Mineralization Profile of Annexin A6-Harbouring Proteoliposomes: Shedding Light on the Role of Annexin A6 on Matrix Vesicle-Mediated Mineralization

Abstract

The biochemical machinery involved in matrix vesicles-mediated bone mineralization involves a specific set of lipids, enzymes, and proteins. Annexins, among their many functions, have been described as responsible for the formation and stabilization of the matrix vesicles′ nucleational core. However, the specific role of each member of the annexin family, especially in the presence of type-I collagen, remains to be clarified. To address this issue, in vitro mineralization was carried out using AnxA6 (in solution or associated to the proteoliposomes) in the presence or in the absence of type-I collagen, incubated with either amorphous calcium phosphate (ACP) or a phosphatidylserine-calcium phosphate complex (PS–CPLX) as nucleators. Proteoliposomes were composed of 1,2-dipalmitoylphosphatidylcholine (DPPC), 1,2-dipalmitoylphosphatidylcholine: 1,2-dipalmitoylphosphatidylserine (DPPC:DPPS), and DPPC:Cholesterol:DPPS to mimic the outer and the inner leaflet of the matrix vesicles membrane as well as to investigate the effect of the membrane fluidity. Kinetic parameters of mineralization were calculated from time-dependent turbidity curves of free Annexin A6 (AnxA6) and AnxA6-containing proteoliposomes dispersed in synthetic cartilage lymph. The chemical composition of the minerals formed was investigated by Fourier transform infrared spectroscopy (FTIR). Free AnxA6 and AnxA6-proteoliposomes in the presence of ACP were not able to propagate mineralization; however, poorly crystalline calcium phosphates were formed in the presence of PS–CPLX, supporting the role of annexin-calcium-phosphatidylserine complex in the formation and stabilization of the matrix vesicles’ nucleational core. We found that AnxA6 lacks nucleation propagation capacity when incorporated into liposomes in the presence of PS–CPLX and type-I collagen. This suggests that AnxA6 may interact either with phospholipids, forming a nucleational core, or with type-I collagen, albeit less efficiently, to induce the nucleation process.

Shedding Light on the Role of Na,K-ATPase as a Phosphatase during Matrix-Vesicle-Mediated Mineralization

“…For these experiments, when at optimal pH, 10 mM pNPP dissolved in Ampol 50 mM pH 10.0, 2 mM MgCl 2 buffer was used at 37 °C in a final volume of 0.5 mL. The reaction was started by adding an aliquot of the samples and stopped by adding 0.5 mL of 1.0 M NaOH at appropriate time intervals [ 90 , 91 ]. When specified, U/mg was used.…”

AnnexinA6: a potential therapeutic target gene for extracellular matrix mineralization

“…A case-control analysis in the Korean Women’s Cohort (3,570 subjects) has indicated that genetic variation of AnxA6 is significantly associated with OP ( kim, 2011 ). The possible reasons are as follows: First, AnxA6 regulates osteoblast proliferation, differentiation, necrosis, and apoptosis ( Kim et al, 2011 ); second, AnxA6 may interact with either phospholipids or type I collagen to induce the nucleation process in MVs-mediated mineralization ( Veschi et al, 2020 ; Veschi et al, 2022 ). Accordingly, AnxA6 enhances the occurrence and progression of OP by exerting a significant influence on osteoblasts.…”

“…In general, the linking role of AnxA6 should be analyzed from two aspects, including type I collagen and MVs membranes. Recently, AnxA6-loaded liposomes have been used to explore the role of AnxA6 in MVs-mediated mineralization, and the findings suggest that AnxA6 may exert its nucleation and mineralization abilities by necessary anchoring to type I collagen ( Veschi et al, 2022 ). Other studies have reported similar findings ( Chen et al, 2008 ).…”

“…The extracellular matrix (ECM) mineralization is an orchestrated and fine-tuned biological process during which inorganic minerals are produced and deposited in ECM ( Hasegawa et al, 2022 ). The formation of hydroxyapatite (HA), the core element of inorganic minerals, depends on the combined effects of surrounding calcium ions (Ca 2+ ), inorganic phosphate (PO 4 3− ), as well as matrix vesicles (MVs) ( Wuthier and Lipscomb, 2011 ; Bottini et al, 2018 ; Veschi et al, 2022 ). Annexins (Anxs) are Ca 2+ -dependent phospholipid-binding proteins that emerge as a multigene family with a conserved evolutionary origin and are widely distributed in animals and plants.…”

“…AnnexinA6 (AnxA6), a major component of MVs, has been reported to regulate physiological ECM mineralization by participating in Ca 2+ transport and forming mineralized nucleation sites ( Davies et al, 2019 ; Veschi et al, 2020 ; Veschi et al, 2022 ). AnxA6 has a Ca 2+ transport capacity that can mediate Ca 2+ influx into artificial liposomes ( Matsuda et al, 1997 ).…”