Borisenko I et al. (2022), Novel protein from larval sponge cells, ilborin...

ECB-ART-50475

Open Biol 2022 Feb 01;122:210336. doi: 10.1098/rsob.210336.

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Novel protein from larval sponge cells, ilborin, is related to energy turnover and calcium binding and is conserved among marine invertebrates.

Borisenko I , Daugavet M , Ereskovsky A , Lavrov A , Podgornaya O .

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Sponges (phylum Porifera) are early-branching animals, whose outwardly simple body plan is underlain by a complex genetic repertoire. The transition from a mobile larva to an attached filter-feeding organism occurs by metamorphosis, a process accompanied by a radical change of the body plan and cell transdifferentiation. The continuity between larval cells and adult tissues is still obscure. In a previous study, we have produced polyclonal antibodies against the major protein of the flagellated cells covering the larva of the sponge Halisarca dujardini, used them to trace the fate of these cells and shown that the larval flagellated cells transdifferentiate into the choanocytes. In the present work, we identified the sequence of this novel protein, which we named ilborin. A search in the open databases showed that multiple orthologues of the newly identified protein are present in sponges, cnidarians, flatworms, ctenophores and echinoderms, but none of them has been described yet. Ilborin has two conserved domains: triosephosphate isomerase-barrel, which has enzymatic activity against macroergic compounds, and canonical EF-hand, which binds calcium. mRNA of ilborin is expressed in the larval flagellated cells. We suggest that the new protein is involved in the calcium-mediated regulation of energy metabolism, whose activation precedes metamorphosis.

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	Figure 1. . (a) Sexual life cycle of marine sponge (adult/juvenile individual, larva, settled larva transforming into pupa, formation of aquiferous system inside the pupa); (b) marine sponge H. dujardini in vivo (scale bar, 5 mm); (c) swimming larva of Halisarca dujardini (differential interference contrast microscopy; scale bar, 50 µm).
	Figure 2. . (a) Dissociated larval cells separated in Percoll discontinuous gradient. Fractions 1 and 2 are enriched with anterolateral flagellated cells, and fraction 3, with cells of the posterior pole of the larva. Major protein of anterolateral flagellated cells (arrowhead) is absent in posterior cells of fraction 3. (b) SDS-PAGE of whole larval proteins (‘whole’ lane) and corresponding fractions.
	Figure 3. . Protein backbone disorder prediction for ilborin A from Halisarca dujardini. Blue rectangles show borders of homology to particular superfamilies identified using InterPro [21]. The Ca-binding site is marked by a red box. Regions of maximum flexibility inside the amino acid sequence are marked by dashed lines.
	Figure 4. . Ilborin expression and associated structures. WMISH with antisense (a) and sense (b, negative control) probes to ilborin A on whole larva. ap, anterior pole; pp, posterior pole. (c) Paraffin section of juvenile sponge 3 days after settlement, immunostained with antibodies to p68 (ilborin; red) and tubulin (green); arrowheads indicate the apical parts of choanocytes (from [20]). chc, choanocyte chamber formed de novo. TEM of larval anterolateral flagellated cells (d) and adult choanocytes (e). bb, basal body of flagellum; G, Golgi complex; mv, microvilli; m, mitochondria; n, nucleus; nu, nucleolus; v, electron-transparent vesicles; vi, vitelline inclusions. Scale bars: (a) and (b), 25 µm; (c), 10 µm; (d,e), 2 µm.
	Figure 5. . Primmorph development from dissociated sponge cells, and ilborin expression. (a) Scheme of sponge development from dissociated cells. Cells in suspension (i) make contact and form aggregates (ii) with the outer layer of cells developing in the exopinacoderm. Then, the aggregate attaches to the substrate and inner cells dedifferentiate and differentiate into choanocytes, endopynacocytes and mesohylar cells (iii), which then form the adult sponge body (iv). (b) Transmission electron microscopy section of rudimentary choanocyte chamber (outlined with dashed line) consisting of differentiating choanocytes bearing small flagella. (c) Semi-thin section of primmorph, demonstrating development of aquiferous system and rudiments of choanocyte chambers (144 h). (d) WMISH with ilborin probe with developing primmorph (288 h). Main signal, indicated by arrowheads, corresponds to newly formed choanocyte chambers. aG, Golgi apparatus; cc, choanocyte chambers; ccr, rudimentary choanocyte chamber; cn, canal of aquiferous system; exp, exopinacoderm; f, flagella; m, mitochondrion; n, nucleus; o, osculum; ph, phagosomes. Scale bars: (b), 100 µm; (c), 2 µm; (d), 20 μm.
	Figure 6. . (a) Phylogenetic tree of ilborin orthologues from different phyla. Support values of posterior probabilities (top value) and bootstraps (bottom value) are displayed. Adi, Acropora digitifera; Apl, Acanthaster planci; Aqu, Amphimedon queenslandica;; Aru, Asterias rubens; Che, Clytia hemisphaerica; Dgi, Dendronephthya gigantea; Hdu, Halisarca dujardini; Hvu, Hydra vulgaris; Mle, Mnemiopsis leidyi; Mli, Macrostomum lignano; Nve, Nematostella vectensis; Oca, Oscarella carmella; Ofa, Orbicella faveolata; Pda, Pocillopora damicornis; Spi, Stylophora pistillata; Sci, Sycon ciliatum. (b) Domain structure of representative ilborin orthologues for each class. Green, aldolase-type TIM barrel; blue, EF-hand domain; polypeptide length is given as the number of amino acid residues.
	Figure 7. . Position of conserved amino acids responsible for Ca-binding in the EF-hand domain. Top: alignment of EF-hand of different ilborin orthologues. Middle: consensus generated with HMM Logo from top alignment. Bottom: position of conserved residues in the loop and second helix of canonical EF-hand (marked with arrows). Residues X, Y, Z and -Z (numbered 1, 3, 5 and 12, respectively) are responsible for Ca2+ binding (modified from [46, Fig. 2]).
	Figure 8. . Sequence similarity network of EF-hand-containing proteins generated by all-versus-all BLAST and visualized with Cytoscape. Each orange circle is an entry (EF-hand protein sequence); grey lines are edges demonstrating percentage of identity in alignment. Nodes clustered with the Markov cluster (MCL) algorithm. Some major EF-hand protein subfamilies labelled. Ilborin orthologues cluster is at the top right; singletons are below.

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