Localization of Annexin A6 in Matrix Vesicles During Physiological Mineralization

Abstract

Annexin A6 (AnxA6) is the largest member of the annexin family of proteins present in matrix vesicles (MVs). MVs are a special class of extracellular vesicles that serve as a nucleation site during cartilage, bone, and mantle dentin mineralization. In this study, we assessed the localization of AnxA6 in the MV membrane bilayer using native MVs and MV biomimetics. Biochemical analyses revealed that AnxA6 in MVs can be divided into three distinct groups. The first group corresponds to Ca²⁺-bound AnxA6 interacting with the inner leaflet of the MV membrane. The second group corresponds to AnxA6 localized on the surface of the outer leaflet. The third group corresponds to AnxA6 inserted in the membrane's hydrophobic bilayer and co-localized with cholesterol (Chol). Using monolayers and proteoliposomes composed of either dipalmitoylphosphatidylcholine (DPPC) to mimic the outer leaflet of the MV membrane bilayer or a 9:1 DPPC:dipalmitoylphosphatidylserine (DPPS) mixture to mimic the inner leaflet, with and without Ca²⁺, we confirmed that, in agreement with the biochemical data, AnxA6 interacted differently with the MV membrane. Thermodynamic analyses based on the measurement of surface pressure exclusion (π_exc), enthalpy (ΔH), and phase transition cooperativity (Δt_1/2) showed that AnxA6 interacted with DPPC and 9:1 DPPC:DPPS systems and that this interaction increased in the presence of Chol. The selective recruitment of AnxA6 by Chol was observed in MVs as probed by the addition of methyl-β-cyclodextrin (MβCD). AnxA6-lipid interaction was also Ca²⁺-dependent, as evidenced by the increase in π_exc in negatively charged 9:1 DPPC:DPPS monolayers and the decrease in ΔH in 9:1 DPPC:DPPS proteoliposomes caused by the addition of AnxA6 in the presence of Ca²⁺ compared to DPPC zwitterionic bilayers. The interaction of AnxA6 with DPPC and 9:1 DPPC:DPPS systems was distinct even in the absence of Ca²⁺ as observed by the larger change in Δt_1/2 in 9:1 DPPC:DPPS vesicles as compared to DPPC vesicles. Protrusions on the surface of DPPC proteoliposomes observed by atomic force microscopy suggested that oligomeric AnxA6 interacted with the vesicle membrane. Further work is needed to delineate possible functions of AnxA6 at its different localizations and ways of interaction with lipids.

Keywords: Annexin A6; Langmuir monolayers; biomineralization; differential scanning calorimetry.; matrix vesicles; proteoliposomes.

Figure 1

Annexin A6 in matrix vesicles. (A) Western blotting performed using mouse anti-AnxA6 monoclonal antibody to detect two isoforms of annexin A6 (AnxA6) in matrix vesicles (MVs) in the presence or absence of 0.1% trypsin after four freeze-thaw procedures followed by centrifugation. Lanes 1, 2, 3, and 4 corresponded to pellets, while lanes 5 and 6 corresponded to supernatants. MVs were treated with or without trypsin, with 2 mM Ca²⁺ or with 2 mM ethylene glycol tetraacetic acid (EGTA), as indicated. (B) AnxA6 co-localizes with cholesterol. MVs were incubated in synthetic cartilage lymph (SCL) medium containing 10 mM EGTA and an increasing concentration of methyl-β-cyclodextrin (MβCD) for 30 min in room temperature. Then, the samples were centrifuged. Lactate dehydrogenase (▼) and tissue non-specific alkaline phosphatase (TNAP) (●) activities were tested in the pellets and in the supernatants, as described in Materials and Methods. In the pellets, only the content of cholesterol (◆) and protein (■) was measured. MβCD-treated pellets were analyzed to determine AnxA6 (▲) content by Western blotting using a rabbit polyclonal anti-AnxA6 antibody that recognizes a single protein band. (C) Increasing MβCD concentrations led to a decrease of AnxA6 content. The results are expressed as a percentage of release of MV components ± SD.

Figure 2

Changes in the surface pressure (Δπ) due to the injection of AnxA6 as a function of the initial surface pressure (π₀) of Langmuir monolayers in the absence (A) and presence of 2 mM Ca²⁺ (B) for 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (black dots), 9:1 DPPC:1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS) (red dots) and 5:4:1 DPPC:cholesterol (Chol):DPPS (blue dots) monolayers as a consequence of interaction with AnxA6. The intercept with abscissa is the exclusion pressure (π_exc) and is indicated by the arrows. The analyses were adjusted to π₀ close to 10, 15, and 20 mN/m.

Figure 3

Differential scanning calorimetry (DSC) thermograms of DPPC (a,A) and 9:1 DPPC:DPPS (b,B) liposomes (10 mg/mL, total lipid concentration) in the absence (lower case letter) and in the presence (capital letter) of 2 mM Ca²⁺. DSC thermograms were processed in excess heat capacity (C_p) (kcal/K.mol) as function of temperature (°C) of liposomes (black line) and proteoliposome (red line) harboring AnxA6.

Figure 4

DSC thermograms of 6:4 DPPC:Chol (a,A) and 5:4:1 DPPC:Chol:DPPS (b,B) liposomes (10 mg/mL, total lipid concentration) in the absence (lower case) and in the presence (capital letter) of 2 mM Ca²⁺. DSC thermograms were processed in excess heat capacity (C_p) (kcal/K.mol) as function of temperature (°C) of liposomes (black) and AnxA6-harboring proteoliposome (red).

Figure 5

Atomic force microscopy (AFM) images of DPPC (A–C) and 5:4:1 DPPC:Chol:DPPS (D–E) liposomes (1.5 mg/mL): (A,D) Phase image, (B,E) 3D topography, and (C,F) liposome schematic representation. The blue and purple circles in C and F are assigned to the polar heads of DPPC and DPPS, respectively. The brown and yellow colors are assigned to Chol. The chemical structure of DPPC is depicted in C, and the chemical structures of DPPS and Chol are depicted in F.

Figure 6

AFM analysis of DPPC proteoliposomes harboring AnxA6: (A) Phase image, (B) height image, (C) 3D topography, and (D) magnification of a 146.48 × 146.48 nm² surface area showing a protein domain; (E and F) phase and height line analysis (blue and red lines) of an AnxA6 domain inserted in DPPC proteoliposomes (the gray shadow highlights the protrusion). (E) was prepared using WSxM 4.0 Beta 9.1 software developed by Horcas et al. [33].

Figure 7

AFM analysis of 6:4:1 DPPC:Chol:DPPS proteoliposomes harboring AnxA6: (A) Phase image, (B) height image, (C) 3D topographic profile, and (D) magnification of a 109.83 × 109.83 nm² surface area showing protein domains; (E and F) phase and height line analysis (blue and red lines) of an AnxA6 domain inserted into 6:4:1 DPPC:Chol:DPPS proteoliposomes (the gray shadow highlights the protrusions). (E) was prepared using WSxM 4.0 Beta 9.1 software developed by Horcas et al. [33].

Figure 8

Representation of the mechanism of AnxA6 translocation during MV-mediated mineralization and its putative functions. (A) AnxA6 is localized in the lumen of MVs at low calcium concentrations. (B) Accumulation of calcium inside MVs favors the binding of AnxA6 to the inner leaflet of the MV membrane bilayer containing phosphatidylserine (PS) in a Ca²⁺-dependent manner, forming a nucleational core (NC) and initiating apatite formation. (C) During apatite formation, protons (H⁺) are released, inducing the protonation of AnxA6 and rendering it more hydrophobic due to an ionization potential around 5.5. (D) Protonated AnxA6 can translocate within the MV bilayer where it may form an ion pore. (E) The pH outside MVs corresponds to that of the extracellular matrix and is neutral. AnxA6 can be deprotonated and released to the external surface of MVs enriched in phosphatidylcholine (PC). (F) AnxA6 binding to neutral phospholipids is Ca²⁺-independent. AnxA6 may also bind to collagen fibers, contributing to the adhesion of MVs to collagen fibers.