Fungal annexins: a mini review
© Khalaj et al. 2015
Received: 3 June 2015
Accepted: 9 November 2015
Published: 24 November 2015
The large family of annexins is composed of more than a thousand members which are typically phospholipid-binding proteins. Annexins act in a number of signalling networks and membrane trafficking events which are fundamental to cell physiology. Annexins exert their functions mainly through their calcium-dependent membrane binding abilities; however, some calcium-independent interactions have been documented in the literature. Although mammalian and plant annexins have been well characterized, little is known about this family in fungi. This mini review summarizes the available data on fungal annexins.
The basic structure of annexins is composed of both a conserved core domain and a variable N-terminal region. The core is made up of ~70 amino acid repeats which contain the consensus endonexin sequence GXGT….D/E, and normally participates in the formation of type II Ca2+ binding sites (Geisow et al. 1986). Sequences specifying ‘type III’ Ca2+ binding sites as well as a phosphatidylserine-binding sequence have been identified (Weng et al. 1993; Montaville et al. 2002). The variable N-terminal region of annexins is supposed to be involved in regulatory functions, including protein–protein interactions and membrane localization (Hayes et al. 2004).
Annexins have been identified in a wide range of fungi including ascomycetes, basidiomycetes and oomycetes (which are taxonomically distinct from true fungi) (Moss and Morgan 2004; Khalaj et al. 2004b). Despite a large number of studies on mammalian and plant annexins, little is known about fungal annexins. In this review, we sought to focus on the available data regarding the fungal annexins.
The first fungal annexin was identified in ascomycete Neurospora crassa using a bioinformatic approach (Braun et al. 1998). The identified protein displayed ~35 % identity to its orthologue in slime mold Dictyostelium discoideum. In this line, the authors named this gene as anx14 in accordance with D. discoideum. Khalaj et al. (2004a) isolated and characterized an annexin homologue from Aspergillus niger using a degenerative PCR approach. More members of the fungal annexin family were then identified through EST database searches and some of them characterized experimentally (Khalaj et al. 2004b). Although there is no evidence for the presence of these proteins in yeasts Saccharomyces cerevisiae, Candida albicans or Schizosaccharomyces pombe, the annotation of the Yarrowia lipolytica genome has confirmed the presence of an annexin member in this yeast (Khalaj et al. 2004b; Braun et al. 2000; Morgan et al. 2004). In addition, annexins have been found in oomycetes. The first oomycete annexin was isolated from Saprolegnia monoica using biochemical methods, followed by DNA sequence analysis (Bouzenzana et al. 2006). A recent examination of detergent-resistant membrane microdomains from S. monoica has reconfirmed the presence of different isoforms of a 35-kDa annexin in this oomycete (Briolay et al. 2009). Furthermore, the proteome surveys of Phytophthora ramorum and Phytophthora infestans cell walls have identified two annexin homologues in these fungal-like organisms (Meijer et al. 2006; Grenville-Briggs et al. 2010).
In our previous works, we identified three fungal annexins in Aspergillus fumigatus. Two of these proteins were called as ANXC3.1 and ANXC3.2 with 40 % identity in the protein level, while the third member, ANXC4, was identified as a novel annexin with significant differences, when compared to other fungal and human annexins (Khalaj et al. 2004a, b). These newly identified proteins were given the name based on next available number in annexin C group (ANXC1 and ANXC2 previously referred to D. disodium). The presented phylogeny tree in this review, generally separates ANC3.1, ANX C3.2 and ANXC4 and we make corrections on the previous classification. In this sense ANXC3.1 renamed as ANXC1; ANC3.2 as ANXC2 and ANXC4 as ANXC3. A phylogenetic analysis of the annexin sequence isolated from oomycete S. monoica and available EST from other genus of this class, Phytophthora, have shown a well grouped oomycete clade with a high bootstrap support (Bouzenzana et al. 2006). The same situation can be observed for oomycete annexins in the presented tree.
Expression, localization and function
In the higher eukaryotes, depending on the annexin type and the expressing tissue, the gene expression can follow either a constitutive or a highly inducible pattern. In the cellular level, annexins are generally localized in cytosol but partly associated with membranes or cytoskeleton components (Solito et al. 1998; Pratt and Horseman 1998).
Of known members of the ANXC family, two Dictyostelium annexins have been studied in detail (Doring et al. 1991; Greenwood and Tsang 1991; Marko et al. 2006). Based on what Marko et al. (2006) have nicely presented, both Dictyostelium annexins are expressed at a low but constant level during the development. In localization studies, the ANXC1A-GFP fusion protein was present along the cell membrane, in the cytosol, the nucleus and the endosomal compartment, while ANXC1B-GFP fusion labelled the plasma membrane and partially co-localized with Golgi apparatus markers.
A detailed analysis of anxc1 in filamentous fungus, A. niger, confirmed the constitutive expression of this gene (Khalaj et al. 2004a). An ANXC1-GFP fusion construct, driven by an inducible promoter, has been made in our lab and successfully expressed in A. niger (unpublished data). Upon the induction of this expression cassette, a cytosolic distribution of the fusion protein has been observed.
In biochemical studies of Saprolegnia cell wall components, an annexin homologue has been co-purified with a cell wall enzyme, (1→3)-β-d-glucan synthase. This may demonstrate a membrane localization for this protein (Bouzenzana et al. 2006; Briolay et al. 2009).
The proposed functions of annexins are based mainly on Ca2+/phospholipid binding properties in co-operation with various partners (Rescher and Gerke 2004). The most known functions of the annexin family include participation in membrane organization and trafficking events such as endocytosis and exocytosis, ion channel modulation, as well as some extracellular activities like receptor-ligand interactions (Monastyrskaya et al. 2009a, b; Gerke and Moss 2002; Kourie and Wood 2000). However, a number of Ca2+ independent activities have also been assigned (Mortimer et al. 2008; Gerke and Moss 2002).
The examination of amino acid sequences of ANXC1 and ANXC2 in Aspergillus fumigatus have revealed a well-conserved core region compared to other annexins. The calcium-binding repeat regions I and II were found to be similar to those detected in human annexin VII and ANXC1A in D. discoideum. These repeats also contain the consensus phosphatidylserine binding sites. However, for the fourth repeat, there is a very low amino acid identity particularly in the calcium coordinating residues.
The ANXC3 core region has also been examined, showing a little or no conserved endonexin fold, type II Ca2+-binding site or phosphatidylserine-binding sequences. Based on the above analysis, it has been suggested that ANXC3 may not interact with calcium or membranes in a conventional route (Khalaj et al. 2004a, b).
Loss-of-function experiments using gene deletion or RNAi methodology in mammalians resulted in various phenotypes ranging from normal to embryonic lethal (Brachvogel et al. 2003; Clemen et al. 2003). The results are difficult to interpret, partly due to compensatory function of other annexins as well as difference in genetic background of experimental models (Hannon et al. 2003).
Indeed, the first annexin knock out generated for anxc1 in D. discoideum failed to lead to any change in essential cell functions. However, the introduction of low external Ca2+ resulted in defects in growth, motility, and chemotaxis of knock out strain, demonstrating a role for annexin C1 in Ca2+ homeostasis (Doring et al. 1991, 1995).
There is abundant documentation on the role of calcium in fungal tip growth (Jackson and Heath 1993; Garrill et al. 1993; Heath and Geitmann 2000). A variety of imaging and measuring methods have repeatedly shown that the growing mycelia have a high concentration of calcium at their tips (Kim et al. 2012). Likewise, several reports have demonstrated the role of calcium in polar growth of pollen tubes, root hairs and fern rhizoids. (Steer 1989; Hepler 2005; Clark et al. 2005; Bushart and Roux 2007). These observations along with the localization of annexins at the tip of latter cells can emphasize the role of these calcium binding proteins in growth physiology of plants (Konopka-Postupolska et al. 2011).
To investigate the possible role of fungal annexins in tip growth and protein secretion, an anxc1 knock out strain of A. niger has been generated (Khalaj et al. 2004a). Normal growth phenotype and intact secretion capacity of the KO strain, even in low Ca2+ concentrations, suggest a non-essential function of this gene in growth and protein secretion. Disruption of other member of fungal annexins, anxc3, in human pathogen A. fumigatus resulted in no obvious phenotype related to the growth or protein secretion. A comparative protein profiling of δanxc3 mutant revealed the modification of respiratory chain proteins and stress response proteins in this mutant. In this sense, a possible anti-stress function has been proposed for ANXC3 in A. fumigatus (Khalaj et al. 2011). In this line, several studies have highlighted the protective role of annexins against various stresses such as oxidative stress, heavy metal stress and osmotic stress in plants (Jami et al. 2008; Konopka-Postupolska et al. 2009; Laohavisit et al. 2010).
The study of annexin-interacting proteins has been an approach in elucidation of the annexin biological functions. As an example, co-purification of a 34-kD annexin protein with (1→3)-β-d-glucan synthase in Saprolegnia suggests its role in modulation of this cell wall synthesis enzyme. Add-back experiments using purified annexin have confirmed a positive regulatory effect on glucan synthase activity which needs an optimal ratio of enzyme/annexin (Bouzenzana et al. 2006). Similarly, the presence of two cell-wall associated annexins in the plant pathogen P. infestans may indicate the importance of these proteins in mycelia growth and penetration of this pathogen into the host tissue (Grenville-Briggs et al. 2010).
Shirakawa et al. (2005) have identified two aminoacyl-tRNA synthases as annexin binding proteins in true slime mold, Physarum polycephalum. They have suggested that there is a possible relationship between membrane trafficking and protein machinery. More studies are needed to confirm such activities in fungi.
Several potential functions have been assigned to the fungal annexins. The presence of this family in fungi may suggest a role in providing the particular needs of these organisms. The advantage of low numbers of fungal annexins (e.g., 3 in ascomycetes), together with fewer cell types, provides an excellent opportunity for elucidating the biological functions of these proteins. In this case, a comprehensive multi-gene deletion study along with omics-based investigations can give us a deeper insight into the possible role of annexins in fungal development, the adaptation of fungi to different environments and also, pathogenesis. Furthermore, the identification of annexin binding partners and characterization of their possible functional complexes may be helpful in understanding the relevant biological processes.
KK reviewed the published data and prepared the initial draft. EA collaborated in manuscript preparation. AB collaborated in phylogeny analysis. VK designed the structure of the review, supervised the manuscript preparation and revised and finalized the manuscript. All authors read and approved the final manuscript.
We would like to express our gratitude to Professor Reginald O Morgan at University of Oviedo for his excellent comments on phylogeny analysis.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
- Bouzenzana J, Pelosi L, Briolay A, Briolay J, Bulone V (2006) Identification of the first Oomycete annexin as a (1→3)-beta-d-glucan synthase activator. Mol Microbiol 62(2):552–565View ArticleGoogle Scholar
- Brachvogel B, Dikschas J, Moch H, Welzel H, von der Mark K, Hofmann C, Poschl E (2003) Annexin A5 is not essential for skeletal development. Mol Cell Biol 23(8):2907–2913View ArticleGoogle Scholar
- Braun EL, Kang S, Nelson MA, Natvig DO (1998) Identification of the first fungal annexin: analysis of annexin gene duplications and implications for eukaryotic evolution. J Mol Evol 47(5):531–543View ArticleGoogle Scholar
- Braun EL, Halpern AL, Nelson MA, Natvig DO (2000) Large-scale comparison of fungal sequence information: mechanisms of innovation in Neurospora crassa and gene loss in Saccharomyces cerevisiae. Genome Res 10(4):416–430View ArticleGoogle Scholar
- Briolay A, Bouzenzana J, Guichardant M, Deshayes C, Sindt N, Bessueille L, Bulone V (2009) Cell wall polysaccharide synthases are located in detergent-resistant membrane microdomains in oomycetes. Appl Environ Microbiol 75(7):1938–1949View ArticleGoogle Scholar
- Bushart TJ, Roux SJ (2007) Conserved features of germination and polarized cell growth: a few insights from a pollen-fern spore comparison. Ann Bot 99(1):9–17View ArticleGoogle Scholar
- Cantacessi C, Seddon JM, Miller TL, Leow CY, Thomas L, Mason L, Willis C, Walker G, Loukas A, Gasser RB, Jones MK, Hofmann A (2013) A genome-wide analysis of annexins from parasitic organisms and their vectors. Sci Rep 3:2893View ArticleGoogle Scholar
- Clark G, Cantero-Garcia A, Butterfield T, Dauwalder M, Roux SJ (2005) Secretion as a key component of gravitropic growth: implications for annexin involvement in differential growth. Gravit Space Biol Bull 18(2):113–114Google Scholar
- Clemen CS, Herr C, Hovelmeyer N, Noegel AA (2003) The lack of annexin A7 affects functions of primary astrocytes. Exp Cell Res 291(2):406–414View ArticleGoogle Scholar
- Doring V, Schleicher M, Noegel AA (1991) Dictyostelium annexin VII (synexin). cDNA sequence and isolation of a gene disruption mutant. J Biol Chem 266(26):17509–17515Google Scholar
- Doring V, Veretout F, Albrecht R, Muhlbauer B, Schlatterer C, Schleicher M, Noegel AA (1995) The in vivo role of annexin VII (synexin): characterization of an annexin VII-deficient Dictyostelium mutant indicates an involvement in Ca(2 +)-regulated processes. J Cell Sci 108(Pt 5):2065–2076Google Scholar
- Garrill A, Jackson SL, Lew RR, Heath IB (1993) Ion channel activity and tip growth: tip-localized stretch-activated channels generate an essential Ca2+ gradient in the oomycete Saprolegnia ferax. Eur J Cell Biol 60(2):358–365Google Scholar
- Geisow MJ, Fritsche U, Hexham JM, Dash B, Johnson T (1986) A consensus amino-acid sequence repeat in Torpedo and mammalian Ca2+-dependent membrane-binding proteins. Nature 320(6063):636–638View ArticleGoogle Scholar
- Gerke V, Moss SE (2002) Annexins: from structure to function. Physiol Rev 82(2):331–371View ArticleGoogle Scholar
- Greenwood M, Tsang A (1991) Sequence and expression of annexin VII of Dictyostelium discoideum. Biochim Biophys Acta 1088(3):429–432View ArticleGoogle Scholar
- Grenville-Briggs LJ, Avrova AO, Hay RJ, Bruce CR, Whisson SC, van West P (2010) Identification of appressorial and mycelial cell wall proteins and a survey of the membrane proteome of Phytophthora infestans. Fungal Biol 114(9):702–723View ArticleGoogle Scholar
- Hannon R, Croxtall JD, Getting SJ, Roviezzo F, Yona S, Paul-Clark MJ, Gavins FN, Perretti M, Morris JF, Buckingham JC, Flower RJ (2003) Aberrant inflammation and resistance to glucocorticoids in annexin 1−/− mouse. FASEB J 17(2):253–255Google Scholar
- Hayes MJ, Rescher U, Gerke V, Moss SE (2004) Annexin–actin interactions. Traffic 5(8):571–576View ArticleGoogle Scholar
- Heath IB, Geitmann A (2000) Cell biology of plant and fungal tip growth—getting to the point. Plant Cell 12(9):1513–1517Google Scholar
- Hepler PK (2005) Calcium: a central regulator of plant growth and development. Plant Cell 17(8):2142–2155View ArticleGoogle Scholar
- Jackson SL, Heath IB (1993) Roles of calcium ions in hyphal tip growth. Microbiol Rev 57(2):367–382Google Scholar
- Jami SK, Clark GB, Turlapati SA, Handley C, Roux SJ, Kirti PB (2008) Ectopic expression of an annexin from Brassica juncea confers tolerance to abiotic and biotic stress treatments in transgenic tobacco. Plant Physiol Biochem 46(12):1019–1030View ArticleGoogle Scholar
- Khalaj V, Hey P, Smith L, Robson GD, Brookman J (2004a) The Aspergillus niger annexin, anxC3.1 is constitutively expressed and is not essential for protein secretion. FEMS Microbiol Lett 239(1):163–169View ArticleGoogle Scholar
- Khalaj V, Smith L, Brookman J, Tuckwell D (2004b) Identification of a novel class of annexin genes. FEBS Lett 562(1–3):79–86View ArticleGoogle Scholar
- Khalaj V, Azarian B, Enayati S, Vaziri B (2011) Annexin C4 in A. fumigatus: a proteomics approach to understand the function. J Proteomics 74(10):1950–1958View ArticleGoogle Scholar
- Kim HS, Czymmek KJ, Patel A, Modla S, Nohe A, Duncan R, Gilroy S, Kang S (2012) Expression of the Cameleon calcium biosensor in fungi reveals distinct Ca(2+) signatures associated with polarized growth, development, and pathogenesis. Fungal Genet Biol 49(8):589–601View ArticleGoogle Scholar
- Kodavali PK, Dudkiewicz M, Pikula S, Pawlowski K (2014) Bioinformatics analysis of bacterial annexins—putative ancestral relatives of eukaryotic annexins. PLoS One 9(1):e85428View ArticleGoogle Scholar
- Konopka-Postupolska D, Clark G, Goch G, Debski J, Floras K, Cantero A, Fijolek B, Roux S, Hennig J (2009) The role of annexin 1 in drought stress in Arabidopsis. Plant Physiol 150(3):1394–1410View ArticleGoogle Scholar
- Konopka-Postupolska D, Clark G, Hofmann A (2011) Structure, function and membrane interactions of plant annexins: an update. Plant Sci 181(3):230–241View ArticleGoogle Scholar
- Kourie JI, Wood HB (2000) Biophysical and molecular properties of annexin-formed channels. Prog Biophys Mol Biol 73(2–4):91–134View ArticleGoogle Scholar
- Laohavisit A, Brown AT, Cicuta P, Davies JM (2010) Annexins: components of the calcium and reactive oxygen signaling network. Plant Physiol 152(4):1824–1829View ArticleGoogle Scholar
- Marko M, Prabhu Y, Muller R, Blau-Wasser R, Schleicher M, Noegel AA (2006) The annexins of Dictyostelium. Eur J Cell Biol 85(9–10):1011–1022View ArticleGoogle Scholar
- Meijer HJ, van de Vondervoort PJ, Yin QY, de Koster CG, Klis FM, Govers F, de Groot PW (2006) Identification of cell wall-associated proteins from Phytophthora ramorum. Mol Plant Microbe Interact 19(12):1348–1358View ArticleGoogle Scholar
- Monastyrskaya K, Babiychuk EB, Draeger A (2009a) The annexins: spatial and temporal coordination of signaling events during cellular stress. Cell Mol Life Sci 66(16):2623–2642View ArticleGoogle Scholar
- Monastyrskaya K, Babiychuk EB, Hostettler A, Wood P, Grewal T, Draeger A (2009b) Plasma membrane-associated annexin A6 reduces Ca2+ entry by stabilizing the cortical actin cytoskeleton. J Biol Chem 284(25):17227–17242View ArticleGoogle Scholar
- Montaville P, Neumann JM, Russo-Marie F, Ochsenbein F, Sanson A (2002) A new consensus sequence for phosphatidylserine recognition by annexins. J Biol Chem 277(27):24684–24693View ArticleGoogle Scholar
- Morgan RO, Martin-Almedina S, Iglesias JM, Gonzalez-Florez MI, Fernandez MP (2004) Evolutionary perspective on annexin calcium-binding domains. Biochim Biophys Acta 1742(1–3):133–140View ArticleGoogle Scholar
- Morgan RO, Martin-Almedina S, Garcia M, Jhoncon-Kooyip J, Fernandez MP (2006) Deciphering function and mechanism of calcium-binding proteins from their evolutionary imprints. Biochim Biophys Acta 1763(11):1238–1249View ArticleGoogle Scholar
- Mortimer JC, Laohavisit A, Macpherson N, Webb A, Brownlee C, Battey NH, Davies JM (2008) Annexins: multifunctional components of growth and adaptation. J Exp Bot 59(3):533–544View ArticleGoogle Scholar
- Moss SE, Morgan RO (2004) The annexins. Genome Biol 5(4):219View ArticleGoogle Scholar
- Pratt SL, Horseman ND (1998) Identification of two Y-box binding proteins that interact with the promoters of columbid annexin I genes. Gene 214(1–2):147–156View ArticleGoogle Scholar
- Rescher U, Gerke V (2004) Annexins––unique membrane binding proteins with diverse functions. J Cell Sci 117(Pt 13):2631–2639View ArticleGoogle Scholar
- Shirakawa T, Nakamura A, Kohama K, Hirakata M, Ogihara S (2005) Class-specific binding of two aminoacyl-tRNA synthetases to annexin, a Ca2+- and phospholipid-binding protein. Cell Struct Funct 29(5–6):159–164View ArticleGoogle Scholar
- Solito E, de Coupade C, Parente L, Flower RJ, Russo-Marie F (1998) IL-6 stimulates annexin 1 expression and translocation and suggests a new biological role as class II acute phase protein. Cytokine 10(7):514–521View ArticleGoogle Scholar
- Steer M (1989) Calcium control of pollen tube tip growth. Biol bull 176(2S):18–20View ArticleGoogle Scholar
- Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731–2739View ArticleGoogle Scholar
- Weng X, Luecke H, Song IS, Kang DS, Kim SH, Huber R (1993) Crystal structure of human annexin I at 2.5 A resolution. Protein Sci 2(3):448–458View ArticleGoogle Scholar