Biochemical Society Transactions
Annexin V, a calcium-dependent phospholipid-binding protein J. H. Walker,* C. M. Boustead, J. J. Koster, M. Bewley and D. A. Waller Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT,U.K. 828
Introduction Calcium regulates cellular processes by interacting with calcium-binding proteins. Two major families of calcium-binding proteins have been identified: The E-F hand proteins, which include calmodulin, SlOO and calcyclin; and the annexins (see [ l ] for review, and [2] for annexin nomenclature). Both families constitute major pools of calcium-binding activity. They are found in high concentrations in cells and are highly conserved through evolution, being present in all eukaryotes so far studied. Interestingly these two families are linked in certain cases. For instance, annexin I1 contains an S100-like subunit and recently a novel annexin has been described as a calcyclin binding protein [ 31.
The annexin family of calcium-binding proteins The annexins are a group of at least 12 distinct proteins which bind to phospholipids in a calciumdependent manner. Cloning and sequencing of the proteins has shown approximately 50% sequence homology between different annexins in the same species. In addition to binding calcium and phospholipids, they are associated to varying degrees with the cytoskeleton, again with a requirement for calcium. Certain annexins are also substrates for protein kinases including protein kinase C and the tyrosine kinase activities of the EGF-receptor and pp60”“. Many functions have been proposed for annexins at both intracellular and extracellular locations, including a role in calcium-dependent exocytosis [ 41, regulation of membrane-cytoskeleton interactions [ 51, anticoagulant activity [6], inhibition of phospholipase A2 [7, 81, and the formation or regulation of ion channels [9-111. However, their precise physiological role remains to be determined.
Annexin V Annexin V is a 35 kI>A monomeric protein. It was identified independently by several groups with differing research aims, one of the earliest mentions of the human protein being as a placental anticoagulant [12], and of chicken annexin V as a collagen-binding protein [ 131. Subsequent cloning
*Towhom correspondence should be addressed.
Volume 20
and sequencing showed these proteins to be members of the annexin family [ 14-16]. In bovine tissues, but not in other species studied to date, annexin V exists as two closely related isoforms [ 17-19]. Annexin V resembles other annexins of approximately 35 k l h (annexins I, 11, 111, IV and VIII) in consisting of a unique N-terminal region, followed by four repeats of approximately 70 amino acids, each of which contains the highly conserved sequence -(M or 1,)-K-G-(A or 1,)-G-T. The Nterminal region of several other annexins contains phosphorylation sites for various protein kinases but no evidence has been obtained so far for the phosphorylation of annexin V.
Binding properties of annexin V The binding of calcium to annexin V has been investigated by €Iaigler and coworkers [ 201, whose studies suggested the presence of 3-5 CaL+-binding sites in the protein. This correlates well with the number of binding sites recently determined by X-ray crystallographic studies (see below). As described for other annexins, the presence of anionic phospholipid significantly increases the affinity of Ca”-binding to annexin V [20]. Various groups have investigated the phospholipid-binding properties of annexin V and shown that in common with other annexins, annexin V binds preferentially to acidic phospholipids [ 18, 20-231. The binding of human annexin V to phosphatidylserine-containing liposomes is of high affinity, with a K , in the l o - “ ’ \I range at physiological ionic strength [21, 221. Figure 1 demonstrates the results of phospholipid-binding studies on the two bovine isoforms of annexin V, isolated as described previously [18]. Bovine annexin V was incubated with sucrose-loaded liposomes at varying calcium concentrations. Following centrifugation, the proportion of annexin V in the supernatant and pellet was measured as described 1231. The bovine annexin V isoforms bind to liposomes containing phosphatidic acid, phosphatidylserine. phosphatidylethanolamine and phosphatidylinositol uith halfmaximal binding at calcium concentration of 1 1 p \ ~ , 45 p\i, 70 p\i and 1 1 1 p v respectively. No binding was observed to liposomes of phosphatidylcholine alone at calcium concentrations up to 1 mhi. The binding of annexin V to phospholipids
Lipid-Binding Proteins
Fig. I
Effects of Ca2' concentration on the binding of bovine annexin V isoforms t o phospholipids Protein (10 pg) was incubated with sucrose-loaded liposomes ( 150 peg) formed of I : 1 mixtures of phosphatidylcholine and phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylethanolamine (PE) or phosphatidylinositol in the presence of varying free Ca2+ concentrations. Free and bound protein were separated by centrifugation, and the percentage of protein in the phospholipid-containingpellet measured [23].
cellular protein which bound to type I1 collagen. Subsequent sequencing demonstrated this protein to be the chicken homologue of human annexin V [15, 261. It was then shown that the major proportion of this annexin is intracehlar in these cells and the physiological relevance of collagen binding is uncertain. ~ ~ ~ ~mmunoreact~v~ty ~ ~ h to ~ annexin was Seen On the Outer surface Of chondrocytes and at least a proportion of the protein therefore seems to be secreted [27].
Three-dimensional structure of annexin V
40
80
120 l60 Ca2+ ( p )
2oo
300
appears to be largely ionic in nature since binding affinity is reduced by increasing ionic strength [21], and the bound annexin V can be displaced by the polycation spermine [24]. However, it has been reported that annexins IV and VI are able to bind directly to arachidonic acid and oleic acid in a calcium-dependent manner [25], and it is possible that binding to the fatty acid moieties is also involved in the phospholipid binding of annexin V. In addition to binding phospholipids, annexin V interacts in a calcium-dependent manner with cytoskeletal components. Binding to F-actin has been demonstrated using- centrifugation assays, although millimolar calcium concentrations are required, raising doubts about its physiological significance [ZO]. However, we have evidence from immunohistochemical studies at the level of resolution of the electron microscope for an association of chicken annexin V with the actin-rich terminal web of intestinal epithelial cells, suggesting that there may be a cytoskeletal association in viva Collagen binding has also been reported for annexin V. Chicken annexin V was first isolated and sequenced by the group of von der Mark [ 131 who were studying the extracellular matrix produced by chick chondrocytes. They initially named their protein anchorin CII and considered it to be an extra-
The structure of human annexin V was solved in 1990 by Huber and coworkers [9,28]. In collaboration with Professor Huber we have determined the structure of annexin V isolated from chicken liver. Our results demonstrate that annexin V consists of four distinct domains (see Fig. 2a). Each domain contains the 70 amino acid annexin repeat of five helices arranged in a four helix bundle, with a fifth helix linking the anti-parallel helix-turn-helix substructures. The molecule has essentially a disc shape with convex and concave faces to the disc. The convex surface possesses the calcium binding sites which are probably involved in binding the protein to the lipid bilayer. The N-terminal tail interacts with domain IV, binding close to the Cterminus and linking domains I and IV. It has been suggested that the four domains form a structure resembling a channel protein in that bundles of alpha helices surround a central pore. This pore contains salt bridges which could facilitate the transport of ions. The mechanisms by which annexins may be involved in the transport of ions across membranes will be discussed later, but it is very unlikely that these proteins could be incorporated fully into the lipid bilayer. Annexins may therefore transport ions by a mechanism distinct from that of conventional ion channels which are integral membrane proteins. The three-dimensional structure has enabled the identification of three calcium-binding sites in chicken annexin V. These correspond to the conserved sequence -K-G-X-G-T-(38 residues)-E/Dwithin domains I, I1 and IV. The sequence -K-G-XG-T- forms the central portion of the loop joining the first and second alpha-helices within the domain. Interestingly the equivalent loop in domain I11 shows a much lower conservation of sequence and lacks the ability to bind calcium. Each calcium ion is co-ordinated by the carbonyl groups of three residues in the loop, and the carboxylic acid moiety of either the Glu or Asp residue (see Fig. 2b). This
I992
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Biochemical Society Transactions
Fig. 2 Three-dimensional structure of annexin V ( a ) The structure of chicken annexin V. The three calcium ions are shown (shaded circles) in domains I, II and IV. (b) The calcium
830
binding site in domain IV shown in detail. Residues 26 I and 263 are the glycines of the -K-G-X-G-T-sequence, The unlabelled calcium ligand is a water molecule.
x
represents a calcium-binding site distinct from the 'E-F hand' motif characteristic of the calmodulin family. The only protein apart from the annexins to have a similar calcium-binding site is phospholipase A,, which differs in the number of residues between the calcium-binding loop and the carboxylic acid moiety [29]. Human annexin V bound in a calciumdependent manner to phospholipid bilayers has been analysed by electron image analysis [30]. These studies demonstrated that when bound to artificial phospholipid bilayers, annexin V associates into extensive networks of triskelion-like formations. The three-dimensional structure as determined by X-ray crystallography correlates well with the structure shown by electron image analysis [31].
Tissue distribution and subcellular location Annexin V has a wide, although not ubiquitous, distribution, being present at a high concentration (up to 2.0% of cellular protein) in many tissue and cell types [32-34]. Immunohistochemical studies at the level of resolution of the light microscope [23] demonstrated that in porcine brain, annexin V is located in glial cells where its staining pattern is consistent with a cytosolic location. No annexin V
Volume 20
was observed in neurons. A similar pattern was observed in rat brain [34]. We have also studied the location of annexin V in chick embryo fibroblasts [351. Fibroblasts fixed with glutaraldehyde and stained with a monospecific antiserum to annexin V demonstrate immunoreactivity which extends throughout the cell. Lipid droplets in the cytoplasm of the fibroblasts are unstained but are surrounded by immunostaining which is characteristic of the results expected for cytosolic antigens and previously observed for calmodulin [36]. In primary cultures of chick embryo fibroblasts, immunoreactivity extends into the nuclei of the fibroblasts whereas in secondary cultures derived from these primary cultures up to 25% of the cells do not contain annexin V in the nuclei. A sub-population of annexin V is permanently associated with the cell membrane since immune staining can be seen on ventral membranes remaining attached to the substratum when the upper cell membrane has been removed from the cells after binding to a sheet of nitrocellulose [37]. This is true even for cells disrupted in buffers containing high concentrations of calcium chelators, suggesting a calcium-independent membrane-associated form of annexin V. Subcellular fractionation experiments confirm these results (see below). We have also studied the dis-
Lipid-Binding Proteins
tribution of annexin V in sections of chicken tissues using immunohistochemistry. In chicken liver, annexin V is present in several locations. Most noticeably it is found at very high concentrations in bile duct epithelial cells where it seems to be predominantly cytosolic in location. Donato and coworkers have also used an immunochemical approach to investigate the location of annexin V in rat tissues [34]. They also find a predominantly cytosolic location, although in certain tissues, notably cardiac muscle, it has a membrane localization. W e have used sub-cellular fractionation studies to complement our immunohistochemical results. Two pools of annexin V can be identified within chick heart fibroblasts. In addition to annexin V which binds reversibly to membranes in a calcium-dependent manner, a second pool of annexin V seems to exist in a calcium-independent membrane-associated form. Membranes have been prepared from cells homogenized in the presence of high concentrations of calcium chelators. Extensive washing of these membranes is incapable of solubilizing the membrane-associated annexin V. Addition of Triton X- 100 immediately solubilizes the annexin. Similar results have been obtained by Donato and coworkers with bovine annexin V isoforms [38]. They have demonstrated the existence of hydrophobic forms of annexin V which partition into Triton X-114 at 37°C. Thus from both subcellular fractionation studies and from immunohistochemical studies there is considerable evidence in favour of multiple forms of annexin V within cells. Evidence exists for the relocation of annexins onto membranes as a result of various stimuli [39-411. With human platelets we have obtained preliminary evidence that physiological stimulation of platelets with thrombin results in relocation of annexin V to the membrane in a form which requires non-ionic detergent for its subsequent solubilization. Interestingly, there is also increasing evidence for the secretion of annexin V from cells. Extracellular annexin V has been detected in rat peritoneal lavages [32], and is secreted by the human prostate gland [42]. Here annexins I and V were found in prostatic fluid at concentrations approaching 0.1 mg/ml. The possibility that this resulted from cell lysis seems unlikely since a high concentration of annexin IV was observed within the epithelial cells lining the prostate but a very low concentration of annexin IV was found in the prostatic fluid. As mentioned above there is also evidence for the secretion of annexin V from chicken chondrocytes
[27]. With chick embryo fibroblasts, we have also obtained evidence for the secretion of annexin V. Although the bulk of the protein remains within the cell, immunoblot analysis of tissue culture supernatants demonstrates a significant concentration of secreted annexin V whereas actin, used as a marker for cytosolic release, is present at a much lower level in the culture supernatant. Annexin V does not contain a signal sequence and as yet the mechanism of secretion is unknown. Nevertheless, there are examples of other proteins, including interleukin I and platelet-derived growth factor, which appear to be secreted without having a classical signal sequence [43] and it is clear that novel pathways must be involved.
Possible roles of annexin V As discussed above, the three-dimensional structure of annexin V suggests a protein with the characteristics of an ion channel. Electrophysiological studies on annexin VII have shown that in the presence of calcium, it can form highly selective voltage-gated calcium channels [44]. Similar studies on annexin V have now shown that this protein can also form calcium channels in acidic phospholipid bilayers, which differ from the annexin VII channels in that they are also permeant to other divalent and monovalent cations [ 101. Interestingly, although calcium is required together with annexin V for the initial formation of the channel, both calcium and excess protein can then be removed without affecting channel activity. It is not yet clear how the formation of the channel occurs, but Haigler, Pollard and co-workers [ 101 have found, in agreement with results described above, a form of annexin V which is associated with biological membranes in a calcium-independent manner and requires nonionic detergent for its solubilization. They suggest that this form may be the equivalent of the annexin V channel produced in the artificial bilayer. An apparently unrelated function which has also been suggested for annexin V is the inhibition of protein kinase C [45]. This inhibition is not a consequence of annexin preventing the access of protein kinase C to the phospholipid required to activate it, but rather seems to be a direct effect of annexin V on the kinase. The inhibition by annexin V shows specificity for protein kinase C, phosphorylation by CAMP-dependent protein kinase and epidermal growth factor receptor/kinase being unaffected. However, the biological relevance of this activity remains to be determined. The inhibition of phospholipase A, (PLA,) by annexins is another area which has been investi-
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832
gated since it implicates annexins as key factors in the control of the inflammatory response [32]. Unlike the inhibition of protein kinase C, however, the inhibition of PLAZ is likely to be a relatively non-specific effect resulting from the sequestration of the phospholipid substrate by bound annexin 181. The extent to which annexins function as inhibitors of PI,A, in vivo is therefore disputed. As mentioned earlier, annexin V was first characterized as a protein with anticoagulant activity, and several groups have now confirmed the ability of annexin V to inhibit coagulation in vitro [46, 471. There is general agreement that the anticoagulant action results from the calcium-dependent binding of annexin V to phospholipid surfaces in competition with clotting factors. However, the plasma concentration of annexin V is rather low [16], and it is unlikely to act as a circulating anticoagulant in vivo [48].
Future studies on annexin V As is evident from the preceding discussion, a remarkably diverse range of functions, both intracellular and extracellular, have been proposed for annexin V. In addition to hypotheses specific to annexin V, several other roles, such as regulation of exocytosis and membrane-cytoskeleton interactions have been suggested for annexins in general. Although it is possible that annexins share some common function within the cell, it may be that the common structural motif of annexins exists in a variety of proteins of unrelated function in the same way as the ‘E-F’ hand calcium-binding site regulates various enzyme activities. It is clearly important to determine which, if any, of the proposed functions of annexins are of physiological significance. Practical approaches to the elucidation of the biological roles of annexins may lie in the realms of molecular biological techniques such as studies with antisense oligonucleotides to prevent expression of annexins, site-directed mutagenesis, and experiments in which annexin genes are knocked out. However, even without a clearly defined function it is possible to see practical uses for annexins. Already fluorescent derivatives of annexin V have been used to demonstrate the exposure of phosphatidylserine on the surface of activated endothelial cells [49]. Similarly the anticoagulant and anti-inflammatory properties of annexins could lead to the development of novel drugs even if the true physiological function of annexins remains obscure. For instance, a novel anti-inflammatory peptide has been syn-
Volume 20
thesized based on residues 204-212 of human annexin V [SO]. In conclusion, annexins are a fascinating family of phospholipid-binding proteins which are clearly vital to all eukaryotic cells. They are found in both intracellular and extracellular locations and presumably can therefore function in both of these environments. Given the increasing interest in these proteins it seems unlikely that their true roles can remain obscure for much longer. W e are very grateful to Professor K. Huber (Martinsried, Munich, Germany) for collaboration in determining the three-dimensional structure of chicken annexin V. C.M.H. was supported by grants from the MKC and the Wellcome Trust. J.J.K. and M.H. were supported by studentships from the SEKC and MKC respectivcsly. D.A.W. received an EMHO short-term fellowship t o visit the laboratory of Professor I luber. We thank the SmithKline (1 982) Foundation and the Royal Society for equipment used in the purification of chicken annexin V. 1. Komisch. J. & I’aques. E.-1’. (1001) Med. Microbiol. Immunol. 180, 109-126 2. Crumpton, M. J. & Lkdman. J. K. (1 990) Nature 345,
212 3. Tokumitsu. lL, Mizutani, A,, Minami, H., Kobayashi, K. & IIidaka, H. (1002) J. Hiol. Chem. 267, 8910-8024 4. Ali, S. M., Geisow, M. J. & Hurgoyne, K. 13. (1080) Nature 3 4 0 , 3 13-3 15 5. Glenney, J. K., Tack, H. & I’owell, M. A. (1087) J. Cell Hiol. 104, 503-5 1 1 6. Tait, J. F.,Sakat, M., McMullen, H. A,, Miao, C. }I.. Funakoshi, T., Hendrickson, 1,. E. & Fujikawa, K. (1988) Biochemistry 27, 6268-6276) 7 . Huang, K.-S., Wallner, H. I’.. Mattaliano. K.J., Tizard, R., Hurne, C., Frey. A,, IIession, C., McGray, I]., Sinclair, 1,. K., Chow, E. I’., Iirowning, J. I,., Kamachandran, K. I,., Tang, J., Smart, J. I
Annexin V, a calcium-dependent phospholipid-binding protein J. H. Walker,* C. M. Boustead, J. J. Koster, M. Bewley and D. A. Waller Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT,U.K. 828
Introduction Calcium regulates cellular processes by interacting with calcium-binding proteins. Two major families of calcium-binding proteins have been identified: The E-F hand proteins, which include calmodulin, SlOO and calcyclin; and the annexins (see [ l ] for review, and [2] for annexin nomenclature). Both families constitute major pools of calcium-binding activity. They are found in high concentrations in cells and are highly conserved through evolution, being present in all eukaryotes so far studied. Interestingly these two families are linked in certain cases. For instance, annexin I1 contains an S100-like subunit and recently a novel annexin has been described as a calcyclin binding protein [ 31.
The annexin family of calcium-binding proteins The annexins are a group of at least 12 distinct proteins which bind to phospholipids in a calciumdependent manner. Cloning and sequencing of the proteins has shown approximately 50% sequence homology between different annexins in the same species. In addition to binding calcium and phospholipids, they are associated to varying degrees with the cytoskeleton, again with a requirement for calcium. Certain annexins are also substrates for protein kinases including protein kinase C and the tyrosine kinase activities of the EGF-receptor and pp60”“. Many functions have been proposed for annexins at both intracellular and extracellular locations, including a role in calcium-dependent exocytosis [ 41, regulation of membrane-cytoskeleton interactions [ 51, anticoagulant activity [6], inhibition of phospholipase A2 [7, 81, and the formation or regulation of ion channels [9-111. However, their precise physiological role remains to be determined.
Annexin V Annexin V is a 35 kI>A monomeric protein. It was identified independently by several groups with differing research aims, one of the earliest mentions of the human protein being as a placental anticoagulant [12], and of chicken annexin V as a collagen-binding protein [ 131. Subsequent cloning
*Towhom correspondence should be addressed.
Volume 20
and sequencing showed these proteins to be members of the annexin family [ 14-16]. In bovine tissues, but not in other species studied to date, annexin V exists as two closely related isoforms [ 17-19]. Annexin V resembles other annexins of approximately 35 k l h (annexins I, 11, 111, IV and VIII) in consisting of a unique N-terminal region, followed by four repeats of approximately 70 amino acids, each of which contains the highly conserved sequence -(M or 1,)-K-G-(A or 1,)-G-T. The Nterminal region of several other annexins contains phosphorylation sites for various protein kinases but no evidence has been obtained so far for the phosphorylation of annexin V.
Binding properties of annexin V The binding of calcium to annexin V has been investigated by €Iaigler and coworkers [ 201, whose studies suggested the presence of 3-5 CaL+-binding sites in the protein. This correlates well with the number of binding sites recently determined by X-ray crystallographic studies (see below). As described for other annexins, the presence of anionic phospholipid significantly increases the affinity of Ca”-binding to annexin V [20]. Various groups have investigated the phospholipid-binding properties of annexin V and shown that in common with other annexins, annexin V binds preferentially to acidic phospholipids [ 18, 20-231. The binding of human annexin V to phosphatidylserine-containing liposomes is of high affinity, with a K , in the l o - “ ’ \I range at physiological ionic strength [21, 221. Figure 1 demonstrates the results of phospholipid-binding studies on the two bovine isoforms of annexin V, isolated as described previously [18]. Bovine annexin V was incubated with sucrose-loaded liposomes at varying calcium concentrations. Following centrifugation, the proportion of annexin V in the supernatant and pellet was measured as described 1231. The bovine annexin V isoforms bind to liposomes containing phosphatidic acid, phosphatidylserine. phosphatidylethanolamine and phosphatidylinositol uith halfmaximal binding at calcium concentration of 1 1 p \ ~ , 45 p\i, 70 p\i and 1 1 1 p v respectively. No binding was observed to liposomes of phosphatidylcholine alone at calcium concentrations up to 1 mhi. The binding of annexin V to phospholipids
Lipid-Binding Proteins
Fig. I
Effects of Ca2' concentration on the binding of bovine annexin V isoforms t o phospholipids Protein (10 pg) was incubated with sucrose-loaded liposomes ( 150 peg) formed of I : 1 mixtures of phosphatidylcholine and phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylethanolamine (PE) or phosphatidylinositol in the presence of varying free Ca2+ concentrations. Free and bound protein were separated by centrifugation, and the percentage of protein in the phospholipid-containingpellet measured [23].
cellular protein which bound to type I1 collagen. Subsequent sequencing demonstrated this protein to be the chicken homologue of human annexin V [15, 261. It was then shown that the major proportion of this annexin is intracehlar in these cells and the physiological relevance of collagen binding is uncertain. ~ ~ ~ ~mmunoreact~v~ty ~ ~ h to ~ annexin was Seen On the Outer surface Of chondrocytes and at least a proportion of the protein therefore seems to be secreted [27].
Three-dimensional structure of annexin V
40
80
120 l60 Ca2+ ( p )
2oo
300
appears to be largely ionic in nature since binding affinity is reduced by increasing ionic strength [21], and the bound annexin V can be displaced by the polycation spermine [24]. However, it has been reported that annexins IV and VI are able to bind directly to arachidonic acid and oleic acid in a calcium-dependent manner [25], and it is possible that binding to the fatty acid moieties is also involved in the phospholipid binding of annexin V. In addition to binding phospholipids, annexin V interacts in a calcium-dependent manner with cytoskeletal components. Binding to F-actin has been demonstrated using- centrifugation assays, although millimolar calcium concentrations are required, raising doubts about its physiological significance [ZO]. However, we have evidence from immunohistochemical studies at the level of resolution of the electron microscope for an association of chicken annexin V with the actin-rich terminal web of intestinal epithelial cells, suggesting that there may be a cytoskeletal association in viva Collagen binding has also been reported for annexin V. Chicken annexin V was first isolated and sequenced by the group of von der Mark [ 131 who were studying the extracellular matrix produced by chick chondrocytes. They initially named their protein anchorin CII and considered it to be an extra-
The structure of human annexin V was solved in 1990 by Huber and coworkers [9,28]. In collaboration with Professor Huber we have determined the structure of annexin V isolated from chicken liver. Our results demonstrate that annexin V consists of four distinct domains (see Fig. 2a). Each domain contains the 70 amino acid annexin repeat of five helices arranged in a four helix bundle, with a fifth helix linking the anti-parallel helix-turn-helix substructures. The molecule has essentially a disc shape with convex and concave faces to the disc. The convex surface possesses the calcium binding sites which are probably involved in binding the protein to the lipid bilayer. The N-terminal tail interacts with domain IV, binding close to the Cterminus and linking domains I and IV. It has been suggested that the four domains form a structure resembling a channel protein in that bundles of alpha helices surround a central pore. This pore contains salt bridges which could facilitate the transport of ions. The mechanisms by which annexins may be involved in the transport of ions across membranes will be discussed later, but it is very unlikely that these proteins could be incorporated fully into the lipid bilayer. Annexins may therefore transport ions by a mechanism distinct from that of conventional ion channels which are integral membrane proteins. The three-dimensional structure has enabled the identification of three calcium-binding sites in chicken annexin V. These correspond to the conserved sequence -K-G-X-G-T-(38 residues)-E/Dwithin domains I, I1 and IV. The sequence -K-G-XG-T- forms the central portion of the loop joining the first and second alpha-helices within the domain. Interestingly the equivalent loop in domain I11 shows a much lower conservation of sequence and lacks the ability to bind calcium. Each calcium ion is co-ordinated by the carbonyl groups of three residues in the loop, and the carboxylic acid moiety of either the Glu or Asp residue (see Fig. 2b). This
I992
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~
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Biochemical Society Transactions
Fig. 2 Three-dimensional structure of annexin V ( a ) The structure of chicken annexin V. The three calcium ions are shown (shaded circles) in domains I, II and IV. (b) The calcium
830
binding site in domain IV shown in detail. Residues 26 I and 263 are the glycines of the -K-G-X-G-T-sequence, The unlabelled calcium ligand is a water molecule.
x
represents a calcium-binding site distinct from the 'E-F hand' motif characteristic of the calmodulin family. The only protein apart from the annexins to have a similar calcium-binding site is phospholipase A,, which differs in the number of residues between the calcium-binding loop and the carboxylic acid moiety [29]. Human annexin V bound in a calciumdependent manner to phospholipid bilayers has been analysed by electron image analysis [30]. These studies demonstrated that when bound to artificial phospholipid bilayers, annexin V associates into extensive networks of triskelion-like formations. The three-dimensional structure as determined by X-ray crystallography correlates well with the structure shown by electron image analysis [31].
Tissue distribution and subcellular location Annexin V has a wide, although not ubiquitous, distribution, being present at a high concentration (up to 2.0% of cellular protein) in many tissue and cell types [32-34]. Immunohistochemical studies at the level of resolution of the light microscope [23] demonstrated that in porcine brain, annexin V is located in glial cells where its staining pattern is consistent with a cytosolic location. No annexin V
Volume 20
was observed in neurons. A similar pattern was observed in rat brain [34]. We have also studied the location of annexin V in chick embryo fibroblasts [351. Fibroblasts fixed with glutaraldehyde and stained with a monospecific antiserum to annexin V demonstrate immunoreactivity which extends throughout the cell. Lipid droplets in the cytoplasm of the fibroblasts are unstained but are surrounded by immunostaining which is characteristic of the results expected for cytosolic antigens and previously observed for calmodulin [36]. In primary cultures of chick embryo fibroblasts, immunoreactivity extends into the nuclei of the fibroblasts whereas in secondary cultures derived from these primary cultures up to 25% of the cells do not contain annexin V in the nuclei. A sub-population of annexin V is permanently associated with the cell membrane since immune staining can be seen on ventral membranes remaining attached to the substratum when the upper cell membrane has been removed from the cells after binding to a sheet of nitrocellulose [37]. This is true even for cells disrupted in buffers containing high concentrations of calcium chelators, suggesting a calcium-independent membrane-associated form of annexin V. Subcellular fractionation experiments confirm these results (see below). We have also studied the dis-
Lipid-Binding Proteins
tribution of annexin V in sections of chicken tissues using immunohistochemistry. In chicken liver, annexin V is present in several locations. Most noticeably it is found at very high concentrations in bile duct epithelial cells where it seems to be predominantly cytosolic in location. Donato and coworkers have also used an immunochemical approach to investigate the location of annexin V in rat tissues [34]. They also find a predominantly cytosolic location, although in certain tissues, notably cardiac muscle, it has a membrane localization. W e have used sub-cellular fractionation studies to complement our immunohistochemical results. Two pools of annexin V can be identified within chick heart fibroblasts. In addition to annexin V which binds reversibly to membranes in a calcium-dependent manner, a second pool of annexin V seems to exist in a calcium-independent membrane-associated form. Membranes have been prepared from cells homogenized in the presence of high concentrations of calcium chelators. Extensive washing of these membranes is incapable of solubilizing the membrane-associated annexin V. Addition of Triton X- 100 immediately solubilizes the annexin. Similar results have been obtained by Donato and coworkers with bovine annexin V isoforms [38]. They have demonstrated the existence of hydrophobic forms of annexin V which partition into Triton X-114 at 37°C. Thus from both subcellular fractionation studies and from immunohistochemical studies there is considerable evidence in favour of multiple forms of annexin V within cells. Evidence exists for the relocation of annexins onto membranes as a result of various stimuli [39-411. With human platelets we have obtained preliminary evidence that physiological stimulation of platelets with thrombin results in relocation of annexin V to the membrane in a form which requires non-ionic detergent for its subsequent solubilization. Interestingly, there is also increasing evidence for the secretion of annexin V from cells. Extracellular annexin V has been detected in rat peritoneal lavages [32], and is secreted by the human prostate gland [42]. Here annexins I and V were found in prostatic fluid at concentrations approaching 0.1 mg/ml. The possibility that this resulted from cell lysis seems unlikely since a high concentration of annexin IV was observed within the epithelial cells lining the prostate but a very low concentration of annexin IV was found in the prostatic fluid. As mentioned above there is also evidence for the secretion of annexin V from chicken chondrocytes
[27]. With chick embryo fibroblasts, we have also obtained evidence for the secretion of annexin V. Although the bulk of the protein remains within the cell, immunoblot analysis of tissue culture supernatants demonstrates a significant concentration of secreted annexin V whereas actin, used as a marker for cytosolic release, is present at a much lower level in the culture supernatant. Annexin V does not contain a signal sequence and as yet the mechanism of secretion is unknown. Nevertheless, there are examples of other proteins, including interleukin I and platelet-derived growth factor, which appear to be secreted without having a classical signal sequence [43] and it is clear that novel pathways must be involved.
Possible roles of annexin V As discussed above, the three-dimensional structure of annexin V suggests a protein with the characteristics of an ion channel. Electrophysiological studies on annexin VII have shown that in the presence of calcium, it can form highly selective voltage-gated calcium channels [44]. Similar studies on annexin V have now shown that this protein can also form calcium channels in acidic phospholipid bilayers, which differ from the annexin VII channels in that they are also permeant to other divalent and monovalent cations [ 101. Interestingly, although calcium is required together with annexin V for the initial formation of the channel, both calcium and excess protein can then be removed without affecting channel activity. It is not yet clear how the formation of the channel occurs, but Haigler, Pollard and co-workers [ 101 have found, in agreement with results described above, a form of annexin V which is associated with biological membranes in a calcium-independent manner and requires nonionic detergent for its solubilization. They suggest that this form may be the equivalent of the annexin V channel produced in the artificial bilayer. An apparently unrelated function which has also been suggested for annexin V is the inhibition of protein kinase C [45]. This inhibition is not a consequence of annexin preventing the access of protein kinase C to the phospholipid required to activate it, but rather seems to be a direct effect of annexin V on the kinase. The inhibition by annexin V shows specificity for protein kinase C, phosphorylation by CAMP-dependent protein kinase and epidermal growth factor receptor/kinase being unaffected. However, the biological relevance of this activity remains to be determined. The inhibition of phospholipase A, (PLA,) by annexins is another area which has been investi-
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gated since it implicates annexins as key factors in the control of the inflammatory response [32]. Unlike the inhibition of protein kinase C, however, the inhibition of PLAZ is likely to be a relatively non-specific effect resulting from the sequestration of the phospholipid substrate by bound annexin 181. The extent to which annexins function as inhibitors of PI,A, in vivo is therefore disputed. As mentioned earlier, annexin V was first characterized as a protein with anticoagulant activity, and several groups have now confirmed the ability of annexin V to inhibit coagulation in vitro [46, 471. There is general agreement that the anticoagulant action results from the calcium-dependent binding of annexin V to phospholipid surfaces in competition with clotting factors. However, the plasma concentration of annexin V is rather low [16], and it is unlikely to act as a circulating anticoagulant in vivo [48].
Future studies on annexin V As is evident from the preceding discussion, a remarkably diverse range of functions, both intracellular and extracellular, have been proposed for annexin V. In addition to hypotheses specific to annexin V, several other roles, such as regulation of exocytosis and membrane-cytoskeleton interactions have been suggested for annexins in general. Although it is possible that annexins share some common function within the cell, it may be that the common structural motif of annexins exists in a variety of proteins of unrelated function in the same way as the ‘E-F’ hand calcium-binding site regulates various enzyme activities. It is clearly important to determine which, if any, of the proposed functions of annexins are of physiological significance. Practical approaches to the elucidation of the biological roles of annexins may lie in the realms of molecular biological techniques such as studies with antisense oligonucleotides to prevent expression of annexins, site-directed mutagenesis, and experiments in which annexin genes are knocked out. However, even without a clearly defined function it is possible to see practical uses for annexins. Already fluorescent derivatives of annexin V have been used to demonstrate the exposure of phosphatidylserine on the surface of activated endothelial cells [49]. Similarly the anticoagulant and anti-inflammatory properties of annexins could lead to the development of novel drugs even if the true physiological function of annexins remains obscure. For instance, a novel anti-inflammatory peptide has been syn-
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thesized based on residues 204-212 of human annexin V [SO]. In conclusion, annexins are a fascinating family of phospholipid-binding proteins which are clearly vital to all eukaryotic cells. They are found in both intracellular and extracellular locations and presumably can therefore function in both of these environments. Given the increasing interest in these proteins it seems unlikely that their true roles can remain obscure for much longer. W e are very grateful to Professor K. Huber (Martinsried, Munich, Germany) for collaboration in determining the three-dimensional structure of chicken annexin V. C.M.H. was supported by grants from the MKC and the Wellcome Trust. J.J.K. and M.H. were supported by studentships from the SEKC and MKC respectivcsly. D.A.W. received an EMHO short-term fellowship t o visit the laboratory of Professor I luber. We thank the SmithKline (1 982) Foundation and the Royal Society for equipment used in the purification of chicken annexin V. 1. Komisch. J. & I’aques. E.-1’. (1001) Med. Microbiol. Immunol. 180, 109-126 2. Crumpton, M. J. & Lkdman. J. K. (1 990) Nature 345,
212 3. Tokumitsu. lL, Mizutani, A,, Minami, H., Kobayashi, K. & IIidaka, H. (1002) J. Hiol. Chem. 267, 8910-8024 4. Ali, S. M., Geisow, M. J. & Hurgoyne, K. 13. (1080) Nature 3 4 0 , 3 13-3 15 5. Glenney, J. K., Tack, H. & I’owell, M. A. (1087) J. Cell Hiol. 104, 503-5 1 1 6. Tait, J. F.,Sakat, M., McMullen, H. A,, Miao, C. }I.. Funakoshi, T., Hendrickson, 1,. E. & Fujikawa, K. (1988) Biochemistry 27, 6268-6276) 7 . Huang, K.-S., Wallner, H. I’.. Mattaliano. K.J., Tizard, R., Hurne, C., Frey. A,, IIession, C., McGray, I]., Sinclair, 1,. K., Chow, E. I’., Iirowning, J. I,., Kamachandran, K. I,., Tang, J., Smart, J. I
