Localization of Annexin A6 in Matrix Vesicles During Physiological Mineralization

Author(s):Show less -

Veschi, Ekeveliny Amabile ^[1] ; Bolean, Mayte ^[1] ; Strzelecka-Kiliszek, Agnieszka ^[2] ; Bandorowicz-Pikula, Joanna ^[2] ; Pikula, Slawomir ^[2] ; Granjon, Thierry ^{[3, 4, 5, 6, 7]} ; Mebarek, Saida ^{[3, 4, 5, 6, 7]} ; Magne, David ^{[3, 4, 5, 6, 7]} ; Ramos, Ana Paula ^[1] ; Rosato, Nicola ^[8] ; Milian, Jose Luis ^[9] ; Buchet, Rene ^{[3, 4, 5, 6, 7]} ; Bottini, Massimo ^{[9, 8]} ; Ciancaglini, Pietro ^[1]

Affiliation

^[1] Univ Sao Paulo FFCLRP USP, Fac Filosofia Ciencias & Letras Ribeirao Preto, Dept Quim, BR-14040900 Ribeirao Preto, SP - Brazil

^[2] Nencki Inst Expt Biol, 3 Pasteur St, PL-02093 Warsaw - Poland

^[3] Univ Lyon, F-69622 Villeurbanne - France

^[4] Univ Lyon 1, UFR Chim Biochim, F-69622 Villeurbanne - France

^[5] INSA, F-69622 Villeurbanne - France

^[6] CPE, F-69622 Villeurbanne - France

^[7] CNRS, ICBMS UMR 5246, F-69622 Villeurbanne - France

^[8] Univ Roma Tor Vergata, Dept Expt Med, I-00133 Rome - Italy

^[9] Sanford Burnham Prebys Med Discovery Inst, La Jolla, CA 92037 - USA

• Document type: Journal article
• Source: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES; v. 21, n. 4 FEB 2020.
• Web of Science Citations: 0

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 (pi(exc)), enthalpy (Delta H), and phase transition cooperativity (Delta t(1/2)) 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-beta-cyclodextrin (M beta CD). AnxA6-lipid interaction was also Ca2+-dependent, as evidenced by the increase in pi(exc) in negatively charged 9:1 DPPC:DPPS monolayers and the decrease in Delta H 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 Delta t(1/2) 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. (AU)

• FAPESP's process: 17/08892-9 - Bioactive surfaces designed from Langmuir-Blodgett Films and Biominerals
• Grantee: Ana Paula Ramos
• Support Opportunities: Regular Research Grants
• FAPESP's process: 14/00371-1 - Are the interactions between collagen and proteins/enzymes present in the matriz vesicles responsible for the control in the biomineralization process?
• Grantee: Maytê Bolean Correia
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 15/06814-5 - Do interactions between collagen and proteins/enzymes present in matrix vesicles control biomineralization?
• Grantee: Maytê Bolean Correia
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor