Structural characterisation of Acan-Gal-1
To characterize the structure of Acan-Gal-1, amino acids sequence alignment and structural analysis were performed. Full-length Acan-Gal-1 was composed of 285 amino acids, containing N-terminal CRD (residues 1-150) and C-terminal CRD (residues 158–285) in the manner of tandem repeat, and a short linker (residues 151–157) held them together (Fig. 2a). The amino acids sequence was aligned with Ce-Lec-1 and Tl-Gal-9 (Fig. 2b). The results showed that Acan-Gal-1 had a similarity of 84.4% and 71.6% to Ce-Lec-1 and Tl-Gal-9, respectively. And the conserved motifs HXXXR and WGXEER, involved with carbohydrate binding sites , were located in both NCRD and CCRD. The charged Arg69/Arg203 and Glu88/Glu222 were conserved in the motifs, that are critical amino acids for recognizing carbohydrate binding, and affect protein folding and structure . The shorter linker “GKYYPVP” than other tandem repeat-type galectins, such as galectin-9, that are flexible and susceptible to proteolysis [39–41], may indicate the structural stability of Acan-Gal-1. Structural analysis from homology models revealed that the NCRD of Acan-Gal-1 possessed 11 β-sheets and a small α-helix located between β9 and β10, whereas the CCRD contained 10 β-sheets and no α-helix (Fig. 2b, c). The conserved motifs HXXXR and WGXEER, the carbohydrate binding sites, were located in concave surface surrounded by β4 and β6 of NCRD, and those were also located in the surface formed by β4’ and the loop between β5’and β6’ of CCRD ( Fig. 2c).
Evolutionary relationship of Acan-Gal-1 with its orthologues from other nematode species
To determine the evolutionary relationship between A. cantonensis and other nematodes, the amino acid sequence of Acan-Gal-1 was aligned with its 11 orthologs selected from other 11 nematodes, and subjected to phylogenetic analyses (Fig. 3). Acan-Gal-1 clustered closely with Ce-Lec-1 (from C. elegans) and Hc-Galectin (from Haemonchus contortus) with similarity of 83% and 93%, respectively. Cladistic analysis showed that the Acan-Gal-1 and its homologues were mainly grouped into three clades. C. elegans, H. contortus, Ancylostoma ceylanicum, T. leonine and A. cantonensis were in Clade V; Brugia malayi, Brugia pahangi, Wuchereria bancrofti, Loa loa, Enterobius vermicularis and Toxocara canis were in Clade III; Trichinella spiralis was in Clade I. This result was in agreement with the modern phylogenetic analysis of nematodes .
The expression patterns of Acan-Gal-1
To determine the relative abundance of Acan-gal-1 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-gal-1 was transcribed in larval and adult developmental stages examined in different levels (Fig. 4; Additional file 2: Table S2). The expressions of Acan-gal-1 were greatly up-regulated in both L5 and adult, compared with that in L3; whereas the expression levels were not significantly changed among L5, adult and different genders. This might indicate the important roles of Acan-Gal-1 in L5 and adult, which reside in mammals, such as humans and rats, where a full immune system exist.
For the lack of functional genetic and in vitro culture methods, it is unable to detect the functions of Acan-Gal-1 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 [42–44], was used to investigate the anatomical expression patterns of Acan-Gal-1 for the closely 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-gal-1::gfp and pCe-lec-1::rfp, respectively (Fig. 1a). The results showed that GFP under the promoter pAcan-gal-1 was only expressed in pharyngeal neurons of C. elegans (Fig. 5a–c), in contrast to the situation in worms expressing pCe-lec-1::rfp, where RFP was mainly localized in cuticle, and less in intestine, nervous system and pharynx (Fig. 5d–f). This result was in agreement with the previous report .
Cross-species expressions of Acan-Gal-1 in C. elegans worms
The different activity of pAcan-gal-1 and pCe-lec-1 might be due to the heterologous expression, with the low promoter sequences similarity (data not shown). Therefore, pCe-lec-1 was used as the promoter in this research on the functions of Acan-Gal-1 in C. elegans.
To clarify the role of Acan-Gal-1, cross-species expression in C. elegans was performed. The expressing constructs containing Acan-gal-1::rfp coding sequences driven by Ce-lec-1 promoter (Fig. 1b), were used to transform C. elegans strains N2, lec-1 (tm1345), ced-3 (ok2734) and fat-6;fat-7 (BX156) respectively, and pCe-lec-1::rfp transforming was used as control. In worms, transformed with pCe-lec-1::Acan-gal-1::rfp, RFP was expressed widely, and mainly in cuticle (Fig. 6), consistent with the pCe-lec-1::rfp expression pattern (Fig. 5e, f). The level of lipid storage in lec-1 mutant worms expressing pCe-lec-1::Acan-gal-1::rfp was lower (Fig. 6d) than that in lec-1 mutant worms expressing pCe-lec-1::rfp (Fig. 6g). This might suggest the function of Acan-Gal-1 in reducing lipid deposition in C. elegans, and this morphological change would be investigated further in the following research.
Functional role of Acan-Gal-1 in oxidative stress
To investigate the role of Acan-Gal-1 in regulating oxidative stress resistance, we performed oxidative stress assays using H2O2. We found that the incidence of rapid death among lec-1 deletion mutants was significantly higher than that among the N2 worms; and this oxidative stress susceptibility phenotype could be rescued by expressing pCe-lec-1::Acan-gal-1::rfp in lec-1 mutant worms. The N2 worms expressing pCe-lec-1::Acan-gal-1::rfp were significantly more resistant to H2O2 than the N2 worms expressing pCe-lec-1::rfp (Fig. 7a; Additional file 2: Table S2). This might suggest the regulating role of Acan-Gal-1 in increasing oxidative stress tolerance.
As we have demonstrated that down-regulated Acan-RPS-30 in A. cantonensis L5 could resist oxidative stress damage through inhibiting worm apoptosis , and oxidative stress is thought to be one of the major factors that promote apoptosis , we next determined whether Acan-Gal-1 increased oxidative stress tolerance via inhibiting worm apoptosis. Then, the expression levels of apoptosis genes were detected in C. elegans. The results showed that all the apoptosis genes were not significantly changed in lec-1 mutant worms expressing pCe-lec-1::Acan-gal-1::rfp, compared with those in lec-1 mutant worms expressing pCe-lec-1::rfp (Fig. 7b). Furthermore, oxidative stress assays were performed to detect the effects of expressing Acan-Gal-1 on the oxidative stress damage in ced-3 mutant worms (ced-3 is the core apoptosis executive genes ). The results showed that the incidence of rapid death among ced-3 mutant worms expressing pCe-lec-1::Acan-gal-1::rfp was significantly lower than that among that among the ced-3 mutant worms (Fig. 7c; Additional file 2: Table S2). And ced-3 mutant worms, with lec-1 RNAi, exhibited greatly more susceptibility to oxidative stress than ced-3 mutant worms (Fig. 7d; Additional file 2: Table S2). This might indicate that Acan-Gal-1 could not regulate worm cell apoptosis intracellularly, and the oxidative stress resistance function of Acan-Gal-1 was not via regulating apoptosis in C. elegans.
Functional role of Acan-Gal-1 in lipid storage
Morphological changes were further detected under a light microscope, and we found the level of lipid storage in lec-1 mutant worms expressing pCe-lec-1::Acan-gal-1::rfp was lower than that in lec-1 mutant worms expressing pCe-lec-1::rfp (Fig. 6d, g). Then, Oil Red O fat staining was performed to detect the lipid storage in worms. The results showed that lec-1 mutant worms stored significantly more lipid than N2 worms, and this lipid accumulation phenotype could be rescued by expressing pCe-lec-1::Acan-gal-1::rfp in lec-1 mutant worms. The N2 worms, expressing pCe-lec-1::Acan-gal-1::rfp exhibited greatly less lipid storage than the N2 worms expressing pCe-lec-1::rfp (Fig. 8a, b; Additional file 2: Table S2). This might suggest the function of Acan-Gal-1 in reducing lipid deposition in C. elegans.
Fatty-acid metabolism is involved in the oxidative stress resistance in C. elegans [47, 48]. Therefore, we next investigated whether Acan-Gal-1 increased oxidative stress tolerance via reducing lipid deposition in C. elegans. fat-6;fat-7 double-mutant worm was selected. Because fat-6 and fat-7 genes encode stearoyl-CoA desaturases (SCDs), that are key lipogenic enzymes, and the fat-6;fat-7 double-mutant worms have decreased fat stores . Then, oxidative stress assays were performed to determine the effects of expressing Acan-Gal-1 on the oxidative stress damage in fat-6; fat-7 double-mutant worms. The results showed that the incidence of rapid death was not significantly changed among fat-6;fat-7 double-mutant worms expressing pCe-lec-1::Acan-gal-1::rfp, compared with that among fat-6;fat-7 double-mutant worms expressing pCe-lec-1::rfp (Fig. 8c). And the susceptibility to oxidative stress was not influenced by lec-1RNAi in fat-6;fat-7 double-mutant worms (Fig. 8c). This might suggest the regulating role of Acan-Gal-1in increasing oxidative stress tolerance was played via reducing lipid deposition in C. elegans.