Fluorescence evidence of annexin A6 translocation across membrane in model matrix vesicles during apatite formation

Abstract

Matrix vesicles (MVs) are 100-300 nm spherical structures released by mineralization competent cells to initiate formation of apatite, the mineral component in bones. Among proteins present in MVs, annexin A6 (AnxA6) is thought to be ubiquitously distributed in the MVs' lumen, on the surface of the internal and external leaflets of the membrane and also inserted in the lipid bilayer. To determine the molecular mechanism(s) that lead to the different locations of AnxA6, we hypothesized the occurrence of a pH drop during the mineralization. Such a change would induce the AnxA6 protonation, which in turn, and because of its isoelectric point of 5.41, would change the protein hydrophobicity facilitating its insertion into the MVs' bilayer. The various distributions of AnxA6 are likely to disturb membrane phospholipid organization. To examine this possibility, we used fluorescein as pH reporter, and established that pH decreased inside MVs during apatite formation. Then, 4-(14-phenyldibenzo[a,c]phenazin-9(14H)-yl)-phenol, a vibration-induced emission fluorescent probe, was used as a reporter of changes in membrane organization occurring with the varying mode of AnxA6 binding. Proteoliposomes containing AnxA6 and 1,2-Dimyristoyl-snglycero-3phosphocholine (DMPC) or 1,2-Dimyristoyl-sn-glycero-3phosphocholine: 1,2-Dipalmitoyl-sn-glycero-3-phosphoserine (DMPC:DPPS 9:1), to mimic the external and internal MV membrane leaflet, respectively, served as biomimetic models to investigate the nature of AnxA6 binding. Addition of Anx6 to DMPC at pH 7.4 and 5.4, or DMPC:DPPS (9:1) at pH 7.4 induced a decrease in membrane fluidity, consistent with AnxA6 interactions with the bilayer surface. In contrast, AnxA6 addition to DMPC:DPPS (9:1) at pH 5.4 increased the fluidity of the membrane. This latest result was interpreted as reflecting the insertion of AnxA6 into the bilayer. Taken together, these findings point to a possible mechanism of AnxA6 translocation in MVs from the surface of the internal leaflet into the phospholipid bilayer stimulated upon acidification of the MVs' lumen during formation of apatite.

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

“…The presence of acidic phosphate as indicated by the IR bands at around 1150–1090 cm −1 and at 985 cm −1 ( Figure 3 ) may suggest a slight acidification of the SCL buffer due to the formation of apatite. Such acidification during apatite formation induced by MVs was reported [ 24 ]. The pH affected the Ca/P atomic ratio (1.42 at pH 5.8, and 1.48 at pH 6.5) while their XRD and IR spectra remained similar at both pHs [ 49 ].…”

“…The pH affected the Ca/P atomic ratio (1.42 at pH 5.8, and 1.48 at pH 6.5) while their XRD and IR spectra remained similar at both pHs [ 49 ]. AnxA6 at pH 7.4 interacted on the surface of DMPC:DPPS (9:1), while it increased the fluidity of the DMPC:DPPS (9:1) proteoliposome at pH 5.4, suggesting its insertion into the bilayer [ 24 ]. The interactions between AnxA6 and lipids can be affected by a pH variation.…”

“…AnxA6 might locally accumulate Ca 2+ and stabilize Ca 2+ binding PS, promoting a favourable environment for the formation of apatite ( Figure 5 C), albeit in a less effective manner than AnxA5 [ 22 ]. Followed by a local drop in pH during apatite formation, the insertion of hydrophobic AnxA6 into the matrix vesicles’ bilayer can be promoted ( Figure 5 D) [ 24 ]. Although AnxA6 may have univalent K + conductance [ 52 ], it is unclear if the transmembrane AnxA6 has the capacity to transport Ca 2+ [ 23 ] or if another unidentified channel has this ability.…”

“…Although AnxA6 may have univalent K + conductance [ 52 ], it is unclear if the transmembrane AnxA6 has the capacity to transport Ca 2+ [ 23 ] or if another unidentified channel has this ability. The outer pH is neutral, rendering AnxA6 more hydrophilic, which can expel AnxA6 towards the extracellular medium, where AnxA6 may bind to outside-leaflet phosphatidylcholine (PC) [ 24 ] ( Figure 5 E). AnxA6 could associate with AnxA5 on the vesicles′ surface and contribute to reinforce the interactions of MVs with collagen fibers ( Figure 5 F).…”

“…It has been proposed that AnxA2, AnxA5, and AnxA6 may be also involved in the formation of a nucleational core associated with phosphatidylserine in the MVs’ lumen [ 22 ]. AnxA6 is thought to tightly bind or insert into the MVs’ lipid bilayer, since a part of the protein cannot be extracted by calcium chelators (e.g., Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) or Ethylenediaminetetraacetic acid (EDTA)) [ 20 , 23 , 24 ]. One part corresponds to Ca 2+ -bound AnxA6 that interacts with the inner leaflet of the MVs’ membrane [ 23 , 24 ], a second part corresponds to AnxA6 localized on the surface of the outer leaflet and, lastly, a third part corresponds to AnxA6 inserted in the hydrophobic membrane bilayer and co-localizes with cholesterol [ 23 , 24 ].…”