Biol. Chem. 2017; 398(7): 751–763 Maria-Pilar Fernandez, Montserrat Garciaa, Silvia Martin-Almedinab and Reginald O. Morgan* Novel domain architectures and functional determinants in atypical annexins revealed by phylogenomic analysis DOI 10.1515/hsz-2016-0273 Keywords: 3D modeling; conserved motifs; functional Received August 27, 2016; accepted December 11, 2016; previously determinants; membrane association; molecular phylo- published online December 20, 2016 geny; profile hidden Markov models. Abstract: The fundamental cellular role and molecular interactions of annexins in vesicle trafficking and mem- brane remodeling remain to be further clarified in order to better understand and exploit their contributions to Introduction health and disease. We focused on distinctive features Those who moil for annexins have made significant of atypical annexins from all domains of life using phy- advances during four decades of multidisciplinary logenomic, molecular systematic and experimental research in documenting the molecular properties and approaches, to extend the current paradigm and better cellular roles of this ubiquitous and unique gene super- account for annexin diversity of structure, function and family (Creutz et al., 1978; Gerke et al., 2005). Observa- mechanistic role in membrane homeostasis. The analy- tions and empirical testing of their association with cell sis of gene duplications, organization of domain archi- membrane lipids and cytoskeletal proteins are compatible tectures and profile hidden Markov models of subfamily with their proposed participation in intracellular trans- orthologs defined conserved structural features relevant port processes leading to vesicle translocation, fusion to molecular interactions and functional divergence of and transcytosis (Potez et al., 2011; Tebar et al., 2014). seven family clades ANXA-G. Single domain annexins of These basic processes can be conceptually linked to an bacteria, including cyanobacteria, were frequently cou- integrated physiological role in membrane remodeling pled to enzymatic units conceivably related to membrane and maintenance to promote cellular membrane growth, metabolism and remodeling. Multiple ANX domains repair and to facilitate transmembrane microvesicle trans- (up to 20) and various distinct functional domains were port (Bouter et al., 2015; Draeger et al., 2011; Demonbreun observed in unique annexins. Canonical type 2 calcium and McNally, 2016). The prominent expression and apical binding ligands were well-preserved in roughly half of all trafficking of annexins in epithelial and endothelial ANX domains, but alternative structural motifs comprised tissues support this interpretation of a protective homeo- of ‘KGD’, cysteine or tryptophan residues were promi- static role, but they have additional, distinct actions in nently conserved in the same strategic interhelical loops. immune responses (D’Acquisto et al., 2008), secretory, Selective evolutionary constraint, site-specific location apoptotic and chemoresistance events, antithrombotic, and co-occurrence in all kingdoms identify alternative anti-inflammatory and tumor suppressor roles, and in modes of fundamental binding interactions for annexins. muscle contraction and neuronal activity. Such functional diversity argues for even broader roles as scaffolding com- ponents of multifunctional complexes involved in cellular a Current address: Foundation for Ophtalmologic Investigation, Avda. signal transduction networks of multicellular organisms Doctores Fernández-Vega 34, E-33012 Oviedo, Spain and other coordinated molecular interactions at the level bCurrent address: Cardiovascular and Cell Sciences Institute, St. George’s University of London, London SW17 0RE, UK of unicellular organisms. *Corresponding author: Reginald O. Morgan, Department of Annexin subfamily specificity in calcium affinity is Biochemistry and Molecular Biology, Faculty of Medicine, University influenced by individual structural variation under differ- of Oviedo, Edificio Santiago Gascon 4.3, E-33011 Oviedo, Spain, ent proteoglycolipid environments and intracellular pH e-mail: [email protected] that modulate membrane binding kinetics (Gerke et al., Maria-Pilar Fernandez, Montserrat Garcia and Silvia Martin-Almedina: Department of Biochemistry and Molecular Biology, Faculty of 2005; Potez et al., 2011). This basic paradigm defines Medicine, University of Oviedo, Edificio Santiago Gascon 4.3, annexins as calcium-mediated, membrane lipid-associ- E-33011 Oviedo, Spain ated proteins, but does not consider calcium-independent Bereitgestellt von | De Gruyter / TCS Angemeldet Heruntergeladen am | 09.11.17 18:15 752 M.-P. Fernandez et al.: Annexin phylogenomics annexins, other conserved structural motifs, distinct Results functional domains, unique amino-termini and varied architectures to adequately postulate integrated roles Annexin molecular phylogenetics and mechanisms for individual annexin function. A unifying concept for annexins must recognize their The 12 annexins common to vertebrates (ANXA1-ANXA13, commonly shared characteristics as well as the unique excluding unassigned ANXA12) extend from the earliest features that define individual members. The advent of diverging extant species, the jawless fish lamprey and high throughput, low-cost sequencing has already sup- hagfish, whilst annexins A5, A6 and A10 appeared later plied ample genomic, transcriptomic and proteomic data in cartilaginous fish and ANXA8 in coelacanth (Figure 1). to permit the characterization and comparative analysis The annexin A2-A1-A9 clade also separated early from a of some 4000 individual annexins. These data provide lamprey common ancestor and ANXA9 first appeared in new evidence for ‘atypical’ annexins from diverse phyla cartilaginous fish. Earlier metazoan origins of annexins in which the fundamental relationships between struc- A13 and A7 were apparent from their close association ture and function are expected to be preserved despite with known nonvertebrate outgroups and from other mechanistic variance. similarities in genetic linkage and gene exon organization Phylogenetic reconstruction of the annexin gene (Fernandez and Morgan, 2003). Among the 10 remaining superfamily from bacterial, protist, plant, fungal and vertebrate annexins, ANXA11 can be regarded as the most metazoan sequences (Morgan and Fernandez, 1995, 1997; basal member from Agnatha with a gene organization Weiland et al., 2005) can chart the complete gene family congruous among all annexins except A7 and A13 (Moss history and effectively distinguish paralogs and orthologs and Morgan, 2004). Copy number variation is important for a comprehensive classification of individual gene for assessing associated phenotypes, so the documenta- subfamilies (Moss and Morgan, 2004; Clark et al., 2012). tion of extra paralogous gene duplications is noteworthy It can also provide informative evidence for positive or (Figure 1). ANXA1 has two to five copies in certain fish, negative selection and the dates, rates and patterns of birds and amphibians, ANXA7 and ANXA13 duplicated in divergence or conservation. This is essential for compara- lamprey, like most other annexins in teleost fish, and a tive studies because species orthologs vary by essentially second ANXA8 gene has emerged only in humans, con- silent changes in primary structure without significant sistent with a genome propensity for segmental chromo- functional differences, whereas the multiple paralogous somal duplications. genes within single species diverge at semi-conserved The fact that major taxonomic groups ‘approximate’ sites responsible for subfamily functional specificity. The monophyletic distributions of species groups must have evolutionary distance between paralogs duplicated from been the result of evolutionary selection by which annex- a common ancestor depends not only on the timing and ins have adapted and diverged within at least six major rate of divergence but also on functional adaptation perti- families; e.g. the ANXA family consisting of 12 gene sub- nent to each species group, so it can be insightful to follow families of more than 2000 known vertebrate annexins. these features throughout an extensive period of evolu- Distinct clades form other families, including ANXB from tionary change that the annexin superfamily has under- nonvertebrate metazoa, ANXC with fungal-related annex- gone. Molecular fingerprints of each subfamily can be ins, ANXD comprising 40 + subfamilies in plants (Clark generated from multiple sequence alignments as profile et al., 2012), ANXE in diverse protists and ANXF now hidden Markov models (pHMM) to predict amino acid pro- represented by more than 200 bacterial annexins (this files with statistical precision and to decipher conserved study). The loss of annexin genes from certain genomes sequence patterns that can be used to infer functional was deduced from more extensive analyses to document importance. This evolutionary, structural and physico- the absence of ANXA3 from Archosauria (crocodiles, chemical property information can be represented in 3D dinosaurs and birds) and Squamata (snakes), ANXA8 models to substantiate those inferences and to define the from Afrotherian mammals, Amphibia, most fish and spatial context of key functional domains, motifs and Lepidosauria (salamanders and snakes), ANXA9 from amino acids responsible for mediating physiological and Archosauria, ANXA7 and ANXA10 from Teleostei (bony pharmacological interactions of individual annexins. The