Roth MO et al. (2014), Characterization of the highly variable immune ...

ECB-ART-43664

PLoS One 2014 Oct 17;910:e62079. doi: 10.1371/journal.pone.0062079.

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Characterization of the highly variable immune response gene family, He185/333, in the sea urchin, Heliocidaris erythrogramma.

Roth MO , Wilkins AG , Cooke GM , Raftos DA , Nair SV .

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This study characterizes the highly variable He185/333 genes, transcripts and proteins in coelomocytes of the sea urchin, Heliocidaris erythrogramma. Originally discovered in the purple sea urchin, Strongylocentrotus purpuratus, the products of this gene family participate in the anti-pathogen defenses of the host animals. Full-length He185/333 genes and transcripts are identified. Complete open reading frames of He185/333 homologues are analyzed as to their element structure, single nucleotide polymorphisms, indels and sequence repeats and are subjected to diversification analyses. The sequence elements that compose He185/333 are different to those identified for Sp185/333. Differences between Sp185/333 and He185/333 genes are also evident in the complexity of the sequences of the introns. He185/333 proteins show a diverse range of molecular weights on Western blots. The observed sizes and pIs of the proteins differ from predicted values, suggesting post-translational modifications and oligomerization. Immunofluorescence microscopy shows that He185/333 proteins are mainly located on the surface of coelomocyte subpopulations. Our data demonstrate that He185/333 bears the same substantial characteristics as their S. purpuratus homologues. However, we also identify several unique characteristics of He185/333 (such as novel element patterns, sequence repeats, distribution of positively-selected codons and introns), suggesting species-specific adaptations. All sequences in this publication have been submitted to Genbank (accession numbers JQ780171-JQ780321) and are listed in table S1.

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Species referenced: Heliocidaris erythrogramma
Genes referenced: 185-333 dll1.b LOC100887844 LOC100893907 LOC105438357 LOC115919910 LOC115925415 LOC590297 LOC594261
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	Figure 1. Element patterns of He185/333 cDNAs. A. A total of 26 distinct elements (1â€“26 and leader (L) â€“ see consensus pattern in the top row of the alignment) have been identified amongst He185/333 sequences, and these are arranged into 28 unique element patterns. A horizontal line indicates elements that are â€œmissingâ€. The locations of sequence repeats are shown at the bottom of the figure. The numbers at the bottom of the alignments indicate nucleotide positions. B. Element patterns of He185/333 cDNAs with mutations. Sequences with the element patterns E, I, P, Q, U, L, M contained insertions, deletions or point mutations, resulting in frame shifts and/or early stop codons. Mutations are marked by black lined yellow arrowheads, early stop codons as red bars across an element. Arrowheads falling together with bars are cases of stop codons caused by point mutations, as seen in the three lower element patterns. Arrowheads and bars that are located apart from each other are cases of insertions or deletions, resulting in frame shifts and downstream early stop codons. Elements between mutations and early stop codons contain black central lines to point out missense translations, due to the frame shift. Elements after early stops are patterned with white centres to highlight non-translated regions of the cDNAs.

	Figure 3. Structural overview of deduced consensus of He185/333 polypeptides.Approximate amino acid positions are given at the top, amino/carboxy-terimini are labeled with N and C to the left and right of the consensus, respectively. Polypeptides carry a signal sequence at the N-terminus, followed by glycine-rich and histidine-rich regions. Potential phosphorylated serine, threonine and tyrosine residues are marked with S, T and Y respectively and potential N- and O-linked glycosylations with N and O, respectively. Repeats, as shown for cDNA element patterns in figure 2A and in table S2, are shown at the bottom and are labeled with repeat type followed by copy number.
	Figure 4. Phylogenetic relationship between 25 He185/333 and Sp185/333 cDNAs. He185/333 clades are highlighted in a darker shade than Sp185/333 clades. The phylogenetic tree shows clearly that 185/333 sequences cluster according the species they originated from. The main branch separating the two clades was shortened for display purposes (dashed line). It is representative of the corrected genetic distance of 0.27949 and is supported by 100% of bootstraps. Hence, all sequences within one species had lower genetic distances to each other (highest value 0.07705 for He185/333 and 0.12781 for Sp185/333, respectively) than to any sequence from the opposite species. Boot Strap values are shown next to the branches with the ordering NJ/MP. Values below 50 are not displayed.

	Figure 6. Total coelomocyte proteins from five sea urchins were analysed by Western blotting (A) and SDS-PAGE (B).Coelomocyte proteins were extracted from animals either before (pre-) or after (post-) immunological challenge with heat killed bacteria. Other treatments included injections with filtered sea water (FSW) and injury (pricked with a sterile needle). In both panels A and B, lanes with odd numbers (1, 3, 5, 7 and 9) show pre-challenged protein profiles while lanes with even numbers (2, 4, and 6) show post-challenge protein profiles. Lanes 8 and 10 show the profiles after filtered sea water injection and Injury, respectively. Asterisks to the right of the figures indicate regions that are not stained with Coomassie Blue (B), which contain He185/333 + bands in the Western blot. A. The Western blot shows a diverse pattern of He185/333 proteins between animals but also changes within individuals before and after immunological challenge, FSW injection and injury. Bands on the blot are not as discrete and sharp as their corresponding bands on the Coomassie Blue stained gel, but appear to be rather diffuse and large. Arrows between pre- and post-challenged samples indicate bands that change in intensity or are present/absent as a result of the experimental treatment. B. The Coomassie Blue stained gel shows discreet, sharp protein bands, some of which differ in size and intensity between animals and within individuals before and after immune challenge. None of the bands, however, could unambiguously be identified as He185/333 + band when compared to the Western blot.
	Figure 7. Cellular expression of He185/333 proteins. Aâ€“D. Immunofluorescence and differential intereference contrast (DIC) microscopic images of different coelomocyte types expressing He185/333 proteins (red) and actin (green). Nuclear DNA appears in blue. A. A small filopodial amoebocyte expressing He185/333 proteins. He185/333 staining is found on the cell surface and the clustering of He185/333-associated fluorescence in knobs is evident in a filopodium (white arrows). B. A large filopodial amoebocyte expressing He185/333 proteins in dense knobs. He185/333 signals are not uniformly distributed but are found in patches. C. A large filopodial amoebocyte expressing He185/333 within the cell body in perinuclear areas. D. A lamellipodial amoebocyte expressing He185/333 as knobs on the cell surface.

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