Que Lan

The Structural Determination of an Insect Sterol Carrier Protein-2 with a Ligand-bound C16 Fatty Acid at 1.35-Å Resolution

Yellow fever mosquito sterol carrier protein (SCP-2) is known to bind to cholesterol. We report here the three-dimensional structure of the complex of SCP-2 from Aedes aegypti with a C16 fatty acid to 1.35-Å resolution. The protein fold is exceedingly similar to the human and rabbit proteins, which consist of a five-stranded β-sheet that exhibits strand order 3-2-1-4-5 with an accompanying layer of four α-helices that cover the β-sheet. A large cavity exists at the interface of the layer α-helices and the β-sheet, which serves as the fatty acid binding site. The carboxylate moiety of the fatty acid is coordinated by a short loop that connects the first α-helix to the first β-strand, whereas the acyl chain extends deep into the interior of the protein. Interestingly, the orientation of the fatty acid is opposite to the observed orientation for Triton X-100 in the SCP-2-like domain from the peroxisomal multifunctional enzyme (Haapalainen, A. M., van Aalten, D. M., Merilainen, G., Jalonen, J. E., Pirila, P., Wierenga, R. K., Hiltunen, J. K., and Glumoff, T. (2001) J. Mol. Biol. 313, 1127-1138). The present study suggests that the binding pocket in the SCP-2 family of proteins may exhibit conformational flexibility to allow coordination of a variety of lipids.

Isolation and expression of a sterol carrier protein‐2 gene from the yellow fever mosquito, Aedes aegypti

Trafficking of cholesterol in insects is a very important process due to the fact that insects depend on dietary cholesterol to fulfil their physiological needs. We identified a putative mosquito sterol carrier protein‐2 (SCP‐2) cDNA from fourth instar subtracted cDNA library. The AeSCP‐2 protein has high degree homology in the sterol transfer domain to both rat and human SCP‐2. Transcripts of AeSCP‐2 in fourth instars were detected strongly in the midgut, and weakly in the head and hindgut. In the early pupae, AeSCP‐2 transcription was observed in the thorax, head and body wall of abdomen, but not in the gut.

Functional analysis of AeSCP‐2 using gene expression knockdown in the yellow fever mosquito, Aedes aegypti

The effect of gene expression knockdown was used to study the function of the sterol carrier protein‐2 (AeSCP‐2) in the yellow fever mosquito, Aedes aegypti. Injection of small double stranded AeSCP‐2 RNAs into mosquito larvae resulted in the knockdown of gene products. The lack of AeSCP‐2 in larvae coincided with a reduction in accumulated cholesterol in pupae, supporting the hypothesis that AeSCP‐2 may be involved in cholesterol uptake in mosquito larvae. Knockdown of AeSCP‐2 caused a high mortality rate in developing adult and reduced egg viability. Results from this study indicate that AeSCP‐2 is important for adult development and for the viability of the eggs.

The Biological Activity of α-Mangostin, a Larvicidal Botanic Mosquito Sterol Carrier Protein-2 Inhibitor

ABSTRACT &agr;-Mangostin derived from mangosteen was identified as a mosquito sterol carrier protein-2 inhibitor via high throughput insecticide screening. &agr;-Mangostin was tested for its larvicidal activity against third instar larvae of six mosquito species, and the median lethal concentration values range from 0.84 to 2.90 ppm. The residual larvicidal activity of &agr;-mangostin was examined under semifield conditions. The results indicated that &agr;-mangostin was photolytic with a half-life of 53 min in water under full sunlight exposure. The effect of &agr;-mangostin on activities of major detoxification enzymes such as P450, glutathione S-transferase, and esterase was investigated. The results showed that &agr;-mangostin significantly elevated activities of P450 and glutathione S-transferase in larvae, whereas it suppressed esterase activity. Toxicity of &agr;-mangostin against young rats was studied, and there was no detectable adverse effect at dosages as high as 80 mg/kg. This is the first multifaceted study of the biological activity of &agr;-mangostin in mosquitoes. The results suggest that &agr;-mangostin may be a lead compound for the development of a new organically based mosquito larvicide.

Three-dimensional structure/function analysis of SCP-2-like2 reveals differences among SCP-2 family members

Mosquito sterol carrier protein-2 (AeSCP-2) and sterol carrier protein-2-like2 (AeSCP-2L2) are members of the SCP-2 protein family with similar expression profiles in the mosquito life cycle. In an effort to understand how lipids can be transported by different SCP-2 proteins, the three-dimensional crystal structure of AeSCP-2L2 was solved at 1.7 Å resolution. AeSCP-2L2 forms a dimer and binds three fatty acids, one of which resides in a position within the internal cavity at a right angle to the others. This first report of ligand-bound dimerized protein in the SCP-2 protein family indicates that the family has a much more divergent mode of interaction with ligands than previously reported. The potential function of AeSCP-2L2 was investigated via in vivo incorporation of [3H]cholesterol and [3H]palmitic acid. Overexpression of AeSCP-2L2 in mosquito cells leads to an increased uptake of free fatty acid, whereas knockdown of AeSCP-2L2 in adult females decreases the accumulation of free fatty acid in the fat body from a blood meal. In contrast, overexpression or knockdown of AeSCP-2L2 has no effect on cholesterol uptake. Our results suggest that the main function of AeSCP-2L2 is as a general intracellular fatty acid carrier, as opposed to having a dedicated role in cholesterol transport.

Expression of a sterol carrier protein-x gene in the Yellow fever mosquito, Aedes aegypti

The sterol carrier protein‐x (SCP‐x), a peroxisomal thiolase/nonspecific lipid binding protein, was characterized in the yellow fever mosquito, Aedes aegypti. The Aedes aegypti SCP‐x (AeSCP‐x) has 83% and 75% similarities to Drosophila and mammalian SCP‐x, respectively. However, the AeSCP‐x gene did not produce multiple transcripts, which is characteristic of the vertebrate SCP‐x gene. Levels of AeSCP‐x transcription were higher in larvae and pupae. Gut tissue showed the highest level of AeSCP‐x mRNA in larvae. In adults, low levels of AeSCP‐x transcription were detected in both sexes. Polyclonal antibodies against the sterol carrier protein‐2 (SCP‐2) domain of AeSCP‐x detected two proteins of 62 kDa and 13 kDa. The results indicate that AeSCP‐x is proteolytically cleaved after translation to produce a smaller protein that contains only the SCP‐2 domain, which is similar to post‐translational modification of the vertebrates SCP‐x to produce multiple products.

Use of subtracted libraries and macroarray to isolate developmentally specific genes from the mosquito, Aedes aegypti

Subtracted cDNA libraries were screened with cDNA macroarrays to isolate larval and pupal stage-specific genes from Aedes aegypti. Of 103 partial cDNAs sequenced from the 4th instar subtracted cDNA library, 62 have counterpart genes in other organisms while 41 of them have no significant similarity to any known genes. Sequences of 116 partial cDNA clones from the pupal subtracted library revealed that 57 belong to unknown genes and 59 have homologous genes in other organisms. Results of cDNA macroarrays showed that 42-50% of randomly selected genes in the subtracted cDNA libraries were differentially expressed. Of the unknown genes, transcripts of 15-19% of the genes were detected in larval or pupal stages, respectively. The results indicate that a subtracted cDNA library in combination with a cDNA macroarray can be used effectively to identify genes expressed in a particular stage.

Larvicidal Activity of Sterol Carrier Protein-2 Inhibitor in Four Species of Mosquitoes

A previous report has shown that mosquito sterol carrier protein-2 inhibitors (SCPIs) are larvicidal to larvae of the yellowfever mosquito, Aedes aegypti (L.) (J. Lipid Res. 46: 650-657, 2005). In the current study, we tested SCPI-1 in an additional four mosquito species for larvicidal activities: Culex pipiens pipiens, Anopheles gambiae, Culex restuans, and Aedes vexans. Cholesterol accumulation in SCPI-treated Ae. aegypti fourth instars was examined. SCPI-1 is lethal to all tested mosquito species, with the LC50 value ranging from 5.2 to 15 microM when treatments started at the first to third instar. However, LC50 values increase to from 5.2 to 38.7 microM in treatments started at first and fourth instar, respectively. The results indicate that the lethal effect of SCPI-1 decreases with the growth of larvae, which suggests that SCPI-1 is more effective before the larvae reach final growth period (the last instar). SCPI-1 suppressed cholesterol uptake in Ae. aegypti fourth instars, suggesting that one of the modes of action of SCPI-1 is via reduction in cholesterol absorption.

Effects of mutations in Aedes aegypti sterol carrier protein-2 on the biological function of the protein.

Sterol carrier protein-2 (SCP-2) is a nonspecific intracellular lipid carrier protein. However, the molecular mechanism of ligand selectivity and the in vivo function of SCP-2 remain unclear. In this study, we used site-directed mutagenesis to investigate the ligand selectivity and in vivo function of the yellow fever mosquito sterol carrier protein-2 protein (AeSCP-2). Mutations to amino acids in AeSCP-2 known to interact with bound ligand also weakened NBD-cholesterol binding. Substitution of amino acids in the ligand cavity changed the ligand specificity of mutant AeSCP-2. Overexpressing wild-type AeSCP-2 in the Aedes aegypti cultured Aag-2 cells resulted in an increase in the level of incorporation of [(3)H]cholesterol. However, overexpressing mutants that were deleterious to the binding of NBD-cholesterol in AeSCP-2 showed a loss of ability to enhance uptake of [(3)H]cholesterol in cultured cells. Interestingly, when [(3)H]palmitic acid was used as the substrate for incorporation in vivo, there was no change in the levels of incorporation with overexpression of wild-type protein or mutated AeSCP-2s. The in vivo data suggest that AeSCP-2 is involved in sterol uptake, but not fatty acid uptake. This is the first report that the cholesterol binding ability may directly correlate with AeSCP-2s in vivo function in aiding the uptake of cholesterol.

Identification of two sterol carrier protein-2 like genes in the yellow fever mosquito, Aedes aegypti

Two genes encoding sterol carrier protein‐2 like proteins are identified from fourth instar cDNAs of the yellow fever mosquito, Aedes aegypti. The predicted AeSCP‐2like1 (AeSCP‐2L1) and AeSCP‐2like2 (AeSCP‐2L2) proteins are small, acidic and lacking the peroxisomal targeting sequence at the C‐termini. Purified recombinant AeSCP‐2L1 and ‐2L2 bind to cholesterol with a Kd of 5.4 × 10−6 M and 2.6 × 10−6 M, respectively. The Kd values of AeSCP‐2L1 and ‐2L2 to palmitic acid are 3.7 × 10−7 M and 2.6 × 10−7 M, respectively. Both genes are expressed predominantly in gut tissues. The transcripts of the AeSCP‐2L1 gene are only detected in larval stages, whereas AeSCP‐2L2 is expressed in larval and adult stages. AeSCP‐2L2 transcription increases within 5 h after a bloodmeal and stays at high levels during vitellogenesis. In in vitro larval gut tissue cultures, AeSCP‐2L1 transcripts were increased in the presence of juvenile hormone III, whereas AeSCP‐2L2 mRNA levels increased in the presence 20‐hydroxylecdysone. The results suggest that transcription of AeSCP‐2L1 and ‐2L2 genes are regulated differently through the mosquito life cycle.