The sterol carrier protein 2/3-oxoacyl-CoA thiolase (SCPx) is involved in cholesterol uptake in the midgut of Spodoptera litura: gene cloning, expression, localization and functional analyses

Abstract

BackgroundSterol carrier protein-2/3-oxoacyl-CoA thiolase (SCPx) gene has been suggested to be involved in absorption and transport of cholesterol. Cholesterol is a membrane component and is a precursor of ecdysteroids, but cannot be synthesized de novo in insects. However, a direct association between SCPx gene expression, cholesterol absorption and development in lepidopteran insects remains to be experimentally demonstrated.ResultsAn SCPx cDNA (SlSCPx) cloned from the common cutworm, Spodoptera litura, was characterized. The SlSCPx cDNA encoded a 535-amino acid protein consisting of a 3-oxoacyl-CoA thiolase (SCPx-t) domain and a SCP-2 (SCPx-2) domain. SlSCPx mRNA was expressed predominately in the midgut, while SlSCPx-2 mRNA was detected in the midgut, fat body and epidermis and no SlSCPx-t mRNA was detected. A 58-kDa full-length SCPx protein and a 44-kDa SCPx-t protein were detected in the midgut of sixth instar larvae when the anti-SlSCPx-t antibody was used in western blotting analysis; a 16-kDa SCP-2 protein was detected when anti-SlSCPx-2 antibody was used. SlSCPx protein was post-translationally cleaved into two smaller proteins, SCPx-t and SCPx-2. The gene appeared to be expressed into two forms of mRNA transcripts, which were translated into the two proteins, respectively. SlSCPx-t and SlSCPx-2 proteins have distinct and different locations in the midgut of sixth instar larvae. SlSCPx and SlSCPx-t proteins were detected predominately in the cytoplasm, whereas SlSCPx-2 protein was detected in the cytoplasm and nuclei in the Spli-221 cells. Over-expression of SlSCPx and SlSCPx-2 proteins enhanced cholesterol uptake into the Spli-221 cells. Knocking-down SlSCPx transcripts by dsRNA interference resulted in a decrease in cholesterol level in the hemolymph and delayed the larval to pupal transition.ConclusionSpatial and temporal expression pattern of this SlSCPx gene during the larval developmental stages of S. litura showed its specific association with the midgut at the feeding stage. Over-expression of this gene increased cholesterol uptake and interference of its transcript decreased cholesterol uptake and delayed the larval to pupal metamorphosis. All of these results taken together suggest that this midgut-specific SlSCPx gene is important for cholesterol uptake and normal development in S. litura.

Characterization of the sterol carrier protein-x/sterol carrier protein-2 gene in the cotton bollworm, Helicoverpa armigera

“…The dsRNA-treated H. armigera larvae were smaller in the body size (Fig. 6B), which is consistent with the observations in SlSCP-x dsRNA-treated S. litura larvae (Guo et al, 2009). In the 4th instar treatment group, the trend of lowered pupal weight was not significant, which may be due to decreasing in the effectiveness of the dsRNA with the rapid growth of the larvae from 4th through 5th stadium.…”

“…4) suggests that either HaS-CP-x was rapidly processed post-translationally or HaSCP-2 expression is transcribed using an alternative promoter. The insect SCP-x protein appears to be processed post-translationally to produce 2/ 3-oxoacyl-CoA thiolase and the SCP-2 (Takeuchi et al, 2004;Guo et al, 2009). Therefore, regardless of which transcript of the SCPx/SCP-2 gene is predominant in the midgut; high levels of SCP-2 domain protein may be the end result (Fig.…”

“…In S. littoralis, a single SCPx/SCP-2 gene is mainly transcribed as a fusion mRNA and the translated product is posttranslationally cleaved into two proteins: 3-oxoacyl-CoA thiolase and SCP-2 (Takeuchi et al, 2004). S. litura SlSCP-x gene produces an mRNA transcript that contains both a SlSCPx-t domain and a SlSCPx-2 domain for a SCP-x protein, and a SlSCP-2 mRNA transcript for the SlSCP-2 protein by splicing (Guo et al, 2009). In the B. mori and M. sexta, there is evidence of alternative promoters for the transcription of either full length SCP-x or the shorter transcript for SCP-2 (Gong et al, 2006;Kim and Lan, 2010).…”

“…The AeSCP-x gene produces a single transcript encoding SCP-x, but does not encode the AeSCP-2 mRNA (Lan and Wessley, 2004). In lepidopteran insects, such as Spodoptera littoralis (Takeuchi et al, 2004), Spodoptera litura (Guo et al, 2009), Bombyx mori (Gong et al, 2006) and Manduca sexta (Kim and Lan, 2010) the homologous gene encoding SCP-x/SCP-2 has been reported. A single SCP-x/SCP-2 gene encodes both lepidepteran SCPx and SCP-2 mRNAs, which is similar to that of vertebrates rather than that of dipterans (Lan and Wessley, 2004).…”

Immuno-histochemical localization of cholesterol binding proteins in Schistocerca gregaria (Forskal)

“…These sterol carrier proteins are also characterized in different insects like cotton bollworm, Helicoverpa armigera (Du et al, 2012), caterpillar of Helicoverpa zea. (Jing et al, 2012), Spodoptera litura (Guo et al, 2009), and Manduca sexta and immunocytochemically localized in insect tissues (Dyer et al, 2003;Horton and Shimomura, 1999). It suggests that CBPs are very similar to SCPs and FABPs in function and might have belong to family of sterol carrier or lipid carrier proteins which are evolved in different insect groups during long evolution.…”