Structural characterisation of Acan-RPS-30
The complete cDNAs of Acan-rps-30 was isolated by RACE from Angiostrongylus cantonensis. Acan-rps-30 cDNA was 1,209 bp in length, including an open reading frame (ORF) of 393 bp (including stop codon), a 5’-untranslated region (UTR) of 190 bp, and a 3’-UTR of 626 bp (Fig. 2A). 5’ UTR harbored the consecutive pyrimidines (TTTCTTTTC), which are commonly found at the 5’ end of eukaryotic ribosomal protein mRNAs , and may play a role in regulating translation . The 3’ UTR contained the hexamer AATAAA (positions, 612 bp downstream of the TAA). The complete Acan-rps-30 gene, isolated by Genome Walking from genomic DNA of A. cantonensis was 2,967 bp in length, consisting of 4 exons and 3 introns (Fig. 2A).
To characterize the structure of Acan-RPS-S30, sequence alignment and structural analysis were performed. The cDNA of Acan-rps-30 encoded predicted proteins of 130 amino acids (Fig. 2B), which contained the potential cleavage sites (Gly-Gly) of the fusion protein (ubiquitin-like; UBiL-ribosome protein S30; S30). The amino acids sequence was aligned with Ce-RPS-30 and Hs-RPS-30 (Fig. 2B). The results showed that the C-terminal S30 domains were conserved (Acan-RPS-S30 versus Ce-RPS-S30 and Hs-RPS-30, 87.9% and 77.6% similarity, respectively), whereas the N-terminal UBiL domains were divergent (37.5% and 30.4% similarity, respectively). The S30 domain contained a nuclear location signal (NLS), KQEKKKKKK, with which RPS-30 can go into the nucleus and involves itself in the small subunit assembly of ribosome. Structural analysis from homology models revealed that UBiL region possessed 3 β-sheets and 2 α-helixes (Fig. 2C), and S30 region contained 2 α-helixes (Fig. 2D). The UBiL region did not harbor the K48 and K63 residues, sites of poly ubiquitin chain formation, consisted with the orthologues from other species, indicated the different functions , though the structure of UBiL was similar to that of ubiquitin.
Figure 2 Structure and sequence analysis of Acan-RPS-S30. (A) The exon-intron organization of Acan-rps-30 from Angiostrongylus cantonensis. The Acan-rps-30 gene spans 2,967 bp, consisting of 4 exons and 3 introns. Narrow bar represented untranscribed sequences or introns; wide bars represented exons; brown blocks, coding regions; grey blocks, non-coding 5’- and 3’- untranslated region (UTR). (B) Alignment of amino acid sequences of Acan-RPS-S30 from Angiostrongylus cantonensis with those from Homo sapiens and Caenorhabditis elegans. The accession numbers of sequences available from current databases are: NP_505007.1 (Ce-RPS-S30) and NP_001988.1 (Hs-RPS-30). Identical and similar residues are shown in black and grey blocks, respectively. The potential cleavage sites (Gly-Gly) of the fusion protein (ubiquitin-like; UBiL-ribosome protein S30; S30) are indicated with green arrows (upstream and downstream sequences are UBiL and S30 regions, respectively). The nuclear location signals in the S30 regions are indicated with a green block. The secondary structural elements of Acan-RPS-S30 are shown above the alignment. (C) Predicted tertiary structure of UBiL region, showing 3 β-sheets and 2 α-helixes. (D) Predicted tertiary structure of S30 region, showing 2 α-helixes.
Evolutionary relationship of Acan-RPS-30 with RPS-30 orthologues from other nematode species
To determine the evolutionary relationship between A. cantonensis and other nematodes, the predicted amino acid sequence of Acan-RPS-30 was aligned with orthologs from other nematodes, and subjected to phylogenetic analyses (Fig. 3). Acan-RPS-30 clustered closely with Dv-RPS-30 from Dictyocaulus viviparus with similarity of 89.2%. Cladistic analysis showed that the DAF-2 homologues selected from seven parasitic nematodes were mainly grouped into two clades. C. elegans, H. contortus, Necator americanus, D. viviparus and A. cantonensis were in Clade V; Wuchereria bancrofti, Brugia malayi and Loa loa were in Clade III. This result was in agreement with modern phylogenetic analysis of nematodes . When sequences from the S30 regions only were analysed, bootstrapping did not support the clusters (data not shown). This might indicate that the divergences of the UBiL regions are likely related to species specificity.
Figure 3 Neighbour-joining phylogenetic tree of RPS-30 proteins from several nematodes. The tree is calculated using the Jones–Taylor–Thornton model in the MEGA program version 5.0. Bootstrap values above the branches (1000 iterations) are shown for robust clades (> 70%). Abbreviation for species names are as follows: Ce: Caenorhabditis elegans; Acan: Angiostrongylus cantonensis; Dv: Dictyocaulus viviparous; Na: Necator americanus; Hc: Haemonchus contortus; Ll: Loa loa; Wb: Wuchereria bancrofti; Bm: Brugia malayi. The corresponding accession numbers are listed on the right of each species. Clade numbers were noted in Roman numerals.
The expression patterns of Acan-rps-30
To determine the relative abundance of Acan-rps-30 transcript in different developmental stages (L3, L5 and adult) and genders [females (F) and males (M)] of the life cycle of A. cantonensis, qRT-PCR was performed with the 18S ribosomal RNA gene as an internal loading control. The results showed that Acan-rps-30 was transcribed in larval and adult developmental stages examined in different levels (Fig. 4). The expressions of Acan-rps-30 were significantly down-regulated in both L5 and adult, compared with that in L3; furthermore, the expression level in L5 was greatly lower than that in adult. This might indicate the important roles of Acan-RPS-30 in different developmental stages (L3, L5 and adult), which reside in different host.
Figure 4 Transcriptional profile of Acan-rps-30 in different developmental stages (L3, L5 and
adult) and genders [females (F) and males (M)] of A. cantonensis, determined by real-time PCR analysis. Data shown are mean ± S.E.M derived from three technical replicates with two biological replicates. Relative transcription of the Acan-rps-30 gene in each sample was calculated by normalisation of the raw data, followed by the determination of abundance relative to the 18S ribosomal RNA gene (GenBank: AY295804), which was served as an internal loading control. Statistical analysis was conducted using a one-way ANOVA. *P < 0.05; **P < 0.01.
For the lack of functional genetic and in vitro culture methods, it is unable to detect the functions of Acan-RPS-30 directly in A. cantonensis. Here, C. elegans, proposed by numerous authors as a general model for many aspects of basic molecular, cellular and developmental biology in the less tractable parasitic nematodes [34–36], was used to investigate the anatomical expression patterns of Acan-rps-30 for the closed evolutionary relationship between A. cantonensis and C. elegans, both belonging to Clade V according to Cladistic analysis . Wild type C. elegans (N2 strain) were transformed with the construct pAcan-rps-30::gfp and pCe-rps30::gfp, respectively (Fig. 1A). Plasmid pRF4 was included in all transformations as a behavioural marker. Transgenic worms showing the roller phenotype were selected. The results showed that GFP under the promoter pAcan-rps-30 was only expressed in intestine of C. elegans, mainly in the anterior end (Fig. 5A–C), which is the major tissue for lifespan regulation in C. elegans , in contrast to the situation in worms expressing pCe-rps-30::gfp, where GFP was expressed in almost all cells, including intestine, nervous system, pharynx, muscle (Fig. 5D–F). The different activity of pAcan-rps-30 and pCe-rps-30 might be due to the heterologous expression, with the low promoter sequences similarity (data not shown). Therefore, pCe-rps-30 was used as the promoter in following research on the functions of Acan-RPS-30 in C. elegans.
Figure 5 Expression pattern of Angiostrongylus cantonensis Acan-rps-30 promoter in Caenorhabditis elegans. (A-C) The promoter activity of Acan-rps-30 in C. elegans. pAcan-rps-30::gfp is only expressed in intestine, mainly in the anterior end. (D-F) The promoter activity of Ce-rps-30. pCe-rps-30::gfp is expressed ubiquitously. Arrows indicate the following tissues: i, intestinal; m, muscle; n, neuron; p, pharynx.
Cross-species expressions of Acan-RPS-30 in N2 and the rps-30 deletion mutant worms
In order to clarify the role of Acan-RPS-30, cross-species expression of Acan-rps-30 in C. elegans was performed. The expressing constructs containing Acan-rps-30::rfp coding sequences driven by Ce-rps-30 promoters (Fig. 1B), were used to transform C. elegans N2 strain and rps-30 deletion mutant strain (tm6034), respectively. In N2 worms, transformed with pCe-rps30::Acan-rps-30::rfp, RFP was expressed widely (Fig. 6B and C), consistent with the pCe-rps30::gfp expression pattern(Fig. 5D and F). In addition, RFP mainly focused on the nucleus for the existence of NLS in S30 region. The “button-like” morphology, corpses arising from developmental apoptosis and the gold standard for quantification of apoptosis in C. elegans , was seen in the anterior pharynx (Fig. 6A and D). This might suggest the pro-apoptotic effect of Acan-RPS-30, consistent with the pro-apoptotic regulatory role of Hs-RPS-30 [19, 23].
In trans-heterozygous worms (tm6034), the GFP fluorescence positive animals (pharynx), carrying nT1 are heterozygous rps-30+/− (Fig. 6E-G), and animals without GFP (nT1) are mutation homozygous rps-30− /− (Fig. 6H). After transformation of pCe-rps30::Acan-rps-30::rfp in rps-30+/− worms, the offspring contained rps-30+/− expressing pCe-rps30::Acan-rps-30::rfp (Fig. 6I-L) and rps-30− /− expressing pCe-rps30::Acan-rps-30::rfp (Fig. 6M-P), with the rps-30− /− expressing pCe-rps30::gfp (Fig. 6Q-S) as the control in the following assay.
Figure 6 Cross-species expressions of Acan-RPS-30 in N2 and the rps-30 deletion mutant worms. (A-D) Expression of pCe-rps-30::Acan-rps-30::rfp in N2. RFP was expressed widely; RFP mainly focused on the nucleus; the “button-like” corpses were seen in the anterior pharynx. Arrow heads indicate the corpses. (E-G) The heterozygous rps-30+/− worm. The GFP fluorescence positive worms (pharynx) carried the balancer nT1. (H) The homozygous rps-30− /− worm. Worms without GFP (nT1) were mutation homozygous rps-30− /−. (I-L) Expression of pCe-rps-30::Acan-rps-30::rfp in the heterozygous rps-30+/− worm. RFP was expressed widely, and GFP fluorescence was positive in pharynx. (M-P) Expression of pCe-rps-30::Acan-rps-30::rfp in the homozygous rps-30− /− worm. RFP was expressed widely, and GFP fluorescence was negative in pharynx. (Q-S) Expression of pCe-rps-30::gfp in the homozygous rps-30− /− worm.
Functional role of Acan-RPS-30 in oxidative stress
To investigate the role of Acan-RPS-30 in regulating oxidative stress resistance, we performed oxidative stress assays using H2O2. We found that the incidence of rapid death among the N2 worms expressing pCe-rps30::Acan-rps-30::rfp was significantly higher than that among the N2 worms expressing pCe-rps30::gfp; and the rps-30 deletion mutants (rps-30− /−) was significantly more resistant than N2 worms; and this oxidative stress resistance phenotype could be rescued and inhibited by expressing pCe-rps30::Acan-rps-30::rfp in rps-30− /− mutant worms (Fig. 7A). This might suggest the regulating role of Acan-RPS-30 in promoting susceptibility to oxidative stress.
As oxidative stress is considered to be one of the major factors that promote apoptosis , we next detect the expression levels of apoptosis genes in C. elegans. The results showed that all the apoptosis genes were down-regulated in rps-30− /− mutant worms, except akt-1 up- regulated (Fig. 7B), which inhibits CEP-1 and decreases DNA damage-induced apoptosis ; and ced-3 and ced-4, the core apoptosis executive genes , were both up-regulated in N2 worms expressing pCe-rps30::Acan-rps-30::rfp; Whereas, ced-9 down-regulated (Fig. 7C), which encodes the homologous protein to the anti-apoptotic B-cell lymphoma 2 (Bcl-2) family of proteins .This might indicate the role of Acan-RPS-30 in promoting apoptosis in C. elegans.
To further determine the effect of apoptosis regulated by Acan-RPS-30 on oxidative stress susceptibility, we constructed the C. elegans strain ced-3−/−(ok2734) expressing pCe-rps30::Acan-rps-30::rfp and the strain N2; pCe-rps30::Acan-rps-30::rfp with ced-3 knocked down using RNA interference. Then, the survival percentages were detected with the strains ced-3−/− expressing pCe-rps30::gfp and N2; pCe-rps30::Acan-rps-30::rfp as controls, respectively. We found that the incidence of rapid death among the worms ced-3−/− expressing pCe-rps30::Acan-rps-30::rfp was almost the same as that among the worms ced-3−/− expressing pCe-rps30::gfp; and that among the worms N2 expressing pCe-rps30::Acan-rps-30::rfp was significantly higher than that among the worms N2 expressing pCe-rps30::Acan-rps-30::rfp with ced-3 knocked down (Fig. 7D). This might suggest that the regulating role of Acan-RPS-30 in promoting susceptibility to oxidative stress was played through CED-3, which is the core executive effector in the worm cell apoptosis .
Figure 7 Down-regulated RPS-30 plays a defensive role against oxidative stress by regulating ced-3. (A) Oxidative stress assays using H2O2 in N2 and rps-30 mutant worms expressing pCe-rps30::Acan-rps-30::rfp. (B) The expression level of apoptosis genes in homozygous rps-30− /− worm. (C) The expression level of apoptosis genes in N2 worms expressing pCe-rps30::Acan-rps-30::rfp. (D) Oxidative stress assays using H2O2 in ced-3 mutant worms and worms expressing pCe-rps30::Acan-rps-30::rfp. The worms were counted as described in the “Methods” section. The error bars indicate standard deviation. *P < 0.05; **P < 0.01.