Annexin-A5 assembled into two-dimensional arrays promotes cell membrane repair.
Bottom Line: Compared with wild-type mouse perivascular cells, AnxA5- cells exhibit a severe membrane repair defect.In contrast, an AnxA5 mutant that lacks the ability of forming 2D arrays is unable to promote membrane repair.We propose that AnxA5 participates in a previously unrecognized step of the membrane repair process: triggered by the local influx of Ca(2+), AnxA5 proteins bind to torn membrane edges and form a 2D array, which prevents wound expansion and promotes membrane resealing.
Affiliation: Molecular Imaging and NanoBioTechnology, IECB, UMR-5248 CBMN CNRS-University Bordeaux1-ENITAB, Talence F-33402, France.Show MeSH
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Mentions: By which mechanism does AnxA5 promote cell membrane repair? The most remarkable in vitro property of AnxA5 is to self-assemble into trimers and 2D ordered arrays of trimers on PS-exposing membranes2935 (Fig. 1b). To investigate whether AnxA5 2D self-assembly had an influence on membrane repair, we produced AnxA5 mutants that lack the ability to form 2D arrays. The choice of the mutations was based on the fact that the monomer–monomer interface of AnxA5 trimers (Fig. 5a) presents several pairs of amino acids of opposite charges situated at distances close enough to form salt bridges39 (Fig. 5b). In addition, Mo et al.39 have shown that mutations of single amino acids were not sufficient to abolish the ability of AnxA5 to form trimers. Consequently, we produced multiple mutants in which several positively charged residues involved in salt bridging were replaced by negatively charged ones. The ability of AnxA5 mutants to form trimers was first assessed by transmission electron microscopy (TEM), according to established procedures4041 (Supplementary Fig. S5). We found that all mutants with one, two or three mutations were still able to form 2D crystals, and thus trimers. Contrarily, all quadruple and quintuple mutants have lost the property of trimer and 2D crystal formation. The quintuple mutant mtT-AnxA5 (R16E, R23E, K27E, K56E, K191E) was selected for further analyses. To further ascertain that mtT-AnxA5 molecules are unable to form trimers, three-dimensional crystals of mtT-AnxA5 were grown in high Ca2+ concentration and their structure was determined by X-ray crystallography. Indeed, all AnxA5 structures hitherto reported from three-dimensional crystals grown at high Ca2+ concentration contain AnxA5 trimers, which are structurally homologous to membrane-bound trimers394243. MtT-AnxA5 molecules crystallize in the P21 space group (Supplementary Table S1; Supplementary Methods) in which they pack as dimers with upside-down orientations (Fig. 5c). This result confirms that mtT-AnxA5 is unable to form trimers.
Affiliation: Molecular Imaging and NanoBioTechnology, IECB, UMR-5248 CBMN CNRS-University Bordeaux1-ENITAB, Talence F-33402, France.