Annexins

Every sentence written [about annexins] must be understood not as an affirmation, but as a question! (adapted slightly from Niels Bohr)

Birth of the Annexins

Typical annexin protein chain fold. Calcium ions and membranes bind to the upper surface
Click here for my 1980's work on the recognition and naming of the Annexin protein family
EM section of adrenal chromaffin cell

In 1979 I started an (optimistic!) research project to determine changes at the level of the membrane of the catecholamine-containing secretory granules of adrenal medulla with an aim to determine molecular changes underlying exocytosis. It appeared to be the case that an increase in adrenal cell cytosol concentration of Ca2+ ions from normally low micromolar levels was essential for secretion. I separated the secretory granules by centrifugation at fixed Ca2+ concentrations. There were no evident changes in the granule proteins themselves, but I was able to recover a number of cytosolic proteins which reversibly associated with the secretion granules (or secretion granule membrane "ghosts").

Isolated secretion granules

A researcher in the USA, Carl Creutz, had also identified an analogous granule binding protein which he named synexin. As we searched the literature it was evident that a number of labs had isolated proteins of very similar molecular weights (32KDa - 36KDa and 70KDa) which bound reversibly to membranes isolated from a range of tissue types; not just secretory organelles. I compared the adrenal proteins with those from other tissues by 2-dimensional gel electrophoresis and found identities or close similarities with analogous proteins from other tissues.

An "Eureka" moment came when we found that a number of our adrenal gland calcium-regulated membrane binding proteins strongly cross-reacted with antibodies to 'calelectrin': a calcium dependent membrane-binding protein isolated by researchers in Göttingen, Germany from Torpedo marmorata: a marine electric ray. The blue arrows in the 2D gel image indicate particularly strong cross reaction with anti-calelectrin.

Several of these proteins were later shown to be substrates for kinases and phosphatases. In particular, the isoelectric splitting of the spot A2 may be due to phosphorylated and dephospho - forms as A2 is known to be a substrate for several different kinases.

2d gel-electrophoresis of annexins. Blue arrows indicate proteins which cross react strongly with anti-calelectrin.

Now with other collaborators joining forces with me I determined peptide amino acid sequences of the adrenal proteins and other analogous proteins. I found extensive sequence homologies; in particular a 17 residue sequence that was highly conserved and repeated 4 times within the proteins examined, suggesting similar protein domains. We also confirmed that the proteins recognised membrane phospholipids: specifically those with acidic head groups like phosphatidylserine and polyphosphoinositides. Shortly after this BIOGEN in the USA published the cDNA sequences several of the proteins which confirmed the conserved domain structure. I suggested the generic name for this new family of calcium and phospholipid binding proteins: ANNEXINS which has been taken up by the scientific community. I have accordingly labelled the proteins on the 2-D gel image above and on the peptide sequences on the left as "A" followed by an internationally agreed identifying number. It was soon evident that the 70KDa Annexins have 8 rather than 4 similar polypeptide domains

Peptide sequences from different annexins
  1. Common domain structure of Ca2+ and lipid-binding proteins (Introduced the term “Annexin”) Michael J Geisow FEBS Lett. 203 99-103 (1986)
  2. New proteins involved in cell regulation by Ca2+ and phospholipids Geisow M J & Walker J H Trends in Biochemical Sciences 11 420-423 (1986)
Typical annexin protein chain fold. Calcium ions and membranes bind to the upper surface

The Annexin structure. With Willie Taylor, then at Birkbeck College, London, I predicted the conserved fold of the annexins in 1986. The x-ray crystal structure was published later by Robert Huber in Germany. In the image (left) the convex upper molecular surface interacts with phospholipid membranes. The extended chain loops (red) are the Ca2+ and phospholipid head group-binding sites. We had correctly predicted the annexin domain secondary structure and location of the calcium and phospholipid binding site but our fold (tertiary structure) was not correct as we had based this on the 'EF' hand: the only then known Ca2+- binding protein fold. Had we had access to Deep Mind's "Alphafold2" protein structure prediction, things would have been different! Since that time many annexin structures have been published in the Protein Data Bank. All have the typical domain structure shown but with very distinct amino termini which appear to impart different functions to the proteins. So for example Annexin A2 combines with plasma membrane and F-actin as can be seen dramatically by specifically immunostaining F-actin-rich brush border membranes in the image below left. Also included are immunostained bovine sperm cells clearly showing the different cytochemical locations of annexins A2, A4 and A6. These images have never previously been published.


Cytochemical Immunolocalisation of Annexins A2, A4 and A6. The sperm images have never previously been published by me

Immunostaining of Annexin A2 in rat jejunum. Note the co-localisation with F-actin in the brush border membrane
Immunostaining of Annexin A2 in bovine sperm. Note the staining of the annulus: a cytoskeletal structure built of septin proteins (marked in the phase contrast micrograph by arrowheads). Some staining of the apical plasma membrane can also be seen.

Immunostaining of Annexin A4 in sperm. The protein appears specifically associated with the acrosomal membrane
Immunostaining of Annexin A6 (70kDa) in sperm. The protein appears to be localised in the tail and the apical plasma membrane.

Annexins in the literature

Since their discovery the literature on annexins has grown extensively. Many questions have arisen about both their biology and biochemistry. One striking example is that all annexins lack signal sequences that would allow them to be secreted by the normal exocytotic route; yet they appear (and seem to have roles) in blood plasma. The mechanism of release appears to be real and not just due to damaged cell cytosol leaking. It has been termed unconventional or anomalous secretion and this is rare but not unique to the annexins.

Annexin genes have been identified by genomic sequencing in all eukaryotic organisms so far except yeast ( but are apparently absent from prokaryotes ). Further nomenclature has been necessary! Annexin categories: A for vertebrates; B for invertebrates; C for fungi and some other unicellular organisms; D for plants & E for protists. There are 12 human annexin genes ranging in size between 15kb to 96 kb. Their tissue distribution has been most intensively studied in mammals. Extensive studies using Annexin gene knock out or knock down have been carried out in mice. especially mice to try to address function. This work has been thoroughly reviewed and the findings related to normal or abnormal phenotypes for each annexin. See the reference: Annexin Animal Models—From Fundamental Principles to Translational Research below

All cells may well express every annexin at some stage of their development or life cycle, but preferential levels have been found in tissues as follows: A1 is widely expressed in somatic tissues and particularly prominent in monocytes: macrophages & neutrophils, the neuronal and endocrine system. A2, A3 & A4 are prominent in lung, pancreas, colon, ileum and adrenal gland. A5 appears universal in tissue types apart from neurones. A6 is widely expressed in cell types), except in ileal epithelial cells & parathyroid gland. (A6 has 8 of the characteristic Type II calcium binding domains) A7 has a uniquely large amino terminal region terminus and is present in alternatively spliced versions: 47 kD isoform present in all tissues apart from skeletal muscle, and 51 kD isoform in heart, brain and muscle. A8 is expressed in lung, liver, kidney, skin and placenta. A9 was detected first of all in fetal liver and spleen expression libraries. It is highly homologous with A2, but the type II-Ca2+ binding sites in its core domain are not able to bind calcium and its membrane and phospholipid-binding properties are calcium-independent. A10 is mainly found mainly in GI tract epithelia. A11 is widely expressed in tissues with cytoplasmic and nuclear localisation. During the cell cycle A11 appears to translocate to permit midbody formation and the completion of cytokinesis.

Such a wide distribution in eukaryotic cells and tissues suggests that annexins supply critical functions to both multicellular and unicellular organisms. However, despite their ubiquity, they have so far resisted clear functional categorisation unlike most other widespread protein families, although many specific functions have been proposed. Biochemically they appear to be acting as "adaptor or scaffold" molecules, reversibly bridging between plasma and organelle membranes and cytoskeletal proteins like filamentous actin.

There are reports of early and specific expression of annexins in the early embryo and annexins may be playing a key role in pattern formation during embryogenesis. The recognition of clear functions for specific proteins acting both extracellularly and in the cell cytosol is unusal, if not unprecedented. My best guess is that, first arising in a very ancient progenitor, evolution has since adapted them for many intracellular and even extracellular tasks. Regulated reversible membrane lipid binding appears to be an extremely useful property, with so many other membrane-associated proteins being permanently membrane embedded. Annexin A13, on genomic considerations, appears to be the closest representative to a very early ancestral molecule. Perhaps the appearance of an ancestral annexin occurred during the transition of nucleated cells to multicellularity or even, like present day slime moulds, promoting reversible aggregation of the isolated cells?

Intracellular interactions

Cytoskeleton
Interactions of annexins with cell cytoskeletal proteins have been reported. This is most clear from both in vitro and in vivo observations for Annexin A2 which binds F-actin. Annexin A1 apparently binds both F-actin and profilin. Annexins A5, 6 and 11 also may interact with cytoskeleton associated proteins. My own cytochemistry of A2 in sperm suggests an interaction with septins or septin associated proteins in the sperm annulus. These interactions with cell cytoskeleton indicates a role as membrane scaffolding. Annexin A5 clearly self-associates to form a 2-dimensional lattice through trimer formation ( an analogous property of proteins like clathrin and COPI and COPII proteins that function in intracellular membrane traffic )
Vesicle traffic
Annexins do seem to be involved in both triggered and constitutive membrane vesicle trafficking, participating in different ways in both phagocytosis, receptor mediated and basal endocytosis and vesicle export from the Golgi apparatus and late endosomes.
Cell signalling
Annexins interact with membrane lipid microdomains containing phosphoinositide head groups in both calcium ion dependent and independent manners. These lipids are the precursors to the cytoplasmic signalling molecules: inositol triphosphate (IP3) and diacylglycerol. Certain annexin sequences are myristolated suggesting an alternative way of associating with cell membranes. Certain annexins are themselves subject to regulation by phosphorylation: these annexins are substrates for tyrosine and serine kinases. The amino-terminus of A1 contains a sequence analogous with the SH2 recognition domain which plays a central role in the processing of external effectors.
Membrane repair
As already noted Annexin A5 participates in plasma membrane repair: a process which also requires phosphatidyl serine (or PIP2 membrane vesicles and calcium ions.
Membrane bridging, lipid & small molecule exchange across subcellular membranes
A1 and A6 control cholesterol transport from the sites of synthesis in the cell endoplasmic reticulum ER to endolysosomes (LE) which are one of the the multivesicular bodies (MVB) within the cell cytosol, via intramembrane contact sites (MCS). A1 mediates MCS formation between the ER and EGF-receptor containing MVB.

Extracellular interactions

Extracellular matrix
Annexin 5 has been shown to bind to type X and II collagens and other extracellular matrix (ECM) components and this may underlie some of the complex cell - ECM assembly and its regulation.
Inflammation
Annexin A1 can inhibit enzymes involved in the inflammatory process and combines with the formyl peptide receptor (FPR2). This can inhibit leucocyte migration and affect cell apoptosis by triggering neutrophils. Amino terminal A1 peptides can also substitute for the full length protein. Annexin A5 appears to inhibit macrophage phagocytosis of apoptotic cells
Coagulation and clearance of fibrin
As already mentioned, Annexin A5 self-associates bound to the membrane lipid phosphatidyl serine (PS) so masking this procoagulation trigger from clotting proteins. Annexin A1 can inhibit enzymes involved in the inflammatory process and combines with the formyl peptide receptor. This can inhibit leucocyte migration and affect cell apoptosis by triggering neutrophils. Annexin A5 appears to inhibit macrophage phagocytosis of apoptotic cells

Clinical indications (not exhaustive!)

  • Vascular tissue repair
  • Inflammation: can be both pro- and anti-inflamatory!
  • Autoimmune disorders
  • Recurrent placental loss in pregnancy
  • Apoptosis
  • Sarcoidosis
  • Diagnostics
  • Drug targeting
  • Coagulation and fibrinolysis
  • Metastasis & tumour progression

Pharmaceutical developments

Mention must be made of companies developing therapeutic interventions related to annexins. The anticoagulant properties of a protein fraction present in blood plasma, previously named lipocortin was identified back in the 70's and investigated as a potential therapeutic. This was an annexin, but which one(s) is retrospectively less clear. Today, the identities and sub and extracellular locations of annexins is much clearer! Exposure of the normally inner membrane resident phosphaphotidyl serine (PS) on the surface of endothelial cells is an indication of poor cell health (leaking or dying). This has suggested its use in various disorders and diseases. Currently the Swedish biotechnology company ANNEXIN PHARMA has recombinant Annexin 5 in clinical trials for Retinal Vascular Occlusion (RVO).

Blood clots form at sites of inflamed and/or damaged endothelial cells which provide the lining of all our blood vessels. In the eye, Central retinal vein occlusion (CRVO) is the blockage of the main retinal vein and branch retinal vein occlusion (BRVO) represents blockage of one of the smaller branch veins. (see the figure above). If presistent (especially in older people) the swelling due to vascular fluid leakage at the centre of the retina (macula) can cause permanent loss of central vision. Swelling of the retinal artery presses on the retinal veins that all exit in the same area.

Presently medicines such as anti- vascular endothelial growth factor (anti-VEGF) or steroids may be are used to combat swelling and leakage. These medicines ahave to be periodically injected into the eye which entails obvious anxiety discomfort and work absences. Systemic Annexin 5 administration is aimed to suppress these localised blood clots without intraocular injection.

Sites of retinal vein occlusion in the eye

As Annexin A5 rapidly combines with the membrane lipid PS and probably other acidic phospholipids normally not exposed on healthy endothelial cells, its therapeutic use is indicated on other conditions. For example it has been directly proposed as a drug candidate to reduce the damage observed in the vascular system and lungs of patients with severe COVID-19 disease.

Tumour and apoptotic cells (sick and dying) externalise intracellular phosphatidyl serine (PS). Tumour cells especially display PS through poor vascularisation and chemotherapies. PS has widely been used as a marker for this. However, unlike phagocytosis of pathogens, the process of apoptotic cell clearance is immunologically silent, avoiding the normal inflammatory response. But this process (efferocytosis) contributes to a localised immunosupressive environment which can be exploited by tumor cells to avoid immune attack. Cell surface molecules (usually proteins) which normally act to suppress immune attack are known as "checkpoints" Annexin A5 (AnxA5) binds membrane externalised PS with high affinity hence can act as a so called "immune checkpoint inhibitor" hence can increase the effectiveness of anti-tumour therapies.

PS also appears on aging erthyrocytes, which of course lack metabolism. Normally these senescent cells are phagocytosed by macrophages and cleared from the circulation. But in sickle cell disease (SCD) crisis events a large number of erythocytes are senescent, can adhere to endothelial cells and can overwhelm this clearance system. The high level of exposed PS acts to initiate clot formation. Administration of annexin 5 in binds to the exposed PS and administration may be indicated to calm the crisis and minimise coagulation

References

Note: Annexins have up to date received so many reviews that it would be overwhelming and probably counterproductive to list them. Instead I have picked out several that cover the actual or potential roles of annexins in biology. These refer back to many other key papers and reviews. As other key papers appear I will add them here.

  1. The Annexins (Review) Moss, S.E. & Morgan, R.O. (2004) Genome Biology 5 219-227
  2. Annexin-phospholipid interactions - Functional Implications. Lizarbe, M A, Barraza, J I, Olmo, N, Gavilanes, F and Turnay, J. (2013) Int J Mol Sci 14, 2652-2683
  3. Annexins: From Structure to Function. Gerke,V and Moss, S E (2002) Physiol Rev 10 331-371
  4. Annexins family: insights into their functions and potential role in pathogenesis of sarcoidosis. Mirsaeidi, M. Gidfar, S. Vu, A. & Schrsunagel, D. (2016) J Transl Med 14 89-98
  5. Annexins Bridging the Gap: Novel Roles in Mambrane Contact Site Formation Carlos Enrich, Albert Lu1, Francesc Tebar, Carles Rentero and Thomas Grewal in Frontiers in Cell & Developmental Biology (2022) 9 doi: 10.3389/fcell.2021.797949
  6. Annexin Animal Models—From Fundamental Principles to Translational Research Thomas Grewal, Carles Rentero, Carlos Enrich, Mohamed Wahba, Carsten A. Raabe and Ursula Rescher (2021) Int. J. Mol. Sci. 22, 3439

My own work at the MRC Labs

We ran some detailed studies of receptor-mediated protein phosphorylation in isolated chromaffin (adrenal medullary cells). although we did not persue this further at the time, protein phosphorylation and dephosphorylation mediated by receptor coupled protein kinase C, tyrosine kinases and phosphatases represent major steps in cell signalling pathways!

Key papers leading to the recognition and naming of the Annexin protein family are highlighted in red

  1. Specific binding of 125I-calmodulin to and protein phosphorylation in adrenal chromaffin granule membranes Burgoyne R.D. & Geisow M.J. FEBS Lett., 131, 127-131 (1981)
  2. Interaction of calmodulin with adrenal chromaffin granule membranes Geisow M.J., Burgoyne R.D. & Harris A. FEBS Lett., 143, 69-72 (1982)
  3. Phosphoproteins of the adrenal chromaffin granule membrane Burgoyne R.D. & Geisow M.J. J. Neurochem, 39, 1387-1396 (1982)
  4. Effect of Ca2+, calmodulin and trifluoperazine on protein phosphorylation in adrenal chromaffin granule membranes. Burgoyne R.D. & Geisow M.J. Biochem. Soc. Trans., 10, 267-268 (1982)
  5. Effect of monensin on chromaffin cells and the mechanism of organelle swelling Geisow M.J. & Burgoyne R.D. Cell. Biol. Int. Rep., 6, 933-939 (1982)
  6. Cation-dependent lysis of chromaffin granules - an alternative hypotheses for osmotically-driven exocytosis Geisow M.J. & Burgoyne R.D. Cell. Biol. Int. Rep., 6, 353-359 (1982)
  7. Calcium-dependent binding of cytosolic proteins by chromaffin granules from adrenal medulla Geisow, M J & Burgoyne, R D Journal of neurochemistry 38, 1735-1741 (1982)
  8. Cytochemical localisation of calcium binding sites in adrenal chromaffin cells and their relation to secretion R D, Barron, J & Geisow, M J Cell Tissue Research 229 207-217 (1983)
  9. Cholinergic stimulation of chromaffin cells induces rapid coating of the plasma membrane Geisow, M J, Childs, J & Burgoyne, R D European Journal of Cell Biology 38, 51-56 (1985)
  10. Cellular distribution of three mammalian Calcium binding proteins related to Torpedo calectrin Geisow M, Childs J, Dash. B, Harris A, Panayoutou G, Südhof T and Walker J H The EMBO journal 3 2969-2974 (1984)
  11. A consensus amino acid sequence repeat in Torpedo and mammalian Ca2+ – dependent membrane-binding proteins Geisow MJ, Fritsche, U Hexham J M, Dash, B and Johnson, T Nature 320 636-638 (1986)
  12. Common domain structure of Ca2+ and lipid-binding proteins (Introduced the term “Annexin”) Michael J Geisow FEBS Lett. 203 99-103 (1986)
  13. New proteins involved in cell regulation by Ca2+ and phospholipids Geisow M J & Walker J H Trends in Biochemical Sciences 11 420-423 (1986)
  14. Predicted structure for the calcium dependent membrane-binding (annexins) Taylor, W R and Geisow M J Protein Engineering 1 183-187 (1987)
  15. An integrated approach to secretion: Phosphorylation and Ca2+-dependent binding of proteins associated with chromaffin granules Geisow M.J. & Burgoyne R.D. Ann. New York Academy of Sciences, 493, 563-576 (1987)
  16. Isolation and characterisation of two novel (annexins) from bovine lung Boustead, C M, Walker, J H and Geisow M J FEBS Lett. 233 233-238 (1988)
  17. Annexins – A new family of Ca2+- regulated phospholipid binding proteins Geisow M J, Walker, J H, Boustead, C and Taylor, W in Molecular Mechanisms of secretion Alfred Benzon Symposium 25 596-608 (1988)
  18. The annexin family of calcium-binding proteins (review ) Burgoyne R D & Geisow M J Cell Calcium 10 1-10 (1989).