Kirsch T, Nah H D, Demuth D R, Harrison G, Golub E E, Adams S L, Pacifici M
Department of Anatomy and Histology, School of Dental Medicine, University of Pennsylvania, Philadelphia 19104, USA.
Biochemistry. 1997 Mar 18;36(11):3359-67. doi: 10.1021/bi9626867.
Annexin V is a major component of matrix vesicles and has a role in mediating the influx of Ca2+ into these vesicles, thus promoting the initiation of hypertrophic cartilage matrix mineralization. However, the mechanisms and factors regulating annexin V-mediated Ca2+ influx into these vesicles are not well understood. Since the lipid composition of matrix vesicles differs from that of the plasma membrane of chondrocytes and is rich in phosphatidylserine, we asked whether the lipid composition may regulate annexin V function. We prepared liposomes containing different concentrations of phosphatidylserine and determined how the lipid composition affected (a) the interactions between annexin V and liposomes and (b) annexin V-mediated Ca2+ influx into the liposomes. We found that annexin V was able to bind to every liposome tested. However, we observed the most prominent increases in tryptophan 187 emission intensity, a measure of the degree of interactions between annexin V and lipid bilayers, only with liposomes containing a high concentration of phosphatidylserine. In addition, a significant fraction of annexin V associated with phosphatidylserine-rich liposomes was not extractable by EDTA treatment but required a detergent, indicating that annexin V inserts into bilayers of these liposomes. Chemical cross-linking analysis revealed that matrix vesicles and phosphatidylserine-rich liposomes induced the formation of the annexin V hexamer. Interestingly, a significant Ca2+ influx in the presence of annexin V occurred only in liposomes containing a high phosphatidylserine content. Moreover, annexin V-mediated Ca2+ influx into these liposomes was inhibited (i) by anti-annexin V antibodies and (ii) by treatment with zinc and cadmium, indicating the essential role of the protein in Ca2+ influx. The results of this study indicate that phosphatidylserine-rich bilayers induce the formation of a hexameric annexin V, possibly leading to a Ca2+-dependent insertion of annexin V into the bilayer and establishment of annexin V-mediated Ca2+ influx into matrix vesicles or liposomes. The phosphatidylserine-rich membrane of matrix vesicles in vivo may thus offer an ideal specialized environment in which the biological function of annexin V is optimized, leading to rapid Ca2+ influx, intralumenal crystal growth, and cartilage matrix mineralization.
膜联蛋白V是基质小泡的主要成分，在介导Ca2+流入这些小泡中发挥作用，从而促进肥大软骨基质矿化的起始。然而，调节膜联蛋白V介导的Ca2+流入这些小泡的机制和因素尚未完全清楚。由于基质小泡的脂质组成不同于软骨细胞的质膜，且富含磷脂酰丝氨酸，我们推测脂质组成是否可能调节膜联蛋白V的功能。我们制备了含有不同浓度磷脂酰丝氨酸的脂质体，并确定脂质组成如何影响（a）膜联蛋白V与脂质体之间的相互作用以及（b）膜联蛋白V介导的Ca2+流入脂质体。我们发现膜联蛋白V能够与所测试的每个脂质体结合。然而，只有在含有高浓度磷脂酰丝氨酸的脂质体中，我们才观察到色氨酸187发射强度最显著的增加，这是膜联蛋白V与脂质双层相互作用程度的一种度量。此外，与富含磷脂酰丝氨酸的脂质体结合的相当一部分膜联蛋白V不能通过EDTA处理提取出来，而是需要一种去污剂，这表明膜联蛋白V插入到这些脂质体的双层中。化学交联分析表明，基质小泡和富含磷脂酰丝氨酸的脂质体诱导了膜联蛋白V六聚体的形成。有趣的是，只有在磷脂酰丝氨酸含量高的脂质体中，在膜联蛋白V存在的情况下才会发生显著的Ca2+流入。此外，膜联蛋白V介导的Ca2+流入这些脂质体受到（i）抗膜联蛋白V抗体和（ii）锌和镉处理的抑制，这表明该蛋白在Ca2+流入中起着重要作用。这项研究的结果表明，富含磷脂酰丝氨酸的双层诱导了六聚体膜联蛋白V的形成，可能导致膜联蛋白V以Ca2+依赖的方式插入双层，并建立膜联蛋白V介导的Ca2+流入基质小泡或脂质体。因此，体内基质小泡富含磷脂酰丝氨酸的膜可能提供了一个理想的特殊环境，在其中膜联蛋白V的生物学功能得到优化，导致快速的Ca2+流入、腔内晶体生长和软骨基质矿化。
