Structure of Membrane-bound Annexin A5 Trimers: A Hybrid Cryo-EM - X-ray Crystallography Study

Oling, Frank; Santos, Jana Sopková−de Oliveira; Govorukhina, Natalia; Mazères-Dubut, Christine; Bergsma-Schutter, Wilma; Oostergetel, Gert T.; Keegstra, Wilko; Lambert, Olivier; Lewit-Bentley, Anita; Brisson, Alain

doi:10.1006/jmbi.2000.4183

Cited by 81 publications

(79 citation statements)

References 53 publications

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“…7). From the experiments with M23 and annexin A1 we concluded that the process of invagination is driven nanomechanically by the formation of an annexin A5 two-dimensional crystal that bends the membrane, which is likely due to the bent shape of the trimer building blocks (19). The mechanism of membrane bending differs from that operating in clathrin-coated pits.…”

Section: Discussionmentioning

confidence: 96%

“…The tertiary structure of annexin A5 shows that its phospholipid binding side has a convex shape (27). When bound to the membrane annexin A5 forms trimers of which each monomer retains the convex shape at its phospholipid binding side (19,28). We hypothesized that the trimers bend the membrane and provide the driving force for the reversion of membrane movement.…”

Section: A Novel Pinocytic Pathway Internalizes Annexin A5 Into Cellsmentioning

confidence: 99%

“…We hypothesized that the trimers bend the membrane and provide the driving force for the reversion of membrane movement. To test the hypothesis we generated the annexin A5 mutant M23 (21), which by prediction from the available structural data (19,27) binds to PtdSer but lacks the ability to form trimers. Using a novel FRET assay we showed that annexin A5 but not M23 develops FRET when bound to a phospholipid surface (Fig.…”

Section: A Novel Pinocytic Pathway Internalizes Annexin A5 Into Cellsmentioning

confidence: 99%

“…It binds reversibly to PtdSer-expressing membranes with high affinity in a calcium-dependent manner (15,16) such that ambient extracellular calcium conditions in vivo promote binding to apoptotic cells (17). Binding to PtdSer is accompanied by the formation of a two-dimensional network of annexin A5 molecules through protein-protein interactions (18,19). This phenomenon was explored using model membranes but is believed to occur on the apoptotic cell surface also (20).…”

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confidence: 99%

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Cell Surface-expressed Phosphatidylserine and Annexin A5 Open a Novel Portal of Cell Entry

Kenis¹,

Genderen²,

Bennaghmouch³

et al. 2004

Journal of Biological Chemistry

141

138

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Expression of phosphatidylserine (PtdSer) at the cell surface is part of the membrane dynamics of apoptosis. Expressed phosphatidylserine functions as an "eat me" flag toward phagocytes. Here, we report that the expressed phosphatidylserine forms part of a hitherto undescribed pinocytic pathway. Annexin A5, a phosphatidylserine-binding protein, binds to and polymerizes through protein-protein interactions on membrane patches expressing phosphatidylserine. The two-dimensional protein network of annexin A5 at the surface prevents apoptotic body formation without interfering with the progression of apoptosis as demonstrated by activation of caspase-3, PtdSer exposure, and DNA fragmentation. The annexin A5 protein network bends the membrane patch nanomechanically into the cell and elicits budding, endocytic vesicle formation, and cytoskeleton-dependent trafficking of the endocytic vesicle. Annexin A1, which binds to PtdSer without forming a two-dimensional protein network, does not induce the formation of endocytic vesicles. This novel pinocytic pathway differs from macropinocytosis, which is preceded by membrane ruffling and actin polymerization. We clearly showed that actin polymerization is not involved in budding and endocytic vesicle formation but is required for intracellular trafficking. The phosphatidylserine-annexin A5-mediated pinocytic pathway is not restricted to cells in apoptosis. We demonstrated that living tumor cells can take up substances through this novel portal of cell entry. This opens new avenues for targeted drug delivery and cell entry.

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Section: Discussionmentioning

confidence: 96%

Section: A Novel Pinocytic Pathway Internalizes Annexin A5 Into Cellsmentioning

confidence: 99%

Section: A Novel Pinocytic Pathway Internalizes Annexin A5 Into Cellsmentioning

confidence: 99%

mentioning

confidence: 99%

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Cell Surface-expressed Phosphatidylserine and Annexin A5 Open a Novel Portal of Cell Entry

Kenis¹,

Genderen²,

Bennaghmouch³

et al. 2004

Journal of Biological Chemistry

141

138

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“…The four AB calcium (35,36). From a structural standpoint, the independence of binding sites in different domains seems reasonable, because the sites are much further separated in space than are binding sites within the same domain, and each domain forms a fairly autonomous folding unit.…”

Section: Role Of Individual Sites In Membranementioning

confidence: 99%

Essential Role of B-helix Calcium Binding Sites in Annexin V-Membrane Binding

Jin¹,

Smith²,

Hsieh³

et al. 2004

Journal of Biological Chemistry

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Crystal structures of annexin V have shown up to 10 bound calcium ions in three different types of binding sites, but previous work concluded that only one of these sites accounted for nearly all of the membrane binding affinity of the molecule. In this study we mutated residues contributing to potential calcium binding sites in the AB and B helices in each of the four domains (eight sites in total) and in DE helices in the first, second, and third domains (three sites in total). We measured the affinity of each protein for phospholipid vesicles and cell membranes by quantitative calcium titration under low occupancy conditions (<1% saturation of available membrane binding sites). Affinity was calculated from the midpoint and slope of the calcium titration curve and the concentration of membrane binding sites. The results showed that all four AB sites were essential for high affinity binding, as were three of the four B sites (in domains 1, 2, and 3); the DE site in the first domain made a slight contribution to affinity. Multisite mutants showed that each domain contributed additively and independently to binding affinity; in contrast, AB and B sites within the same domain were interdependent. The number of functionally important sites identified was consistent with the Hill coefficient observed in calcium titrations. This study shows an essential and previously unappreciated role for B-helix calcium binding sites in the membrane binding of annexins and indicates that all four domains of the molecule are required for maximum membrane binding affinity.The annexins are a family of calcium-dependent membranebinding proteins with diverse functions (1-4). Annexin V is a typical member of this protein family, which is becoming important as a means to measure cell death in vivo in cancer chemotherapy, organ transplant rejection, and myocardial infarction (5-10), creating a need to better understand structural and functional properties relevant to its clinical use. Novel engineered forms of annexin V are being developed to improve its utility for imaging (11,12), and there has been a recent report of a single-domain version of annexin V that may be useful for imaging cell death in vivo (13).Calcium binding sites in annexins are of three structural types: AB (or Type II), DE (or Type III), and B or ABЈ (14). In these binding sites, calcium ligands consist of one to three carbonyl oxygens from the protein backbone, and one carboxyl group from an Asp or Glu residue; sites are often named based on the domain (1 through 4) and ␣-helices (A through E), which contribute the backbone carbonyl ligands. However, the number of calcium ions observed in different structures varies widely; for example, anywhere from 2 or 3 (15, 16) to 10 (17) calcium ions have been observed in different crystal forms of annexin V. Thus, it has been difficult to determine which calcium binding sites are functionally important based on structural data alone. Previous work with annexin V mutants indicated that the AB site in domain 1 was the most impor...

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Internalization of annexin A5‐functionalized iron oxide particles by apoptotic Jurkat cells

Tilborg

Geelen

Duimel

et al. 2009

Contrast Media & Molecular

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Apoptosis plays an important role in the etiology of various diseases. Several studies have reported on the use of annexin A5-functionalized iron oxide particles for the detection of apoptosis with MRI, both in vitro and in vivo. The protein annexin A5 binds with high affinity to the phospholipid phosphatidylserine, which is exposed in the outer leaflet of the apoptotic cell membrane. When co-exposed to apoptotic stimuli, this protein was shown to internalize into endocytic vesicles. Therefore in the present study we investigated the possible internalization of commercially available annexin A5-functionalized iron oxide particles (r1 = 34.0 +/- 2.1 and r2 = 205.0 +/- 10.4 mm(-1) s(-1) at 20 MHz), and the effects of their spatial distribution on relaxation rates R2*, R2 and R1. Two different incubation procedures were performed, where (1) Jurkat cells were either incubated with the contrast agent after induction of apoptosis or (2) Jurkat cells were simultaneously incubated with the apoptotic stimulus and the contrast agent. Transmission electron microscopy images and relaxation rates showed that the first incubation strategy mainly resulted in binding of the annexin A5-iron oxide particles to the cell membrane, whereas the second procedure allowed extensive membrane-association as well as a small amount of internalization. Owing to the small extent of internalization, only minor differences were observed between the DeltaR2*/DeltaR2 and DeltaR2/DeltaR1 ratios of cell pellets with membrane-associated or internalized annexin A5 particles. Only the increase in R1 (DeltaR1) appeared to be diminished by the internalization. Internalization of annexin A5-iron oxide particles is also expected to occur in vivo, where the apoptotic stimulus and the contrast agent are simultaneously present. Where the extent of internalization in vivo is similar to that observed in the present study, both T2- and T2*-weighted MR sequences are considered suitable for the detection of these particles in vivo.

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Structure of Membrane-bound Annexin A5 Trimers: A Hybrid Cryo-EM - X-ray Crystallography Study

Cited by 81 publications

References 53 publications

Cell Surface-expressed Phosphatidylserine and Annexin A5 Open a Novel Portal of Cell Entry

Cell Surface-expressed Phosphatidylserine and Annexin A5 Open a Novel Portal of Cell Entry

Essential Role of B-helix Calcium Binding Sites in Annexin V-Membrane Binding

Internalization of annexin A5‐functionalized iron oxide particles by apoptotic Jurkat cells

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