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 (πexc), enthalpy (ΔH), and phase transition cooperativity (Δt1/2) 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 πexc 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 Δt1/2 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.

Annexins A2, A6 and Fetuin-A Affect the Process of Mineralization in Vesicles Derived from Human Osteoblastic hFOB 1.19 and Osteosarcoma Saos-2 Cells

“…Calcium nodules indicated the presence of calcium precipitates, but not necessarily the presence of apatite, as reported below. Inhibition of the activity of Ca channels by K201 seemed to induce more calcium nodules in hFOB 1.19 cells and in Saos-2 cells under resting conditions because resting cells (without stimulators like AA and β-GP) produce more amorphous calcium phosphate complexes, while it was without effect in stimulated hFOB 1.19 ( Figure 2 A) and Saos-2 ( Figure 2 B) cells maintained in conditions favoring conversion to apatite (with stimulators such as AA resulting in acidic pH and β-GP being a source of P i ) [ 27 ]. TNAP activity in hFOB 1.19 osteoblastic cells was much lower than that in Saos-2 osteosarcoma cells, being almost on the threshold of detection ( Figure 2 C,D) [ 9 ].…”

“…Acidic pH-induced formation of AnxA6 ion channels [ 26 ]. A fraction of cellular Anx6 seems to be tightly bound or inserted into the bilayer of MV [ 27 ] since it could not be extracted by calcium chelators such as EGTA or EDTA [ 21 ]. AnxA6 present in MVs can be divided into three distinct pools.…”

“…The second pool corresponds to AnxA6 localized on the surface of the outer leaflet. The third pool corresponds to AnxA6 inserted in the hydrophobic membrane bilayer and co-localized with cholesterol [ 27 ]. K201 (a benzodiazepine derivative), a potential inhibitor of annexin calcium channel activity, reduced the ability of MVs or extracellular vesicles from VSMCs to mineralize collagen fibers independently of whether it was added during or after MV formation by VSMC [ 23 ].…”

Matrix Vesicles: Role in Bone Mineralization and Potential Use as Therapeutics

“…For the isolation of MtVs, centrifugation-based methods are commonly used. Around two-thirds of the reviewed papers which included MtV isolation in their methodology used differential centrifugation (DC) combined with a final ultracentrifugation (UC) (>10,000× g ) step [ 17 , 102 , 107 , 132 , 133 , 134 ]. Even though DC is most often used to isolate MtVs, the exact execution differs in the number of steps, centrifugation speed, and centrifugation time used.…”

“…Collagenase breaks down collagen fibers and is believed to release the MtVs that are embedded in the matrix [ 142 , 143 ]. Therefore, to isolate MtVs from tissue, a digestion step with collagenase should be performed prior to MtV isolation [ 107 , 132 , 133 ]. MtVs can also be isolated from cell culture samples [ 17 , 102 , 144 ].…”