. Arch Biochem Biophys. .

Abstract

We describe the production of stable DPPC and DPPC:DPPS-proteoliposomes harboring annexin V (AnxA5) and tissue-nonspecific alkaline phosphatase (TNAP) and their use to investigate whether the presence of AnxA5 impacts the kinetic parameters for hydrolysis of TNAP substrates at physiological pH. The best catalytic efficiency was achieved in DPPS 10%-proteoliposomes (molar ratio), conditions that also increased the specificity of TNAP hydrolysis of PPi. Melting behavior of liposomes and proteoliposomes was analyzed via differential scanning calorimetry. The presence of 10% DPPS in DPPC-liposomes causes a broadening of the transition peaks, with AnxA5 and TNAP promoting a decrease in ΔH values. AnxA5 was able to mediate Ca(2+)-influx into the DPPC and DPPC:DPPS 10%-vesicles at physiological Ca(2+) concentrations (∼2 mM). This process was not affected by the presence of TNAP in the proteoliposomes. However, AnxA5 significantly affects the hydrolysis of TNAP substrates. Studies with GUVs confirmed the functional reconstitution of AnxA5 in the mimetic systems. These proteoliposomes are useful as mimetics of mineralizing cell-derived matrix vesicles, known to be responsible for the initiation of endochondral ossification, as they successfully transport Ca(2+) and possess the ability to hydrolyze phosphosubstrates in the lipid-water interface.

Keywords: Alkaline phosphatase; Annexin V; Calorimetry; DPPC; DPPS; Liposome; Matrix vesicles; Proteoliposome.

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Figures

Fig. 1
Fig. 1
Effect of increasing DPPS concentration into DPPC-liposomes on TNAP activity incorporation.
Fig. 2
Fig. 2
Effect of increasing concentrations of (●) ATP, (■) ADP and (▴) PPi substrates on the Pi-generating activity of DPPC-proteoliposomes harboring TNAP. Assays were done at37 °C, buffered with 50 mM Tris–HCl, pH 7.4, containing 2 mM MgCl2, and the Pi released was measured. Inset: Hill coefficient (n).
Fig. 3
Fig. 3
Effect of the liposome composition ((■) DPPC 100%; (●) DPPC:DPPS 5%; (▴) DPPC:DPPS 10% and (◆) DPPC:DPPS 15%, molar ratio) on the kinetic parameters for substrates hydrolysis by TNAP reconstituted in proteoliposomes carrying AnxA5: (A) ATPase activity; (B) ADPase activity and (C) PPiase activity (U/mg). Inset: Hill coefficient (n).
Fig. 4
Fig. 4
DSC thermograms of liposomes and proteoliposomes. (A) DSC thermograms were registered using a Nano-DSC II calorimeter and processed in Excess heat capacity, Cp (kcal/K mol), as a function of temperature (°C) for vesicles constituted by DPPC and DPPC:DPPS 10% (molar ratio) containing TNAP and AnxA5: [a] DPPC-liposomes; [b] DPPC:DPPS 10%-liposomes; [c] DPPC:DPPS 10%-proteoliposomes containing TNAP + AnxA5 and [d] DPPC:DPPS 10%-proteoliposomes containing TNAP. (B): [a] DPPC-liposomes; [b] DPPC:DPPS 10%-liposomes and [c] DPPC:DPPS 10%-proteoliposomes containing AnxA5.
Fig. 5
Fig. 5
Ca2+ influx into the proteoliposomes of different lipid compositions (○) DPPC-proteoliposomes and (●) DPPC:DPPS 10%-proteoliposomes: (A) Proteoliposomes carrying AnxA5 and (B) Proteoliposomes carrying AnxA5 and TNAP. The background values of liposomes were discounted from the values obtained for the respective proteoliposomes.
Fig. 6
Fig. 6
Morphological changes of GUVs exposed to an electric field (10 V, 1 MHz) in the presence or absence of the AnxA5. Images obtained in phase contrast, objective 60x and scale bar corresponds to 20 μm. (A) GUVs constituted by DOPC with 0.5 mM CaCl2 in the inner compartment exhibit spherical shape; (B) Under the electric field the GUVs assume prolate shapes; (C) GUVs incubated with AnxA5 (8.22 μM) and subjected to the electrical field display oblate shapes; (D) GUVs recovered its initial spherical shape with time (∼20 min) after the outer and inner solution conductivities are equilibrated.
Fig. 7
Fig. 7
Morphological changes of GUVs exposed to an electric field (10 V, 1 MHz) in the presence or absence of the AnxA5. Images obtained in phase contrast, objective 60× and scale bar corresponds to 20 μm. (A) GUVs constituted by DOPC:DPPS 10% (molar ratio) with 0.5 mM CaCl2 in the inner compartment exhibit spherical shape; (B) Under the electric field the GUVs assume prolate shapes; (C) GUVs incubated with AnxA5 (8.22 μM) and subjected to the electrical field display oblate shapes; (D) GUVs recovered its initial spherical shape with time (∼20 min) after the outer and inner solution conductivities are equilibrated.
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