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Open Biol
2017 Apr 01;74:. doi: 10.1098/rsob.160300.
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A puzzling homology: a brittle star using a putative cnidarian-type luciferase for bioluminescence.
Delroisse J
,
Ullrich-Lüter E
,
Blaue S
,
Ortega-Martinez O
,
Eeckhaut I
,
Flammang P
,
Mallefet J
.
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Bioluminescence relies on the oxidation of a luciferin substrate catalysed by a luciferase enzyme. Luciferins and luciferases are generic terms used to describe a large variety of substrates and enzymes. Whereas luciferins can be shared by phylogenetically distant organisms which feed on organisms producing them, luciferases have been thought to be lineage-specific enzymes. Numerous light emission systems would then have co-emerged independently along the tree of life resulting in a plethora of non-homologous luciferases. Here, we identify for the first time a candidate luciferase of a luminous echinoderm, the ophiuroid Amphiura filiformis Phylogenomic analyses identified the brittle star predicted luciferase as homologous to the luciferase of the sea pansy Renilla (Cnidaria), contradicting with the traditional viewpoint according to which luciferases would generally be of convergent origins. The similarity between the Renilla and Amphiura luciferases allowed us to detect the latter using anti-Renilla luciferase antibodies. Luciferase expression was specifically localized in the spines which were demonstrated to be the bioluminescent organs in vivo However, enzymes homologous to the Renilla luciferase but unable to trigger light emission were also identified in non-luminous echinoderms and metazoans. Our findings strongly indicate that those enzymes, belonging to the haloalkane dehalogenase family, might then have been convergently co-opted into luciferases in cnidarians and echinoderms. In these two benthic suspension-feeding species, similar ecological pressures would constitute strong selective forces for the functional shift of these enzymes and the emergence of bioluminescence.
Figure 1. The brittle star Amphiura filiformis. (a) Arms of A. filiformis emerging out of the sediment (in aquarium; scale bar, 1 cm). (b) Oral view of A. filiformis (scale bar, 2 mm).
Figure 2. Multiple sequence alignment of the Renilla luciferase (RLuc), deuterostomian luciferase-like proteins (echinoderms: Amphiura filiformis, Asterias rubens, Strongylocentrotus purpuratus; hemichordate: Saccoglossus kowalevskii; urochordate: Ciona intestinalis), placozoan luciferase-like protein (Trichoplax adhaerens) and bacterial haloalkane dehalogenase proteins (Mycobacterium sp.). Alignment was performed using Geneious software (MAFFT alignment). Conserved amino acids relative to RLuc are coloured in all sequences. The amino acid triad known as the active site in haloalkane dehalogenases and RLuc is framed in black. This amino acid triad is conserved in all luciferase-like sequences we identified except in A. filiformis
Gen853765. Sequence accession numbers: T. adhaerens, XP002116677; R. reniformis, AAA29804.1; S. purpuratus, DspA XP794172.2; A. rubens, Ar_comp22488; B. floridae, XP002611539.1; C. intestinalis, XP002127127.1; S. kowalevskii, XP002738321.1, XPE002730984.1; Mycobacterium sp., WP067006024.
Figure 3. Phylogenetic reconstructions of RLuc-like sequence relationships based on maximum likelihood (a), Bayesian (b) and distance (c) methods. Bacterial haloalkane dehalogenases were taken as outgroup. Bootstrap (for a,c) and posterior probability (for b) values are colour-coded from red (0%) to green (100%). Short/partial sequences were not used for the analyses. The analyses included sequences from Amphiura filiformis (Gen224433, Uni203026, Gen313061 and Gen853765), Renilla reniformis (AAA29804), Renilla muelleri (AAG540941), Strongylocentrotus purpuratus (XP788943, XP794172, XP794218, XP798042 and XP792159), Saccoglossus kowalevskii (XP002730984 and XP002738321), Ciona intestinalis (XP002127127), Branchiostoma floridae (XP002611539), Capitella teleta (ELT94308), Trichoplax adhaerens (XP002116677 and XP002116678) and the haloalkane dehalogenases from Roseobacter (EBA17164.1, and WP009810405), Phaeobacter (WP054461824), Thalassobius mediterraneus (WP058317896.1), Sulfitobacter pontiacus (WP064216788), Shimia (WP054002384), Mycobacterium sp. (WP066929894), Geobacter daltonii (WP012647128), Dehalococcoidia bacterium (KPJ48875), Myxococcales bacterium (KPK14642) and Algiphilus aromaticivorans (WP043766976).
Figure 4. In vitro luciferase activity of echinoderm extracts (a–c) and in vivo light emission in A. filiformis (d,e). Luminometry tests of luciferase activity performed on A. filiformis arm extracts (a), on A. rubens tube foot extracts (b), and on purified RLuc (c), after coelenterazine addition. (d) Light emission of an arm in an adult A. filiformis measured by luminometry (RLU: relative light unit). (e) Bioluminescence spectral emission of A. filiformis measured by microspectrophotometry.
Figure 5. Light emission in A. filiformis and immunodetection of RLuc-like proteins. (a) Aboral view of an individual during KCl stimulation (scale bar, 0.5 cm). (b) Detail of the arm light emission (scale bar, 1 mm). (c) Detail of the spine emission (scale bar, 150 µm). (d) Schematic view of the arm bioluminescence. (e–h) Immunolocalization of RLuc-like proteins (red) and acetylated alpha tubulin (green) in an arm portion of A. filiformis. Nuclear DAPI staining is in blue (scale bar, 200 µm). (h) Immunolocalization of RLuc-like proteins in a single spine (scale bar, 40 µm; a, arm; d, disc; s, spine; bs, luminous blue spot; p, podia.
Figure 6. BLAST cluster map of all known luciferase/photoproteins. Node coloration is based on taxonomy. Lines correspond to BLAST connections of p-value < 1 10−3.
Figure 7. Distribution of haloalkane dehalogenases/RLuc-like proteins in metazoans and associated evolutionary hypotheses. The presence of haloalkane dehalogenases/RLuc-like proteins in specific taxa is shown in red (based on BLAST analyses performed on nr/nt/TSA databases). Arrows illustrated hypothetical gene transfers. Tree based on the Tree of Life Project (http://tolweb.org). Organism silhouettes were extracted from the PhyloPic open database (phylopic.org). Renilla reniformis picture courtesy of Dr Alvaro Esteves Migotto (University of Sao Paulo).
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