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.

Xing-Rong Guo,Qi-Li Feng,Lin Liu,Si-Chun Zheng

doi:10.1186/1471-2199-10-102

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.

Highlights

Sterol carrier protein-2/3-oxoacyl-CoA thiolase (SCPx) gene has been suggested to be involved in absorption and transport of cholesterol
To confirm and obtain the SlSCPx full-length cDNA directly from the S. litura midgut, reverse-transcription PCR (RT-PCR) was performed using a pair of primers designed on the basis of the cloned SlSCPx cDNA sequence (Fig. 1 and 2A) and mRNA extracted from the midgut of 3-day-old 6th instar larvae
CPx-t antibody detected a major 44-kDa protein and a weak 58-kDa protein, which were closely equivalent to the predicted molecular mass of the SlSCPx-t and full-length SlSCPx proteins, respectively and as observed for bacterially expressed equivalents (Fig. 5E). No such proteins were detected in the fat body and epidermis, where another small unidentified protein immunologically reacted with the anti-SlSCPx-t antibody. These results indicated either that SlSCPx was post-translationally and proteolytically cleaved into two smaller proteins SlSCPx-t and SlSCPx-2, or that the transcription of the SlSCPx gene was initiated at two different transcription initiation sites, generating a full-length SlSCPx mRNA, which was translated into a full-length protein that was proteolytically cleaved into SlSCPx-t and SlSCPx-2 proteins post translationally, and a SlSCPx-2 mRNA that was translated into a SlSCPx-2 protein

Summary

Introduction

Sterol carrier protein-2/3-oxoacyl-CoA thiolase (SCPx) gene has been suggested to be involved in absorption and transport of cholesterol. Sterol carrier protein 2/3-oxoacyl-CoA thiolase (SCPx) belongs to a well-characterized SCP-2 gene family [1], whose members encode an intracellular non-specific lipid carrier protein. Cholesterol is required for cellular membranes and ecdysteroid biosynthesis. The SCPx-t protein functions as a 3oxoacyl-CoA thiolase in peroxisomal oxidation of branched-chain fatty acids [13]. The SCP-2 protein is released from the peroxisomes into the cytoplasm and translocated into the nucleus, where it acts as a transcription factor [14]. In the lepidopteran insects Bombyx mori and Spodoptera littoralis, the SCPx gene encodes two fused SCPx-t and SCP-2 domains [21,22]

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