Identification of Novel Binding Partners (Annexins) for the Cell Death Signal Phosphatidylserine and Definition of Their Recognition Motif

Identification and clearance of apoptotic cells prevents the release of harmful cell contents thereby suppressing inflammation and autoimmune reactions. Highly conserved annexins may modulate the phagocytic cell removal by acting as bridging molecules to phosphatidylserine, a characteristic phagocytosis signal of dying cells. In this study five members of the structurally and functionally related annexin family were characterized for their capacity to interact with phosphatidylserine and dying cells. The results showed that AnxA3, AnxA4, AnxA13, and the already described interaction partner AnxA5 can bind to phosphatidylserine and apoptotic cells, whereas AnxA8 lacks this ability. Sequence alignment experiments located the essential amino residues for the recognition of surface exposed phosphatidylserine within the calcium binding motifs common to all annexins. These amino acid residues were missing in the evolutionary young AnxA8 and when they were reintroduced by site directed mutagenesis AnxA8 gains the capability to interact with phosphatidylserine containing liposomes and apoptotic cells. By defining the evolutionary conserved amino acid residues mediating phosphatidylserine binding of annexins we show that the recognition of dying cells represent a common feature of most annexins. Hence, the individual annexin repertoire bound to the cell surface of dying cells may fulfil opsonin-like function in cell death recognition. Identification and clearance of apoptotic cells prevents the release of harmful cell contents thereby suppressing inflammation and autoimmune reactions. Highly conserved annexins may modulate the phagocytic cell removal by acting as bridging molecules to phosphatidylserine, a characteristic phagocytosis signal of dying cells. In this study five members of the structurally and functionally related annexin family were characterized for their capacity to interact with phosphatidylserine and dying cells. The results showed that AnxA3, AnxA4, AnxA13, and the already described interaction partner AnxA5 can bind to phosphatidylserine and apoptotic cells, whereas AnxA8 lacks this ability. Sequence alignment experiments located the essential amino residues for the recognition of surface exposed phosphatidylserine within the calcium binding motifs common to all annexins. These amino acid residues were missing in the evolutionary young AnxA8 and when they were reintroduced by site directed mutagenesis AnxA8 gains the capability to interact with phosphatidylserine containing liposomes and apoptotic cells. By defining the evolutionary conserved amino acid residues mediating phosphatidylserine binding of annexins we show that the recognition of dying cells represent a common feature of most annexins. Hence, the individual annexin repertoire bound to the cell surface of dying cells may fulfil opsonin-like function in cell death recognition. IntroductionDiscrimination of viable from dying cells is a prerequisite for the efficient clearance of dying and dead cells and to suppress uncontrolled cell lysis and the release of potentially harmful cellular compounds to the local environment. During development and under normal physiological conditions, cells are removed through the process of apoptosis and undergo a strictly defined series of morphological and biochemical changes, before being engulfed by professional phagocytes such as macrophages or even by neighboring cells. A signature of specific “eat me” signals are exposed at the cell surface of apoptotic cells, which enable direct or indirect interactions with phagocytes and dying cells to promote the efficient clearance of apoptotic cells. This reduces the risk of accumulation of secondarily necrotic cells and the release of cellular content to the microenvironment. Exposure of apoptotic-cell associated molecular patterns together with recognition and interpretation of these by phagocytes are crucial steps in the appropriate response of phagocytes toward the engulfed cells (1Manfredi A.A. Iannacone M. D'Auria F. Rovere-Querini P. Apoptosis. 2002; 7: 153-161Crossref PubMed Scopus (31) Google Scholar).A hallmark of early apoptotic cells is the loss of membrane asymmetry and the exposure of anionic phosphatidylserine (PS) 3The abbreviations used are: PSphosphatidylserinePCphosphatidylcholinePEphosphatidylethanolaminePIpropidium iodide. on the outer lipid layer of the cells. In most cells PS predominantly resides in the inner leaflet of the plasma membrane, regulating membrane charge and protein localization (2Yeung T. Gilbert G.E. Shi J. Silvius J. Kapus A. Grinstein S. Science. 2008; 319: 210-213Crossref PubMed Scopus (748) Google Scholar). This membrane asymmetry is actively maintained by an inward transporting aminophospholipid translocase (3Tang X. Halleck M.S. Schlegel R.A. Williamson P. Science. 1996; 272: 1495-1497Crossref PubMed Scopus (417) Google Scholar). Upon induction of apotosis, rising cytoplasmic Ca2+ levels cause a loss of translocase activity and an activation of bi-directional transporting proteins of the scramblase family to expose PS at the cell surface (4Zhou Q. Zhao J. Stout J.G. Luhm R.A. Wiedmer T. Sims P.J. J. Biol. Chem. 1997; 272: 18240-18244Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). This phospholipid represents the major recognition cue for the engulfment of dying cells by professional and nonprofessional phagocytes (5van den Eijnde S.M. Boshart L. Baehrecke E.H. De Zeeuw C.I. Reutelingsperger C.P. Vermeij-Keers C. Apoptosis. 1998; 3: 9-16Crossref PubMed Scopus (113) Google Scholar). A number of interaction partners for the “eat me” signal PS have been described, including phagocyte cell surface receptors like members of the scavenger receptor family (6Rigotti A. Acton S.L. Krieger M. J. Biol. Chem. 1995; 270: 16221-16224Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar) or TIM1 and TIM4 (7Miyanishi M. Tada K. Koike M. Uchiyama Y. Kitamura T. Nagata S. Nature. 2007; 450: 435-439Crossref PubMed Scopus (810) Google Scholar), which can directly bind to PS on dying cells. Several PS-binding serum proteins like protein S (8Anderson H.A. Maylock C.A. Williams J.A. Paweletz C.P. Shu H. Shacter E. Nat. Immunol. 2003; 4: 87-91Crossref PubMed Scopus (313) Google Scholar), milk fat globule protein (MFG-E8) (9Hanayama R. Tanaka M. Miwa K. Shinohara A. Iwamatsu A. Nagata S. Nature. 2002; 417: 182-187Crossref PubMed Scopus (1030) Google Scholar) and β2-glycoprotein-1 (10Balasubramanian K. Schroit A.J. J. Biol. Chem. 1998; 273: 29272-29277Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) can act as bridging molecules that bind to the signature of the apoptotic cells. The interpretation of the PS signature in the context of bridging molecules and receptors by phagocytes then modulates the response toward apoptotic cells.Annexin A5 (Anxa5-gene, AnxA5-protein) was identified as a specific interaction partner for PS and is extensively used to detect cell death in vitro and in vivo (11Vermes I. Haanen C. Steffens-Nakken H. Reutelingsperger C. J. Immunol. Methods. 1995; 184: 39-51Crossref PubMed Scopus (4546) Google Scholar, 12van Genderen H. Kenis H. Lux P. Ungeth L. Maassen C. Deckers N. Narula J. Hofstra L. Reutelingsperger C. Nat. Protoc. 2006; 1: 363-367Crossref PubMed Scopus (73) Google Scholar, 13Blankenberg F. Mari C. Strauss H.W. Q. J. Nucl. Med. 2003; 47: 337-348PubMed Google Scholar). This protein can also inhibit the phagocytosis of dying cells (14Kenis H. van Genderen H. Deckers N.M. Lux P.A. Hofstra L. Narula J. Reutelingsperger C.P. Exp. Cell Res. 2006; 312: 719-726Crossref PubMed Scopus (53) Google Scholar, 15Munoz L.E. Franz S. Pausch F. Fürnrohr B. Sheriff A. Vogt B. Kern P.M. Baum W. Stach C. von Laer D. Brachvogel B. Poschl E. Herrmann M. Gaipl U.S. J. Leukoc. Biol. 2007; 81: 6-14Crossref PubMed Scopus (52) Google Scholar) and modulates the cytokine response of phagocytes toward apoptotic and necrotic cells (16Gaipl U.S. Munoz L.E. Rödel F. Pausch F. Frey B. Brachvogel B. von der Mark K. Pöschl E. Autoimmunity. 2007; 40: 254-259Crossref PubMed Scopus (25) Google Scholar). In vivo, AnxA5-deficient mice displayed an increased uptake of necrotic cells and a reduced allogenic immune reaction against these (17Frey B. Munoz L.E. Pausch F. Sieber R. Franz S. Brachvogel B. Poschl E. Schneider H. Rödel F. Sauer R. Fietkau R. Herrmann M. Gaipl U.S. J. Cell Mol. Med. 2009; 13: 1391-1399Crossref PubMed Scopus (23) Google Scholar, 18Brachvogel B. Dikschas J. Moch H. Welzel H. von der Mark K. Hofmann C. Pöschl E. Mol. Cell. Biol. 2003; 23: 2907-2913Crossref PubMed Scopus (80) Google Scholar). Besides AnxA5, two other annexins may play a role in cell death recognition and removal. AnxA1 is externalized upon cell death and can be detected in PS-rich membrane plaques of dying cells (19Arur S. Uche U.E. Rezaul K. Fong M. Scranton V. Cowan A.E. Mohler W. Han D.K. Dev. Cell. 2003; 4: 587-598Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar) where it promotes the removal of dying cells and prevents proinflammatory cytokine production (20Blume K.E. Soeroes S. Waibel M. Keppeler H. Wesselborg S. Herrmann M. Schulze-Osthoff K. Lauber K. J. Immunol. 2009; 183: 8138-8147Crossref PubMed Scopus (64) Google Scholar). Both AnxA1 and AnxA2 are present at the cell surface of macrophages and may adjust the phagocyte response toward dying cells (21Fan X. Krahling S. Smith D. Williamson P. Schlegel R.A. Mol. Biol. Cell. 2004; 15: 2863-2872Crossref PubMed Scopus (110) Google Scholar). Most members of the evolutionary conserved, over 1 billion year old annexin family may be involved in the identification and removal of dying cells by phagocytes, but little is yet known about their individual roles as PS-binding immunomodulatory proteins and the underlying molecular mechanisms of PS recognition.In mammals, the protein family of annexins includes 12 members, with AnxA13 being the phylogenetically oldest annexin (22Moss S.E. Morgan R.O. Genome Biol. 2004; 5: 219Crossref PubMed Scopus (401) Google Scholar). All annexins carry four similar domains defined by five α-helices and each domain contains a characteristic type 2 calcium-binding motif (GXGTD-[X]37-(D/E)). The four domains are arranged in a disc-like structure with antipodal convex and concave surfaces. Upon binding to calcium the convex side of an annexin specifically interacts with anionic phospholipids, as shown for AnxA5 (23Andree H.A. Reutelingsperger C.P. Hauptmann R. Hemker H.C. Hermens W.T. Willems G.M. J. Biol. Chem. 1990; 265: 4923-4928Abstract Full Text PDF PubMed Google Scholar). Because of the structural and functional similarities within the annexin family we speculated that annexins can bind to PS exposed at the apoptotic cell In this we and five annexins and their binding toward PS in and interaction In we characterized their binding to cell surface exposed PS in results show that most annexins interact with PS whereas AnxA8 lacks this ability. alignment identified the conserved amino mediating the PS recognition by annexins and when these amino residues the AnxA8 protein site directed mutagenesis the protein gains the to bind to These results that we have identified the essential amino for binding of annexins toward PS and that annexins may act as to the recognition of PS at the cell surface of dying proteins have been as binding partners for cell PS and annexin A5 is as a specific and used as a for cell death (11Vermes I. Haanen C. Steffens-Nakken H. Reutelingsperger C. J. Immunol. Methods. 1995; 184: 39-51Crossref PubMed Scopus (4546) Google Scholar, 12van Genderen H. Kenis H. Lux P. Ungeth L. Maassen C. Deckers N. Narula J. Hofstra L. Reutelingsperger C. Nat. Protoc. 2006; 1: 363-367Crossref PubMed Scopus (73) Google Scholar, 13Blankenberg F. Mari C. Strauss H.W. Q. J. Nucl. Med. 2003; 47: 337-348PubMed Google Scholar). to this study little was known about the role of other members of the annexin family in cell death recognition. a signal for cellular and in the of annexins were it that annexins can be are in serum and act on and Genderen Kenis H. Hofstra L. Narula J. Reutelingsperger C.P. 2008; PubMed Scopus Google Scholar, M. F. Nat. Immunol. 2009; PubMed Scopus Google Scholar). the individual annexin repertoire of dying cells and phagocytes may to the process of clearance of dying cells in normal and of the PS-binding annexins AnxA3, AnxA4, AnxA5, and AnxA13 with which a to identified the amino acid residues essential for apoptotic cell death recognition. The of two acid residues in in domains two and four is for efficient binding of annexins to the of apoptotic cells at physiological calcium in vitro showed that and within the conserved residues of the type 2 calcium-binding motif of to membrane binding and 1995; PubMed Scopus Google Scholar). was shown that of of the four calcium-binding motifs within the AnxA5 protein the for calcium and M. Smith C. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). The conserved acid residues in the motif in domains two and four in other annexins are in AnxA8 by or residues and the of the calcium binding This reduces the of AnxA8 to PS a for with the cell surface of apoptotic cells at physiological calcium This is by the of AnxA8 to PS as detected in surface binding In binding the of four in is for binding to whereas of or residues with acid in the and bind to a similar to apoptotic cells. The phospholipid of liposomes used in biochemical may the on cell the phylogenetically AnxA13 and the AnxA5 an motif and bind to dying cells (22Moss S.E. Morgan R.O. Genome Biol. 2004; 5: 219Crossref PubMed Scopus (401) Google Scholar). This may to a loss of function in the calcium binding motifs of AnxA8 on the of annexin calcium-binding domains in annexins already the about the functional and physiological roles of within these calcium binding domains R.O. S. 2004; PubMed Scopus Google Scholar). The in the recognition of the PS signal by individual annexins the structural of the motif to roles in the recognition of apoptotic the of the PS signal and the calcium binding motif of annexins it is to that the of the immune to the interpretation of the “eat me” signal PS at the cell surface of dying cells in is by the of calcium binding annexins. This is by the that AnxA1 and AnxA5 bind to the cell surface of apoptotic cells, but in macrophages upon The of AnxA1 the efficient clearance of apoptotic cells (19Arur S. Uche U.E. Rezaul K. Fong M. Scranton V. Cowan A.E. Mohler W. Han D.K. Dev. Cell. 2003; 4: 587-598Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar, M. A. C. P. J. Immunol. 2007; PubMed Scopus Google Scholar) and prevents the production of proinflammatory by macrophages (20Blume K.E. Soeroes S. Waibel M. Keppeler H. Wesselborg S. Herrmann M. Schulze-Osthoff K. Lauber K. J. Immunol. 2009; 183: 8138-8147Crossref PubMed Scopus (64) Google Scholar). In AnxA5 with the of apoptotic cells by phagocytosis and an response in macrophages (17Frey B. Munoz L.E. Pausch F. Sieber R. Franz S. Brachvogel B. Poschl E. Schneider H. Rödel F. Sauer R. Fietkau R. Herrmann M. Gaipl U.S. J. Cell Mol. Med. 2009; 13: 1391-1399Crossref PubMed Scopus (23) Google Scholar). of phagocytosis by AnxA5 cells to cells which immune and even against the X. Kern P.M. W. Herrmann M. Cell 7: PubMed Scopus Google Scholar). The of annexins may be to the of the immune to the PS signal in the local to an appropriate response toward dying cells and inflammation and autoimmune on this in vitro a in annexin mice be In the of and AnxA5-deficient mice are (17Frey B. Munoz L.E. Pausch F. Sieber R. Franz S. Brachvogel B. Poschl E. Schneider H. Rödel F. Sauer R. Fietkau R. Herrmann M. Gaipl U.S. J. Cell Mol. Med. 2009; 13: 1391-1399Crossref PubMed Scopus (23) Google Scholar, S. B. M. Full Text Full Text PDF PubMed Scopus Google Scholar). mice show an increased to and whereas in AnxA5-deficient mice on the normal in vivo immune response toward apoptotic cells were L.E. Franz S. Pausch F. Fürnrohr B. Sheriff A. Vogt B. Kern P.M. Baum W. Stach C. von Laer D. Brachvogel B. Poschl E. Herrmann M. Gaipl U.S. J. Leukoc. Biol. 2007; 81: 6-14Crossref PubMed Scopus (52) Google Scholar). when the immune by cells, a of is in AnxA5 mice (17Frey B. Munoz L.E. Pausch F. Sieber R. Franz S. Brachvogel B. Poschl E. Schneider H. Rödel F. Sauer R. Fietkau R. Herrmann M. Gaipl U.S. J. Cell Mol. Med. 2009; 13: 1391-1399Crossref PubMed Scopus (23) Google Scholar). in molecules involved in the recognition and of dying cells show specific but in vivo receptors of the scavenger family directly interact with phosphatidylserine on the cell surface of dying cells (6Rigotti A. Acton S.L. Krieger M. J. Biol. Chem. 1995; 270: 16221-16224Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar, N. S. Cell Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) and macrophages of scavenger receptor mice a in the uptake of apoptotic the of dying cells in the of is by the N. H. T. S. J. Immunol. PubMed Scopus Google Scholar). is that clearance of dying cells in AnxA5-deficient mice is the functional AnxA5 and other of their annexins may also play a role and are to be involved in the and of autoimmune like AnxA1 of the and have been detected in with F. R. C.A. J. E. A. De M. V. M. S. I. PubMed Scopus (110) Google Scholar) and may be a but also a may with AnxA1 bound to apoptotic cell and the removal of dying cells by The of AnxA1 together with the of the PS signal on dying cells may promote uptake by cells and the of which then are for AnxA5 (16Gaipl U.S. Munoz L.E. Rödel F. Pausch F. Frey B. Brachvogel B. von der Mark K. Pöschl E. Autoimmunity. 2007; 40: 254-259Crossref PubMed Scopus (25) Google Scholar). that annexins and in binding and clearance of dying cells, the may be The annexin repertoire at the surface of apoptotic cells as as phagocytes to the interpretation of apoptotic patterns by macrophages to an appropriate response of the immune a against annexin with the capacity to bind to dying cells may the immune response toward dying cells and a risk for and autoimmune IntroductionDiscrimination of viable from dying cells is a prerequisite for the efficient clearance of dying and dead cells and to suppress uncontrolled cell lysis and the release of potentially harmful cellular compounds to the local environment. During development and under normal physiological conditions, cells are removed through the process of apoptosis and undergo a strictly defined series of morphological and biochemical changes, before being engulfed by professional phagocytes such as macrophages or even by neighboring cells. A signature of specific “eat me” signals are exposed at the cell surface of apoptotic cells, which enable direct or indirect interactions with phagocytes and dying cells to promote the efficient clearance of apoptotic cells. This reduces the risk of accumulation of secondarily necrotic cells and the release of cellular content to the microenvironment. Exposure of apoptotic-cell associated molecular patterns together with recognition and interpretation of these by phagocytes are crucial steps in the appropriate response of phagocytes toward the engulfed cells (1Manfredi A.A. Iannacone M. D'Auria F. Rovere-Querini P. Apoptosis. 2002; 7: 153-161Crossref PubMed Scopus (31) Google Scholar).A hallmark of early apoptotic cells is the loss of membrane asymmetry and the exposure of anionic phosphatidylserine (PS) 3The abbreviations used are: PSphosphatidylserinePCphosphatidylcholinePEphosphatidylethanolaminePIpropidium iodide. on the outer lipid layer of the cells. In most cells PS predominantly resides in the inner leaflet of the plasma membrane, regulating membrane charge and protein localization (2Yeung T. Gilbert G.E. Shi J. Silvius J. Kapus A. Grinstein S. Science. 2008; 319: 210-213Crossref PubMed Scopus (748) Google Scholar). This membrane asymmetry is actively maintained by an inward transporting aminophospholipid translocase (3Tang X. Halleck M.S. Schlegel R.A. Williamson P. Science. 1996; 272: 1495-1497Crossref PubMed Scopus (417) Google Scholar). Upon induction of apotosis, rising cytoplasmic Ca2+ levels cause a loss of translocase activity and an activation of bi-directional transporting proteins of the scramblase family to expose PS at the cell surface (4Zhou Q. Zhao J. Stout J.G. Luhm R.A. Wiedmer T. Sims P.J. J. Biol. Chem. 1997; 272: 18240-18244Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). This phospholipid represents the major recognition cue for the engulfment of dying cells by professional and nonprofessional phagocytes (5van den Eijnde S.M. Boshart L. Baehrecke E.H. De Zeeuw C.I. Reutelingsperger C.P. Vermeij-Keers C. Apoptosis. 1998; 3: 9-16Crossref PubMed Scopus (113) Google Scholar). A number of interaction partners for the “eat me” signal PS have been described, including phagocyte cell surface receptors like members of the scavenger receptor family (6Rigotti A. Acton S.L. Krieger M. J. Biol. Chem. 1995; 270: 16221-16224Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar) or TIM1 and TIM4 (7Miyanishi M. Tada K. Koike M. Uchiyama Y. Kitamura T. Nagata S. Nature. 2007; 450: 435-439Crossref PubMed Scopus (810) Google Scholar), which can directly bind to PS on dying cells. Several PS-binding serum proteins like protein S (8Anderson H.A. Maylock C.A. Williams J.A. Paweletz C.P. Shu H. Shacter E. Nat. Immunol. 2003; 4: 87-91Crossref PubMed Scopus (313) Google Scholar), milk fat globule protein (MFG-E8) (9Hanayama R. Tanaka M. Miwa K. Shinohara A. Iwamatsu A. Nagata S. Nature. 2002; 417: 182-187Crossref PubMed Scopus (1030) Google Scholar) and β2-glycoprotein-1 (10Balasubramanian K. Schroit A.J. J. Biol. Chem. 1998; 273: 29272-29277Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) can act as bridging molecules that bind to the signature of the apoptotic cells. The interpretation of the PS signature in the context of bridging molecules and receptors by phagocytes then modulates the response toward apoptotic cells.Annexin A5 (Anxa5-gene, AnxA5-protein) was identified as a specific interaction partner for PS and is extensively used to detect cell death in vitro and in vivo (11Vermes I. Haanen C. Steffens-Nakken H. Reutelingsperger C. J. Immunol. Methods. 1995; 184: 39-51Crossref PubMed Scopus (4546) Google Scholar, 12van Genderen H. Kenis H. Lux P. Ungeth L. Maassen C. Deckers N. Narula J. Hofstra L. Reutelingsperger C. Nat. Protoc. 2006; 1: 363-367Crossref PubMed Scopus (73) Google Scholar, 13Blankenberg F. Mari C. Strauss H.W. Q. J. Nucl. Med. 2003; 47: 337-348PubMed Google Scholar). This protein can also inhibit the phagocytosis of dying cells (14Kenis H. van Genderen H. Deckers N.M. Lux P.A. Hofstra L. Narula J. Reutelingsperger C.P. Exp. Cell Res. 2006; 312: 719-726Crossref PubMed Scopus (53) Google Scholar, 15Munoz L.E. Franz S. Pausch F. Fürnrohr B. Sheriff A. Vogt B. Kern P.M. Baum W. Stach C. von Laer D. Brachvogel B. Poschl E. Herrmann M. Gaipl U.S. J. Leukoc. Biol. 2007; 81: 6-14Crossref PubMed Scopus (52) Google Scholar) and modulates the cytokine response of phagocytes toward apoptotic and necrotic cells (16Gaipl U.S. Munoz L.E. Rödel F. Pausch F. Frey B. Brachvogel B. von der Mark K. Pöschl E. Autoimmunity. 2007; 40: 254-259Crossref PubMed Scopus (25) Google Scholar). In vivo, AnxA5-deficient mice displayed an increased uptake of necrotic cells and a reduced allogenic immune reaction against these (17Frey B. Munoz L.E. Pausch F. Sieber R. Franz S. Brachvogel B. Poschl E. Schneider H. Rödel F. Sauer R. Fietkau R. Herrmann M. Gaipl U.S. J. Cell Mol. Med. 2009; 13: 1391-1399Crossref PubMed Scopus (23) Google Scholar, 18Brachvogel B. Dikschas J. Moch H. Welzel H. von der Mark K. Hofmann C. Pöschl E. Mol. Cell. Biol. 2003; 23: 2907-2913Crossref PubMed Scopus (80) Google Scholar). Besides AnxA5, two other annexins may play a role in cell death recognition and removal. AnxA1 is externalized upon cell death and can be detected in PS-rich membrane plaques of dying cells (19Arur S. Uche U.E. Rezaul K. Fong M. Scranton V. Cowan A.E. Mohler W. Han D.K. Dev. Cell. 2003; 4: 587-598Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar) where it promotes the removal of dying cells and prevents proinflammatory cytokine production (20Blume K.E. Soeroes S. Waibel M. Keppeler H. Wesselborg S. Herrmann M. Schulze-Osthoff K. Lauber K. J. Immunol. 2009; 183: 8138-8147Crossref PubMed Scopus (64) Google Scholar). Both AnxA1 and AnxA2 are present at the cell surface of macrophages and may adjust the phagocyte response toward dying cells (21Fan X. Krahling S. Smith D. Williamson P. Schlegel R.A. Mol. Biol. Cell. 2004; 15: 2863-2872Crossref PubMed Scopus (110) Google Scholar). Most members of the evolutionary conserved, over 1 billion year old annexin family may be involved in the identification and removal of dying cells by phagocytes, but little is yet known about their individual roles as PS-binding immunomodulatory proteins and the underlying molecular mechanisms of PS recognition.In mammals, the protein family of annexins includes 12 members, with AnxA13 being the phylogenetically oldest annexin (22Moss S.E. Morgan R.O. Genome Biol. 2004; 5: 219Crossref PubMed Scopus (401) Google Scholar). All annexins carry four similar domains defined by five α-helices and each domain contains a characteristic type 2 calcium-binding motif (GXGTD-[X]37-(D/E)). The four domains are arranged in a disc-like structure with antipodal convex and concave surfaces. Upon binding to calcium the convex side of an annexin specifically interacts with anionic phospholipids, as shown for AnxA5 (23Andree H.A. Reutelingsperger C.P. Hauptmann R. Hemker H.C. Hermens W.T. Willems G.M. J. Biol. Chem. 1990; 265: 4923-4928Abstract Full Text PDF PubMed Google Scholar). Because of the structural and functional similarities within the annexin family we speculated that annexins can bind to PS exposed at the apoptotic cell In this we and five annexins and their binding toward PS in and interaction In we characterized their binding to cell surface exposed PS in results show that most annexins interact with PS whereas AnxA8 lacks this ability. alignment identified the conserved amino mediating the PS recognition by annexins and when these amino residues the AnxA8 protein site directed mutagenesis the protein gains the to bind to These results that we have identified the essential amino for binding of annexins toward PS and that annexins may act as to the recognition of PS at the cell surface of dying cells.