Localization of Annexin A6 in Matrix Vesicles During Physiological Mineralization.

José Luis Millán,Massimo Bottini,Slawomir Pikula,Saida Mebarek,David Magne,Pietro Ciancaglini,Rene Buchet,Nicola Rosato,Maytê Bolean,Ekeveliny Amabile Veschi,Agnieszka Strzelecka-Kiliszek,Thierry Granjon,Ana Paula Ramos,Joanna Bandorowicz-Pikula

doi:10.3390/ijms21041367

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.

Highlights

Bone mineralization is a tightly regulated process that involves mesenchymal cells condensing into tissue elements followed by their differentiation into cartilage or bone cells [1]
The presence of a 35 kDa-molecular weight (MW) Annexin A6 (AnxA6) fragment in matrix vesicles (MVs) has been previously reported [22] and is due to the collagenase treatment and/or action of endogenous proteases [23,24]. It indicates that a population of AnxA6 associates with the inner leaflet of the MV membrane, accessible to endogenous proteases, as confirmed by the treatment with (Figure 1A, lane 3) or without (Figure 1A, lane 4) trypsin that did not affect the intensity of the bands
We investigated the association of AnxA6 with the membrane bilayer of natural MVs and with MV biomimetics

Summary

Introduction

Bone mineralization is a tightly regulated process that involves mesenchymal cells condensing into tissue elements followed by their differentiation into cartilage (chondrocytes) or bone (osteoblasts) cells [1]. During long bone formation, occurring throughout endochondral ossification, chondrocytes mineralize their extracellular matrix (ECM) by forming apatite. Osteoblasts are responsible for the formation of bone in both endochondral and endomembranous ossification acting in concert with osteoclasts to maintain the integrity of bone tissues. Both chondrocytes and osteoblasts produce a special class of extracellular vesicles, named matrix vesicles (MVs), that provide a suitable microenvironment for initiating apatite formation during bone mineralization [2,3,4,5,6,7,8]. The most abundant proteins in MVs isolated from hypertrophic chondrocytes [9] and human osteoblast-like cells Saos2 [10] are the annexins, AnxA1, AnxA2, AnxA5, AnxA6, and AnxA7

Methods

Results

Conclusion