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

Abstract

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.

Figure 1. Element patterns of He185/333 cDNAs.

A. A total of 26 distinct elements (1–26 and leader (L) – 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 “missing”. 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 2. He185/333 gene structure.

A. Genes consist of a short exon 1 and a longer exon 2, separated by one intron. The leader is 63 bp in length and is interspersed between positions 55/56 by the Intron. Thus, the leader represents exon 1 (excluding the 5′UTR) but also forms the first eight nucleotides of the second exon. The intron varies in size between 457 bp and 1392 bp. The second, long exon ranges from 749 bp to 905 bp (excluding the 3′UTR) and contains the mosaic organisation of elements. The 5′ and 3′ UTRs of He185/333 genes have only been partially sequenced. B. Element patterns of 39 manually aligned He185/333 gDNA sequences. i. The coding regions (exons 1 and 2) are interspersed by one intron, dividing the leader in two parts, as indicated at the top of the diagram. Genes identified to-date show the five exon element patterns E, I, R, W and Y as described for cDNAs in figure 1A. Introns are simplified by interrupted, checkered boxes. The consensus at the top of the diagram is based on all element patterns, including cDNA element patterns as shown in figure 1A. ii. Similar to exons, introns align optimally with insertion of large gaps, resulting in ten intron elements and four intron element patterns, designated alpha (α), beta (β), gamma (γ) and delta (δ). Individual intron elements are named i1 to i10 and are shown as differently shaded gray boxes. Exons are represented as fading extensions to the left and right of intron element patterns. i & ii. Combinations of exon and intron element patterns define gene element patterns of which the following nine have been identified among the 39 gene sequences (number of individual sequences with according gene element pattern in brackets: E-α (11), I-α (2), W-α (8), Y-a (1), E-β (2), R-β (2), W-β (1), E-γ (10), W-δ (2). The first element pattern is the consensus.

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 5. A comparison of codon diversification between S. purpuratus and H. erythrogramma 185/333 cDNA alignments.

Sequence alignments of 231 S. purpuratus and 112 H. erythrogramma cDNAs were used to investigate recombination and selective pressure at the codon level. Element patterns are shown as black and white banding, while positively- and negatively-selected codons are shown as green and red boxes above and below each alignment, respectively. Negative selection (against codon diversification) appears to be prevalent across 185/333 sequences from both species. Significant (p<0.1) negative selection was detected in 64 codons across the 581 codon-long S. purpuratus alignment, while 15 codons were positively-selected. The 360 codon-long H. erythrogramma alignment contained 17 codons under significant negative selective pressure and nine codons with positive selective pressure. S. purpuratus element patterns adapted from .

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.

A–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.