PDF(Pubmed)
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-sn-glycero-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.
摘要：
基质囊泡（MV）是由矿化感受态细胞释放的100-300nm球形结构，以引发磷灰石的形成，骨骼中的矿物质成分。在MV中存在的蛋白质中，膜联蛋白A6(AnxA6)被认为普遍分布在MV腔中，在膜的内部和外部小叶的表面上，也插入脂质双层中。为了确定导致AnxA6不同位置的分子机制，我们假设在矿化过程中发生pH下降。这种变化会引起AnxA6质子化，反过来，并且由于其等电点为5.41，会改变蛋白质的疏水性，从而促进其插入MV双层。AnxA6的各种分布可能会干扰膜磷脂的组织。为了检查这种可能性，我们用荧光素作为pH报告，并确定在磷灰石形成过程中MV内部的pH值降低。然后，4-(14-苯基二苯并[a，c]菲嗪-9(14H)-基)-苯酚，振动诱导发射荧光探针，用作随AnxA6结合模式变化而发生的膜组织变化的报告子。含有AnxA6和1,2-二炔酰-sn-甘油-3磷酸胆碱（DMPC）或1,2-二炔酰-sn-甘油-3磷酸胆碱的蛋白酶体：1,2-二棕榈酰-sn-甘油-3-磷酸丝氨酸（DMPC：DPPS9：1），模仿内外的MV膜小叶,分别,用作仿生模型来研究AnxA6结合的性质。在pH7.4和5.4的DMPC中添加Anx6，或在pH7.4的DMPC：DPPS（9：1）引起膜流动性降低，与AnxA6与双层表面的相互作用一致。相比之下,在pH5.4下向DMPC:DPPS(9:1)中添加AnxA6增加了膜的流动性。这一最新结果被解释为反映了AnxA6插入到双层中。一起来看,这些发现指出了在磷灰石形成过程中，在MVs管腔酸化时，MVs中的AnxA6从内部小叶表面易位到磷脂双层的可能机制。
