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

Abstract

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.Discrimination 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 (1).A hallmark of early apoptotic cells is the loss of membrane asymmetry and the exposure of anionic phosphatidylserine (PS) 3 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 (2). This membrane asymmetry is actively maintained by an inward transporting aminophospholipid translocase (3). Upon induction of apotosis, rising cytoplasmic Ca 2ϩ levels cause a loss of translocase activity and an activ...

Skin Wound Repair Is Not Altered in the Absence of Endogenous AnxA1 or AnxA5, but Pharmacological Concentrations of AnxA4 and AnxA5 Inhibit Wound Hemostasis

“…Similar to AnxA5, AnxA4 also prolonged plasma coagulation and induced excessive bleeding. This annexin shows the highest affinity [Rosenbaum et al, 2011] to PS and forms ordered 2D arrays on membranes [Kaetzel et al, 2001]. In contrast to AnxA5 and AnxA4, wild-type AnxA8 is not able to delay plasma coagulation.…”

“…Annexins were expressed in bacteria (BL21), purified by affinity chromatography using glutathione Sepharose 4B (GE Healthcare) and the GST-tag removed by cleavage with Factor Xa [Rosenbaum et al, 2011]. Recombinant rat AnxA5 and rat AnxA5 R16E, R23E, K27E, K56E, K191E (AnxA5 mutant) were prepared as described [Bouter et al, 2011].…”

“…Typical annexins carry 4 similar domains defined by 5 α-helices, and each domain contains a characteristic type 2 calcium-binding motif [GXGTD-[ X ] 37 -(D/E)]. Most of the evolutionarily conserved annexins can interact with PS [Kastl et al, 2002;Rosenbaum et al, 2011], and during skin injury they can bind at the site of tissue damage to suppress the binding and activation of coagulation factors at the cell surface but also promote repair [Bouter et al, 2011].…”

MiR-26a modulates extracellular matrix homeostasis in cartilage

“…A similar proportion of PECs in the G1/G0-, S-and G2/M-phase was seen in control, miR-26a mimic-or inhibitor-transfected PECs. Cell viability was assessed by flow cytometry after staining with AnxA5-Alexa488 and PI [7]. No significant difference in the proportion of AnxA5-Alexa488 (+) apoptotic and AnxA5-Alexa488 (+) PI (+) dead cells was found in control-, mimic-or inhibitortransfected PECs.…”

Caspase Activity Is Required for Engulfment of Apoptotic Cells

“…PS represents a major recognition cue for engulfment by professional and nonprofessional phagocytes (29)(30)(31). A number of phagocytic transmembrane receptors and secreted bridging molecules have been shown to bind PS (10-12, 29, 32).…”