Proteoliposomes with the ability to transport Ca(2+) into the vesicles and hydrolyze phosphosubstrates on their surface.
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.
Depto. Química, FFCLRP-USP, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
Maytê
M
Bolean
Depto. Química, FFCLRP-USP, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
Ana Maria S
AM
Simão
Sanford Children's Health Research Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA.
Tina
T
Kiffer-Moreira
Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium.
Marc F
MF
Hoylaerts
Sanford Children's Health Research Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA.
José Luis
JL
Millán
Depto. Física Aplicada, Instituto de Física, IF-USP, São Paulo, SP, Brazil.
Rosangela
R
Itri
Depto. Química, FFCLRP-USP, Universidade de São Paulo, Ribeirão Preto, SP, Brazil. Electronic address: pietro@ffclrp.usp.br.
Pietro
P
Ciancaglini
D000469:Alkaline Phosphatase / Q000737:chemistry*; D017304:Annexin A5 / Q000737:chemistry*; D040761:Biomimetic Materials / Q000737:chemistry*; D002118:Calcium / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D019169:Jurkat Cells; D008081:Liposomes / Q000737:chemistry*; D010718:Phosphatidylserines / Q000737:chemistry
D017304:Annexin A5; D008081:Liposomes; D010718:Phosphatidylserines; C038694:dipalmitoylphosphatidylserine; C548267:ALPL protein, human; D000469:Alkaline Phosphatase; D002118:Calcium
Alkaline phosphatase; Annexin V; Calorimetry; DPPC; DPPS; Liposome; Matrix vesicles; Proteoliposome