Human annexin 31 genetic mapping and origin

doi:10.1016/S0378-1119(98)00597-6

Gene

Volume 227, Issue 1, 4 February 1999, Pages 33-38

https://doi.org/10.1016/S0378-1119(98)00597-6 Get rights and content

Abstract

The cDNA encoding novel human annexin 31 was utilized for chromosomal mapping, structural comparison, and phylogenetic analysis to clarify its genetic relationship to other annexins. The ANX31 gene locus was mapped by fluorescence in situ hybridization to human chromosome 1q21, remote from ten other paralogous human annexins on different chromosomes but near the epidermal differentiation gene complex, the S100A gene cluster and a breast-cancer translocation region. Protein homology testing and characterization of incompletely processed expressed sequence tags identified annexin 2 as the closest extant homologue. Maximum likelihood analysis confirmed its most recent common ancestor with vertebrate annexin 2 and validated its classification, in order of discovery, as annexin 31. This subfamily was formed approx. 500–600 million years ago, subsequent to the gene duplication that produced annexin 1. It has diverged rapidly and extensively, especially in the well-conserved and functionally critical type II calcium-binding sites.

Introduction

Phylogenetic and genetic studies have confirmed that the annexin tetrad is a unique, ancient and highly conserved structure (Morgan and Fernandez, 1995, Morgan and Fernandez, 1997a) with some membrane-related role involving calcium channels, cellular signaling, vesicular transport or extracellular matrix (Raynal and Pollard, 1994). Vertebrate annexins are known to have emanated from a common eukaryotic ancestor since the emergence of metazoa around 800 million years ago (Mya) and their independent evolution at dispersed genomic locations implies differentiated, non-redundant functions (Morgan and Fernandez, 1997b; Morgan et al., 1998). The structural and evolutionary interrelationships of annexin genes provide vital information for establishing prospective links to functionally related genes, shared phenotypes and, ultimately, to specific hereditary diseases. A complete knowledge of all extant annexins and their systematic analysis are essential for precisely identifying conserved regions that have a common, critical function and variable regions that distinguish the individual members by their regulation, properties and subcellular roles.

Ten human annexins have been discovered on the basis of diverse functional assays and sequenced between the years 1986 and 1992 (Raynal and Pollard, 1994). The recent identification of an eleventh human annexin featuring lost calcium-binding sites, an RGD-like cell attachment motif and a distinctive expression pattern has raised key questions about the role of membrane interactions in annexin function and the actual diversity of this multigene family (Morgan and Fernandez, 1998). An examination of this gene's chromosomal location, structural organization and phylogenetic place in relation to other annexins was therefore undertaken here to further characterize its unique identity and to resolve its true genetic origin. Such analyses could reveal some evolutionary pattern in annexin divergence and link this gene to novel, specialized functions or hereditary traits.

Section snippets

Chromosomal mapping by fluorescence in situ hybridization

The A31H hybridization probe for chromosomal mapping was the 1.3 kb insert of expressed sequence tag (EST) clone ID 111373 from the I.M.A.G.E. (Integrated Molecular Analysis of Genome Expression) Consortium (LLNL) (Lennon et al., 1996). It was sequence verified to represent most of the coding and 3′ untranslated region of a novel human annexin cDNA (Morgan and Fernandez, 1998; gb: AJ009985). Metaphase spreads were prepared from phytohemaglutinin-stimulated human lymphocytes of a healthy female

Genetic mapping of ANX31

The A31H cDNA probe hybridized to human metaphase spreads with specific labeling on chromosome 1 (Fig. 1A). Fluorescence signals were detected on chromosome 1 in each of 23 metaphase spreads scored. Among 122 signals observed, 54 (44%) were on 1q with the following distribution: one chromatid (five cells), two chromatids (eight cells), three chromatids (seven cells), four chromatids (three cells). Among the 92 chromosome 1 chromatids scored, 54 (59%) hybridized to 1q. All chromosome-specific

Conclusions

1.
Human annexin 31 resides on chromosome band 1q21, remote from ten other annexins but in proximate linkage to S100A and skin differentiation genes with which it may have some regulatory or functional relationship.
2.
Its coding region and protein structure most closely resemble annexin 2, but its unique lack of calcium-binding sites and lower expression level contrast markedly with the ubiquitous annexin 2 and imply their distinct subcellular location and significant functional divergence.
3.

Acknowledgements

This work was supported by grant PM95-0152 from D.G.E.S. of Spain, National Institutes of Health Grant CA-06927 (USA), and by an appropriation from the Commonwealth of Pennsylvania.

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Naked carp (Gymnocypris przewalskii) is a migratory fish endemic to the Qinghai Lake, the largest inland saline-alkaline lake in China. Based on our previous results of comparative transcriptome sequencing, we found that pathways related to regulation of actin cytoskeleton, plasma membrane damage, and inflammation-related signaling were significantly enriched during saline-alkaline stress, suggesting involvement of annexin (ANN) family proteins in stress adaption in naked carp. Herein, to explore the role of annexin family in naked carp under saline-alkaline stress, twenty GpANNs family members were identified at the genome-wide scale, and bioinformatics and expression analysis under saline-alkaline stress were performed. The predicted subcellular localization showed that most members of GpANNs were distributed in the cytoplasm, and the rest were localized in the cytoskeleton, nucleus, or mitochondria, respectively. GpANNs family proteins contained specific type II Ca²⁺-binding motifs, actin-binding motifs, GTP-binding motifs, etc. Phylogenetic tree analysis showed that the annexin homologues from naked carp, carp (Cyprinus carpio), zebrafish (Danio rerio), and chinese cavefish (Sinocyclocheilus rhinocerous) were divided into eight groups, i.e. ANN1 ∼ 6, ANN11 and ANN13. But homologues of ANN7 ∼ 10 groups were not found in these three fish species. Twenty-eight, nineteen and twenty-nine orthologous gene pairs were identified between naked carp and carp, zebrafish and chinese cavefish, respectively. Six paralogous gene pairs were identified in naked carp, all of which had a Ka/Ks ratio less than one, suggesting that the GpANNs family may have undergone purifying selection pressure during evolution. Expression of GpANN3 was significantly up-regulated in the kidney tissue under saline-alkaline stress, while GpANN2 was significantly down-regulated. Similar trends of protein expression were also observed by Western blot, suggesting involvement of GpANN3 and GpANN2 in stress response. Confocal microscopy revealed that GpANN2 was localized in both cytoplasm and nucleus, but GpANN3 was preferentially localized in the cytoplasm. Our study demonstrated the role of annexins in stress adaption, and provided the basic information required for further function analyses in naked carp.
Evolutionary perspective on annexin calcium-binding domains
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Molecular systematic analysis of the annexin gene superfamily characterized the evolutionary origin, frequency and range of structural variation in calcium interaction domains that are considered intrinsic for membrane targeting and ion channel function. Approximately 36% of annexin repeat domains in an estimated 100 distinct subfamilies contained amino acid changes consistent with the functional loss of type two calcium-binding sites. At least 11% of annexin domains contained a novel K/H/RGD motif conserved in particular subfamilies and manifest in all phyla, apparently via convergent evolution. The first yeast annexin from Yarrowia lipolytica was classified in the ANXC1 subfamily with fungal and mycetozoan representatives. This clade had intact calcium-binding sites but disruption of the normally well-conserved, mid-repeat 4 region implicated in calcium channel regulation. Conversely, a tandem pair of novel annexins from the amphioxus Branchiostoma floridae resembled annexin A13 in gene structure and conserved the charged amino acids associated with the internal hydrophilic pore, but were devoid of external type 2 calcium-binding sites and incorporated K/RGD motifs instead, like annexin A9. The selective erosion of calcium-binding sites in annexin domains and the occurrence of alternate ligands in the same exposed, interhelical loops are pervasive features of the superfamily. This suggests greater complexity than previously appreciated in the mechanisms controlling annexin membrane interaction and calcium channel operation.
Structural and functional characterisation of the mouse annexin A9 promoter
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Annexin A9 is an atypical member of the annexin family of Ca²⁺ and phospholipid-binding proteins, initially identified in EST data bases. Its amino acid sequences responsible for calcium coordination are mutated suggesting an atypical, Ca²⁺-independent cellular function in comparison to other family members. In line with a specialized function of annexin A9 is the restricted presence of its cDNA in EST libraries from different tissues. To identify elements mediating this regulation of annexin A9 transcription, we have cloned the mouse homolog of the human annexin A9 gene and characterised its promoter. By employing 2.5 kb of the most 5′ flanking region of the gene, containing 5′ non-coding sequence, exon I and intron I in luciferase reporter assays in annexin A9 positive HEPA 1–6 cells, we reveal the existence of a minimal promoter located at the 3′ flank of intron I. The sequence covering this minimal promoter contains a binding site consensus for the transcription factor GATA-1 whose binding were verified by electrophoretic mobility shift assays (EMSA). Further mapping analysis also identified two elements in exon I with a negative regulatory function on gene transcription. This suggests that the entire region containing the non-protein coding exon I and the adjacent intron I is involved in the regulation of mouse annexin A9 transcription.
Atypical properties displayed by annexin A9, a novel member of the annexin family of Ca<sup>2+</sup> and lipid binding proteins
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Annexin A9 is a novel member of the annexin family of Ca²⁺ and phospholipid binding proteins which has so far only been identified in EST data bases and whose deduced protein sequence shows mutations in residues considered crucial for Ca²⁺ coordination in other annexins. To elucidate whether the annexin A9 protein is expressed as such and to characterize its biochemical properties we probed cell extracts with specific anti-annexin A9 antibodies and developed a recombinant expression system. We show that the protein is found in HepG2 hepatoma cell lysates and that a green fluorescent protein-tagged form is abundantly expressed in the cytosol of HeLa cells. Recombinant expression in bacteria yields a soluble protein that can be enriched by conventional chromatographic procedures. The protein is capable of binding phosphatidylserine containing liposomes albeit only at Ca²⁺ concentrations exceeding 2 mM. Moreover and in contrast to other annexins this binding appears to be irreversible as the liposome-bound annexin A9 cannot be released by Ca²⁺ chelation. These results indicate that annexin A9 is a unique member of the annexin family whose intracellular activity is not subject to Ca²⁺ regulation.
A new consensus sequence for phosphatidylserine recognition by annexins
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Annexins are abundant and ubiquitous proteins that bind, by their four structurally identical domain cores, to phosphatidylserine-containing membranes in the presence of Ca²⁺. Using molecular simulation and mutagenesis, we have identified a new phosphatidylserine-binding site in annexin V domain 1 and established its structure. The residues involved in this site constitute a consensus sequence highly conserved in all annexins. Remarkably, this consensus sequence is exclusively found in domains 1 or 2, sometimes in both, but never in domains 3 and 4. Such a pattern actually delineates three classes of annexins, shedding new light on the role played by the four-domain core of annexins that could encode specific information discriminating the different annexins that compete within a given cell for membrane binding. Our findings thus provide new strategies for understanding the regulation of the cellular functions of annexins.
Cloning and functional identification of a novel annexin subfamily in Cysticercus cellulosae
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A novel cDNA was isolated from a library of Cysticercus cellulosae by immunological screening. Sequence analysis reveals that it has typical annexin structures. However, since its N-terminus is distinct and the core domain only has 32–44% amino acid identity to the tetrads of other annexins, the cDNA encodes a member of a novel annexin subfamily, which is designed as annexin B1. The recombinant protein, GST-anxB1, has the phospholipid-binding property and anticoagulatant activity common to annexins.

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View full text

Human annexin 31 genetic mapping and origin

Abstract

Introduction

Section snippets

Chromosomal mapping by fluorescence in situ hybridization

Genetic mapping of ANX31

Conclusions

Acknowledgements

Genomics

Gene

Genomics

J. Biol. Chem.

FEBS Lett.

Genomics

Meth. Enzymol.

Biochim. Biophys. Acta

Genomics

Gene

Chromosomal localization, in man and rodents, of a gene (GFI1) encoding a novel zinc finger protein reveals a new syntenic region between mouse and man

Cytogenet. Cell Genet.

dbEST — database for expressed sequence tags

Nat. Genet.

Quality not quantity: the putterfish genome

Hum. Mol. Genet.

Mapping small DNA sequences by fluorescence in situ hybridization directly on banded metaphase chromosomes

Proc. Natl. Acad. Sci. USA