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Identification and functional characterisation of a Schistosoma japonicum insulin-like peptide

View Article: PubMed Central - PubMed

ABSTRACT

Background: Previous studies have shown that insulin receptors in schistosomes, triggered by host insulin, play an important role in parasite growth, development and fecundity by regulating glucose metabolism. However, limited information is available on the recently identified endogenous insulin-like peptide (ILP) in blood flukes.

Results: We isolated ILPs from Schistosoma japonicum (SjILP) and S. recognised (SmILP) and present results of their molecular and structural analysis. SjILP shares 63% amino acid identity with SmILP, but only 18% identity with human insulin. There is high cross immunological reactivity between the S. japonicum and S. mansoni ILPs as observed in western blots using an anti-SjILP polyclonal antibody. ADP binding/hydrolysis ability was observed in both SjILP and SmILP, but not in human insulin, suggesting a parasite-specific role for ILP compared with host insulin. Protein binding assays using the Octet-RED system showed SjILP binds S. japonicum IRs (SjIR1 and SjIR2) strongly. An anti-phospho antibody against extracellular signal-regulated kinase (Erk) recognised a 44-kDa target band in an extract of adult worms after stimulation by rSjILP in vitro, suggesting an important role for SjILP in activating SjIRs and in regulating downstream signal transduction. Immunolocalisation showed SjILP is located on the tegument and the underlying musculature, similar to that observed for SjIR1, but it is also present throughout the parenchyma of males and in the vitelline cells of females, the same locations as SjIR2 described in an earlier published study of ours. The same localisation of SjILP and the SjIRs is suggestive of an interaction between the insulin-like peptide and the IRs. In addition to binding host insulin, schistosomes also can express their own endogenous ILPs, which can activate the parasite insulin signal pathway, thereby playing a critical role in worm growth, development and fertility.

Conclusions: These findings shed new light on ILPs in schistosomes, providing further insight into the distinct and specialised functions of SjIR1 and 2 in S. japonicum and their interaction with host insulin.

Electronic supplementary material: The online version of this article (doi:10.1186/s13071-017-2095-7) contains supplementary material, which is available to authorized users.

No MeSH data available.


Effect of stimulation with rSjILP or human insulin on extracellular signal-regulated kinase (Erk) in adult S. japonicum worms. a Anti-actin antibody (upper) and anti-phospho p44/42 MAPK (Erk) antibody (lower) were used to probe in Western blot analysis a protein extract of adult S. japonicum incubated for 30 min with: rSjILP (Lane 1); both rSjILP and human insulin (Lane 2); human insulin (Lane 3); neither rSjILP nor insulin (Lane 4). b Signal intensities of bands recognised by anti-phospho p44/42 MAPK (Erk) antibody in panel A (lower), were measured using the Odyssey Classic Infrared Imager with a scan intensity setting of 5 and sensitivity of 5. Error bars represent the standard error of the mean (SEM). This experiment was performed twice
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Fig3: Effect of stimulation with rSjILP or human insulin on extracellular signal-regulated kinase (Erk) in adult S. japonicum worms. a Anti-actin antibody (upper) and anti-phospho p44/42 MAPK (Erk) antibody (lower) were used to probe in Western blot analysis a protein extract of adult S. japonicum incubated for 30 min with: rSjILP (Lane 1); both rSjILP and human insulin (Lane 2); human insulin (Lane 3); neither rSjILP nor insulin (Lane 4). b Signal intensities of bands recognised by anti-phospho p44/42 MAPK (Erk) antibody in panel A (lower), were measured using the Odyssey Classic Infrared Imager with a scan intensity setting of 5 and sensitivity of 5. Error bars represent the standard error of the mean (SEM). This experiment was performed twice

Mentions: An anti-phospho p44/42 MAPK antibody was used to detect the phosphorylation of Erk in adult S. japonicum stimulated by host insulin or rSjILP. An Erk band, approximately the same size (44 kDa) as observed previously in S. mansoni [37], was recognised by the anti-phospho antibody in protein extracts of adult S. japonicum worms incubated with or without rSjILP and insulin (Fig. 3). A 44 kDa Erk band was recognised in extracts prepared from worms stimulated with rSjILP and from worms co-stimulated with rSjILP and insulin, with no significant difference evident in band intensity between the two extracts (Fig. 3). The Erk band was also detectable in an extract from worms incubated with human insulin only but, compared with that of the worms co-stimulated with rSjILP and insulin, its intensity was reduced by 58% [t(6) = 31.39, P = 0.001] (Fig. 3). The Erk band was detectable in an extract of worms incubated without human insulin and rSjILP, but its intensity was reduced by 30% [t(6) = 4.353, P = 0.049] compared with that in the worm extract incubated with insulin only (Fig. 3) (*P ≤ 0.05, **P ≤ 0.001, ***P ≤ 0.0001).Fig. 3


Identification and functional characterisation of a Schistosoma japonicum insulin-like peptide
Effect of stimulation with rSjILP or human insulin on extracellular signal-regulated kinase (Erk) in adult S. japonicum worms. a Anti-actin antibody (upper) and anti-phospho p44/42 MAPK (Erk) antibody (lower) were used to probe in Western blot analysis a protein extract of adult S. japonicum incubated for 30 min with: rSjILP (Lane 1); both rSjILP and human insulin (Lane 2); human insulin (Lane 3); neither rSjILP nor insulin (Lane 4). b Signal intensities of bands recognised by anti-phospho p44/42 MAPK (Erk) antibody in panel A (lower), were measured using the Odyssey Classic Infrared Imager with a scan intensity setting of 5 and sensitivity of 5. Error bars represent the standard error of the mean (SEM). This experiment was performed twice
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5391603&req=5

Fig3: Effect of stimulation with rSjILP or human insulin on extracellular signal-regulated kinase (Erk) in adult S. japonicum worms. a Anti-actin antibody (upper) and anti-phospho p44/42 MAPK (Erk) antibody (lower) were used to probe in Western blot analysis a protein extract of adult S. japonicum incubated for 30 min with: rSjILP (Lane 1); both rSjILP and human insulin (Lane 2); human insulin (Lane 3); neither rSjILP nor insulin (Lane 4). b Signal intensities of bands recognised by anti-phospho p44/42 MAPK (Erk) antibody in panel A (lower), were measured using the Odyssey Classic Infrared Imager with a scan intensity setting of 5 and sensitivity of 5. Error bars represent the standard error of the mean (SEM). This experiment was performed twice
Mentions: An anti-phospho p44/42 MAPK antibody was used to detect the phosphorylation of Erk in adult S. japonicum stimulated by host insulin or rSjILP. An Erk band, approximately the same size (44 kDa) as observed previously in S. mansoni [37], was recognised by the anti-phospho antibody in protein extracts of adult S. japonicum worms incubated with or without rSjILP and insulin (Fig. 3). A 44 kDa Erk band was recognised in extracts prepared from worms stimulated with rSjILP and from worms co-stimulated with rSjILP and insulin, with no significant difference evident in band intensity between the two extracts (Fig. 3). The Erk band was also detectable in an extract from worms incubated with human insulin only but, compared with that of the worms co-stimulated with rSjILP and insulin, its intensity was reduced by 58% [t(6) = 31.39, P = 0.001] (Fig. 3). The Erk band was detectable in an extract of worms incubated without human insulin and rSjILP, but its intensity was reduced by 30% [t(6) = 4.353, P = 0.049] compared with that in the worm extract incubated with insulin only (Fig. 3) (*P ≤ 0.05, **P ≤ 0.001, ***P ≤ 0.0001).Fig. 3

View Article: PubMed Central - PubMed

ABSTRACT

Background: Previous studies have shown that insulin receptors in schistosomes, triggered by host insulin, play an important role in parasite growth, development and fecundity by regulating glucose metabolism. However, limited information is available on the recently identified endogenous insulin-like peptide (ILP) in blood flukes.

Results: We isolated ILPs from Schistosoma japonicum (SjILP) and S. recognised (SmILP) and present results of their molecular and structural analysis. SjILP shares 63% amino acid identity with SmILP, but only 18% identity with human insulin. There is high cross immunological reactivity between the S. japonicum and S. mansoni ILPs as observed in western blots using an anti-SjILP polyclonal antibody. ADP binding/hydrolysis ability was observed in both SjILP and SmILP, but not in human insulin, suggesting a parasite-specific role for ILP compared with host insulin. Protein binding assays using the Octet-RED system showed SjILP binds S. japonicum IRs (SjIR1 and SjIR2) strongly. An anti-phospho antibody against extracellular signal-regulated kinase (Erk) recognised a 44-kDa target band in an extract of adult worms after stimulation by rSjILP in vitro, suggesting an important role for SjILP in activating SjIRs and in regulating downstream signal transduction. Immunolocalisation showed SjILP is located on the tegument and the underlying musculature, similar to that observed for SjIR1, but it is also present throughout the parenchyma of males and in the vitelline cells of females, the same locations as SjIR2 described in an earlier published study of ours. The same localisation of SjILP and the SjIRs is suggestive of an interaction between the insulin-like peptide and the IRs. In addition to binding host insulin, schistosomes also can express their own endogenous ILPs, which can activate the parasite insulin signal pathway, thereby playing a critical role in worm growth, development and fertility.

Conclusions: These findings shed new light on ILPs in schistosomes, providing further insight into the distinct and specialised functions of SjIR1 and 2 in S. japonicum and their interaction with host insulin.

Electronic supplementary material: The online version of this article (doi:10.1186/s13071-017-2095-7) contains supplementary material, which is available to authorized users.

No MeSH data available.