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Proc Natl Acad Sci U S A. Aug 6, 2002; 99(16): 10581–10586.
Published online Jul 22, 2002. doi:  10.1073/pnas.162182899
PMCID: PMC124981
Genetics

A class B scavenger receptor mediates the cellular uptake of carotenoids in Drosophila

Abstract

Carotenoids are currently being intensely investigated regarding their potential to lower the risk of chronic disease and vitamin A deficiency. Invertebrate models in which vitamin A deficiency is not lethal allow the isolation of blind but viable mutants affected in the pathway leading from dietary carotenoids to vitamin A. Using a mutant in one of these model systems, Drosophila, the vitamin A-forming enzyme has recently been molecularly identified. We now show that the molecular basis for the blindness of a different Drosophila mutant, ninaD, is a defect in the cellular uptake of carotenoids. The ninaD gene encodes a class B scavenger receptor essential for the formation of the visual chromophore. A loss of this function results in a carotenoid-free and thus vitamin A-deficient phenotype. Our investigations provide molecular insight into how carotenoids may be distributed into cells of target tissues in animals and indicate a crucial role of class B scavenger receptors rendering dietary carotenoids available for subsequent cell metabolism, as needed for their various physiological functions.

Carotenoids are C40 isoprenoids synthesized in plants, certain fungi and bacteria with characteristic molecular structures and properties responsible for light absorption as well as for the inactivation of aggressive radicals (reviewed in ref. 1). Among the various classes of pigments found in nature, the diverse family of yellow to red-colored carotenoids is the most widespread, with important functions not only in carotenoid-producing organisms. Some animals use dietary carotenoids for coloration. Well known examples are the feathers of flamingos and the red color of salmon. Because of their antioxidative properties, beneficial effects have also been reported for carotenoids in the prevention of coronary heart diseases, certain kinds of cancer, and age-related macular degeneration in humans (reviewed in ref. 2).

Most important, certain carotenoids are the precursors (provitamins) for the formation of vitamin A in animals. This vitamin is needed for vision in the entire animal kingdom. The visual pigments (rhodopsins) of animals are composed of a retinoid chromophore (vitamin A derivative) bound to a protein moiety (opsin) embedded in the photoreceptor membranes (3, 4). Light activation of the visual pigments triggers a G protein-coupled receptor cascade leading to changes in the permeability of the photoreceptor cell membranes. Besides being crucial for vision, in vertebrates vitamin A is also important in development and cellular differentiation processes. Here, the vitamin A derivative retinoic acid, together with its nuclear receptors, is involved in the regulation of diverse target genes; consequently, complete vitamin A deficiency leads to early embryonic death (5).

To become biologically active, dietary carotenoids must first be absorbed, then delivered to the site of action in the body and, in the case of provitamin A function, metabolically converted. Despite the general importance of carotenoids in animals, their metabolism is still poorly understood (6). Invertebrates like Drosophila represent excellent models for the genetic dissection of the pathway leading from dietary carotenoids to vitamin A. Here, this vitamin is only needed for vision; therefore, its deficiency has no fatal consequences. Among the various Drosophila mutants affected in their visual performance (4), the two mutants ninaB and ninaD lack the visual chromophore of the fly, 3-hydroxyretinal, when raised on standard media with carotenoids as the sole source for vitamin A formation (7). By analyzing the molecular basis of the blindness of ninaB mutants, we already showed that the phenotype is caused by mutations in a gene coding a carotene-15,15′-oxygenase and molecularly identified the key enzyme for carotenoid conversion to vitamin A in animals (8, 9). By sequence identity, orthologs to this insect gene were cloned from several vertebrate species including man, showing that the enzymes catalyzing vitamin A formation are evolutionarily well conserved (10–13). In Drosophila, mRNA expression of ninaB was exclusively found in the head, in agreement with retinoids being restricted in their distribution to the eyes (8, 14). In vertebrates (with vitamin A needed also for cellular differentiation processes), the vitamin A-forming enzyme is expressed in a variety of different tissues including reproductive tissues and the eyes (10, 12, 13). After dietary absorption, carotenoids must be distributed to these tissues to be converted to vitamin A.

In the second chromophore-less Drosophila mutant, ninaD, the carotenoid content was shown to be specifically and significantly altered compared with wild-type (wt) flies and was ineffective at mediating visual pigment synthesis (14). This phenotype is presumably caused by a defect in the body distribution of dietary carotenoids and makes the ninaD gene an interesting candidate for a molecular player necessary for these transport processes.

Here, we report on the identification of the ninaD gene encoding a scavenger receptor with significant sequence identity to the mammalian class B scavenger receptors, SR-BI and CD36. In ninaDP245 flies, there is a nonsense mutation in the gene coding this receptor, thus abolishing its function. By P element-mediated transformation with a wt ninaD allele by using the UAS/GAL4 system, we could rescue the blind phenotype of ninaDP245 flies. Heat shock (hs)-induced wt transgene expression resulted in carotenoid accumulation as judged by quantitative HPLC analyses and subsequent restoration of visual pigments. The identification of this scavenger receptor as being essential for the cellular uptake of carotenoids in Drosophila promises to elucidate new aspects of class B scavenger receptor functions with respect to carotenoid and vitamin A metabolism also in mammals.

Materials and Methods

Drosophila Strains, Construction of Transgenic Flies, Crosses, and Heat Shock Treatment.

Fly strains were raised on standard corn medium at 22°C. The ninaDP245 strain had the genotype w; ninaDP245/CyO; +/+. As wt control, yellow white flies were used. To obtain a UAS-ninaD(wt) fly strain, we cloned the long splicing variant of the wild-type ninaD cDNA into the vector pUAST and transformed yellow white flies with the resulting plasmid (15). By crosses of the resulting transgenic flies with ninaDP245 and balancer flies, we maintained flies with the genotype w;ninaDP245/CyO;UAS-ninaD(wt)/UAS-ninaD(wt). For heat shock rescue, we crossed these flies with w;ninaDP245/CyO;hs-GAL4/hs-GAL4 flies and obtained w;ninaDP245/ninaDP245;hs-GAL4/UAS-ninaD(wt) flies. As controls, we used w;ninaDP245/ninaDP245;hs-GAL4/+ and w;ninaDP245/ninaDP245;UAS-ninaD(wt)/+ flies and w;ninaDP245/CyO;+/+ flies, respectively. Heat shock was for 1 h at 37°C. The flies were analyzed 72 h later. For the dietary rescues of the ninaDP245 flies, the corn medium (total carotenoid content 1 pmol/mg) was supplemented with retinal (100 pmol/mg corn medium) or carotenoids (100 pmol/mg corn medium), both purchased from Sigma. To place the UAS-ninaD(wt) transgene under the control of the ninaE-GAL4 driver, we crossed ninaE-GAL4 flies having the genotype w;ninaE-GAL4/ninaE-GAL4;TM2/MKRS with UAS-ninaD(wt) flies of the genotype w;CyO/wgSp-1;UAS-ninaD(wt)/UAS-ninaD(wt) and analyzed the offspring.

PCR Analyses To Test for a Mutation in CG5750.

To test for a mutation in the genomic sequence of CG5750 in ninaDP245 flies, we isolated genomic DNA from 20 flies and performed PCR analyses using Taq polymerase (Amersham Pharmacia) and the following sets of oligonucleotide primers: 5′-CCAAACCGAGCTGATTACCAC-3′ and 5′-CCCAGAACCAAGTTCTTCTG-3′, 5′-GAAGATGGATCTTGAGTGGC-3′ and 5′-CCTTCAACGGCACTCCCATC-3′, 5′-CAACCGGAGCCACTGACCTAG-3′ and 5′-GATAAAAGAGTTGGGAGCCAG-3′, 5′-GCCTCAGCCTGGCCAGCC-3′ and 5′-CGGGCACTCGCGATTCTTG-3′, 5′-AAGGCGGCGCCACCCATATG-3′ and 5′-GGTTTTGGAGACTGGGCAG-3′. For the determination of the DNA sequences, the PCR products were directly sequenced.

Reverse Transcription (RT)-PCR and PCR Analyses To Check for Fusion of Two Genes.

Reverse transcription was performed with mRNA isolated from total RNA of 20 wt flies by using an Oligotex mRNA mini kit (Qiagen, Valencia, CA), an oligo(dT)17 primer, and Superscript reverse transcriptase (Invitrogen). PCR analyses were performed with the resulting first strain cDNA, and genomic DNA were isolated from 20 wt flies, respectively. The following sets of oligonucleotide primers were used: 5′-CGAGTCGGATCACTTTGCCTG-3′ and 5′-CGGGCACTCGCGATTCTTG-3′; 5′-CGAGTCGGATCACTTTGCCTG-3′ and 5′-CCTTCAACGGCACTCCCATC-3′; and 5′-AAGGCGGCGCCACCCATATG-3′ and 5′-CGGGCACTCGCGATTCTTG-3′. PCR conditions were 94°C for 2 min, 32 cycles of 94°C for 1 min, 57°C for 1 min, 72°C for 80 s, and finally 72°C for 10 min.

3′- and 5′-Rapid Amplification of cDNA Ends (RACE)-PCR.

RACE-PCRs (3′ and 5′) were performed with mRNA of wt flies by using a FirstChoice RLM-RACE kit (Ambion, Austin, TX). The following gene-specific primers were used: 5′-GAAGATGGATCTTGAGTGGC-3′ and 5′-CAACCGGAGCCACTGACCTAG-3′ for the 3′-RACE and 5′-GAATCACATCCGTGTGCTGCTC-3′ and 5′-CGGCCTGGCCATACCACTTC-3′ for the 5′-RACE. PCR conditions were 94°C for 2 min, 32 cycles of 94°C for 1 min, 58°C for 1 min, 72°C for 100 s, and finally 72°C for 10 min. PCR products were subcloned into the pCR2.1-TOPO vector (Invitrogen) and sequenced.

Northern Blot Analyses.

Northern Blot analyses were carried out according to the DIG Application Manual for Filter Hybridization (Roche Diagnostics). To obtain the riboprobe, PCR was performed using the oligonucleotide primers 5′-GAAGATGGATCTTGAGTGGC-3′ and 5′-CCTTCAACGGCACTCCCATC-3. The PCR product was subcloned into the vector pCR2.1-TOPO (Invitrogen), and the antisense riboprobe was synthesized with T7 RNA polymerase by using a DIG-RNA labeling kit (Roche Diagnostics). RNA (4 μg total) and mRNA corresponding to 4 μg of total RNA, respectively, were used per lane of the 1.2% formaldehyde gel.

Analyses of the Major Rhodopsin of Flies.

Proteins from three fly heads were extracted in 6% β-mercaptoethanol/6% SDS/20% glycerol/0.6% bromphenol blue, separated by SDS/PAGE on a 12% gel, and transferred onto a nitrocellulose membrane (Amersham Pharmacia). Rh1 was determined using a polyclonal antibody (16) and the enhanced chemiluminescence system (Amersham Pharmacia).

Extraction of Carotenoids and HPLC Analyses.

Flies were homogenized in 200 μl of 2M NH2OH with a loose fitting potter. Then 400 μl of methanol and 600 μl of acetone were added. Lipophilic compounds were extracted three times with 500 μl of petroleum ether. The collected organic phases were dried under a stream of N2 and dissolved in the HPLC solvent (diethyl ether/n-hexane/ethanol, 79/20/1). HPLC systems were as described (10).

Lipid Analyses and GC/MS Analyses.

Lipid analyses were carried out using Merckotest 3321 Total Lipids (Merck). For each experiment, 70 flies were homogenized in 0.9% NaCl solution with a loose fitting potter. For GC/MS analyses, 10 flies (5 female and 5 male flies) were homogenized in 500 μl of 50 mM tricine/100 mM NaCl, pH 7.5, and extracted three times with 500 μl of chloroform/methanol. After saponification and silanization, fatty acids were subjected to GC/MS analyses using the conditions described (10).

In Situ Hybridization of Embryos.

Embryo dechorionation, devitellination, and fixation was carried out according to ref. 17. In situ hybridization was performed as described (18). The riboprobe was the same as used in Northern blot analyses. Staging of embryos was performed according to ref. 19.

Results

Characterization of the Phenotype of ninaDP245 Flies.

Because the carotenoid composition of ninaDP245 flies had yet not been analyzed, we first addressed this question. We raised a heterozygous, balanced ninaDP245 stock on standard corn medium containing zeaxanthin, lutein, cryptoxanthin, and β-carotene with a molar composition of approximately 9:4:2:1 (all of which exert provitamin A activity in flies) and collected homozygous ninaDP245 flies. As determined by HPLC analyses, the carotenoid contents of both the trunks and heads of adult homozygous ninaDP245 flies were highly reduced compared with controls (Fig. (Fig.11 A and B). To judge more directly the visual pigment content of ninaDP245 flies, we determined the amount and maturation status of the major rhodopsin Rh1 of the flies by Western blot analysis. It has been previously shown that in ninaDP246 flies, the major opsin Rh1 is only found in its immature glycosylated form, most probably because of the absence of the visual chromophore (20). As expected, in contrast to controls, Rh1 also existed only in its immature form in ninaDP245 flies (Fig. (Fig.11C). Next, in rescue experiments we supplemented the corn medium with vitamin A (retinal) and with additional β-carotene and zeaxanthin and measured Rh1 maturation. Feeding ninaDP245 flies retinal led to Rh1 maturation, showing that preformed vitamin A can compensate the blockade in carotenoid utilization. Additionally, supplementation with very high amounts of β-carotene but not zeaxanthin resulted in Rh1 maturation. Thus, the ninaDP245 mutation seems to differentially affect the utilization of βcarotene and zeaxanthin. As shown above, normal corn medium already contained significant amounts of β-carotene. Hence, Rh1 maturation upon feeding additional β-carotene may be explainable by a passive diffusion when very high amounts of this pure hydrocarbon are available. In conclusion, these analyses indicated that the ninaD mutation interferes with the absorption and body distribution of carotenoids and therefore resulted in vitamin A deficiency.

Fig 1.
The carotenoid and visual pigment content of ninaDP245 flies is drastically reduced. (A) HPLC analyses of the lipohilic compounds of the fly's head are shown. The two major carotenoids were lutein and zeaxanthin. The upper lane gives the HPLC profile ...

Identification of the ninaD Gene.

The ninaD mutation has been cytologically mapped to the genomic position 36E-F on chromosome 2 close to the fly's arrestin1 gene (arr1) (7). The Arr1 protein is involved in the regeneration of meta-rhodopsin in the visual cycle, but it could be shown that this protein is not defective in ninaD flies (21). Therefore, we searched the genomic region next to arr1 and focused our attention on the predicted gene CG5750 (Flybase). The deduced amino acid sequence of CG5750 shares significant sequence similarity to the mammalian class B scavenger receptors CD36 and SR-BI (22). SR-BI mediates bidirectional flux of cholesterol from lipoproteins (high and low density lipoproteins) to target cells in mammals (23, 24). Because carotenoids are isoprenoids like cholesterol, known to be transported in insects by lipophorins, which are structurally related to the mammalian lipoprotein classes (25), we wondered whether a mutation in this gene might be the cause for the blockade in carotenoid utilization of ninaDP245 flies. To test this possibility, we designed several sets of oligonucleotide primers overlapping the entire genomic sequence of CG5750 and performed PCR on genomic DNA isolated from ninaDP245 flies. By direct sequencing of the PCR products, we found a nonsense mutation in the deduced coding region (Fig. (Fig.22A). Thus, we supposed that CG5750 encodes the ninaD gene.

Fig 2.
A nonsense mutation is found in the two splicing variants of the ninaD gene. (A) We performed PCR analyses with genomic DNA from homozygous ninaDP245 and wt flies by using several sets of oligonucleotide primers overlapping the entire genomic region of ...

Molecular Structure of the Putative ninaD Gene.

By computer prediction, CG5750 encodes a protein 861 aa in length (Flybase), approximately double the size of the known type B scavenger receptors. On closer inspection, it turned out that the amino acid sequence consists of two recurring parts, both sharing significant overall similarity to class B scavenger receptors. The most plausible explanation seemed to be that the algorithm of the computer program incorrectly fused the coding regions of two individual genes. This hypothesis could be confirmed by RT-PCR analysis, by which no product was obtained with a pair of primers spanning the predicted coding region (Fig. (Fig.22B). To analyze the correct molecular structure and to clone the cDNA of the putative ninaD gene defined by the nonsense mutation, we performed 3′- and 5′-RACE-PCR experiments. We obtained two different products for the 3′-end (Fig. (Fig.22C) and a single product for the 5′-end. Sequencing of the RACE-PCR products revealed the existence of two different splicing variants for the 3′-end. By a Northern blot we confirmed this result (Fig. (Fig.22 D and E). This analysis also showed that the mRNA levels of the putative ninaD gene were significantly reduced in heterozygous ninaDP245 flies and were hardly detectable in homozygous ninaDP245 flies. This decrease is probably caused by rapid degradation of the mRNA of the ninaDP245 allele due to the nonsense mutation, as previously reported for mutated genes (26). The longer mRNA of the putative ninaD gene was 1,736 nucleotides long, coding a protein of 513 aa residues; the second mRNA lacks the last two exons, thus coding for an identical but C-terminally shortened protein of 415 residues. The deduced amino acid sequences shared overall similarities to the Drosophila CD36-like scavenger receptor proteins croquemort and emp (27, 28) and to mammalian class B scavenger receptors. An N-terminal stretch of hydrophobic residues (residues 15–36) probably serves as an uncleaved signal sequence (Fig. (Fig.22F). There are also five putative N-glycosylation sites (residues 72, 110, 217, 248, and 261) near the C terminus five clustered cysteine residues (residues 286, 326, 328, 337, and 348) and a putative transmembrane domain (residues 457–477). Comparison with human CD36 and SR-BI reveals several conserved cysteine, glycine, and proline residues and 22.5 and 24% sequence identity, respectively. Both NinaD and SR-BI possess a C-terminal extension compared with CD36. Interestingly, a threonine found in a putative protein kinase C consensus sequence (GPYTYR) in CD36 (residues 89–94) is replaced by a valine (GPYVYR) in NinaD (residues 92–97), as in SR-BI. This phosphorylation site has been proposed to be a regulatory domain in CD36, whereas replacement of the threonine by a valine residue as in SR-BI and NinaD may predict constitutive, nonregulated binding activity (29).

Rescue of ninaDP245 Flies by PElement-Mediated Transformation with a cDNA Coding the Wild-Type Allele.

To demonstrate that this scavenger receptor is essential for carotenoid utilization and thus is responsible for the blindness of ninaD flies, we established an in vivo system by using P element-mediated transformation and the UAS/GAL4 expression system (15). For this purpose, we transformed flies with a wt cDNA (representing the longer splice variant) in the vector pUAST, resulting in a fly strain with UAS-ninaD(wt) on chromosome 3. By further crosses, homozygous ninaDP245 flies carrying the UAS-ninaD(wt) and hs-GAL4 transgenes on chromosome 3 were generated and tested for the rescue of the blind ninaD phenotype by heat shock-induced transgene expression. To assay for this rescue, we analyzed again the maturation of the fly's major rhodopsin Rh1. In the ninaDP245 flies carrying the UAS-ninaD(wt) and hs-GAL4 transgenes without heat shock treatment, Rh1 was only found in its immature form. Heat shock-induced ninaD(wt) expression resulted in Rh1 maturation, accompanied by a reduction in its molecular mass to 32 kDa (Fig. (Fig.33A). Furthermore, to show directly that the ninaD(wt) transgene expression resulted in carotenoid uptake in ninaD flies, we determined the content and composition of carotenoids. By quantitative HPLC analyses, we found a significant increase in zeaxanthin and lutein (Fig. (Fig.33B). Thus, the ninaD gene encodes a class B scavenger receptor possessing in vivo an essential role in mediating the cellular uptake of carotenoids for the synthesis of the visual chromophore.

Fig 3.
Expression of a ninaD(wt) transgene restores the vitamin A-deficient phenotype of ninaDP245 flies. Adult homozygous ninaDP245 flies carrying a UAS-ninaD(wt) transgene expressed under the control of a hs-GAL4 driver were compared with heterozygous and ...

Total Lipid Composition of ninaDP245 Flies.

The mammalian class B scavenger receptors are multifunctional, mediating the uptake of lipids such as long chain fatty acids and sterols from lipoproteins. Therefore, to ask whether the ninaD mutation might generally interfere with the lipid metabolism in the fly, we determined the total lipid content in ninaDP245 flies. By this analysis, no differences in the total amounts of lipids were detectable compared with control flies. After saponification and silanylation of fatty acids, we also analyzed the profile of these compounds by GC/MS analysis. Again, we found no differences in ninaDP245 flies compared with controls. The qualitative composition of fatty acids was similar to that previously reported in Drosophila (30) with the major constituents 14:0, 16:0, 16:1, 18:0, 18:1, and 18:2. Thus, we conclude that the NinaD scavenger receptor is crucial for carotenoid uptake but evidently dispensable for the lipid metabolism as a whole.

Temporal Expression Pattern of the ninaD Gene.

Having established this new function in carotenoid uptake of a scavenger receptor, we determined the temporal aspects of the ninaD gene expression. We first analyzed different embryonic stages by whole mount in situ hybridization (Fig. (Fig.44A). Specific staining was found beginning with embryonic stage 9. In stages 9 and 10, ninaD expression occurred in the midgut primordia and in mesodermal cells, from which hemocytes and macrophages as well as fat body cells derive. In the embryonic stages 11–14, a more punctuated staining could be observed resembling that described for hemocytes. Interestingly, expression in hemocytes has also been reported for the Drosophila CD36-like scavenger receptor encoded by the gene croquemort (27) and is characteristic for mammalian class B scavenger receptor counterparts like CD36 (24).

Fig 4.
ninaD gene expression during different developmental stages. (A) Whole mount in situ hybridizations of wt embryos by using a digoxygenin-labeled ninaD antisense riboprobe. Lateral view of stained embryos of different developmental stages. In stage 9 and ...

By Northern blot, we then determined the postembryonic ninaD expression and compared it to the carotenoid contents from the different developmental stages (Fig. (Fig.44 B and C). The ninaD mRNA level rose at larval stage 3 (L3) and was strongest in the pupal stages. After eclosion, the ninaD mRNA level remained high for 1 to 2 days but was lower in older imagoes (>10 days). The increase of the ninaD mRNA level in the late larval stages corresponded to some extent with the accumulation of carotenoids found here. In pupae, the carotenoid content was still high but decreased in young imagoes, evidently correlated with the synthesis of the visual chromophore during metamorphogenesis. The high ninaD mRNA level in the pupae might indicate that the ninaD scavenger receptor is of particular importance for the redistribution of the carotenoids from larval stores to the developing eyes. To address the role of the ninaD scavenger receptor in mediating the cellular uptake of carotenoids into any target tissues, we placed the UAS-ninaD(wt) transgene under the control of ninaE-GAL4. The ninaD(wt) transgene expression should then result in a specific high level expression in the R1-R6 photoreceptor cells and thus in carotenoid accumulation in the fly's head. Indeed, by quantitative HPLC analysis of the carotenoids of the heads of these transgenic flies, a 2.5-fold level in zeaxanthin and lutein was found compared with wt controls.

Discussion

In animals, it is long known that carotenoids exert a variety of physiological functions, but the molecular players involved in their absorption and body distribution have not yet been identified. We now show that the molecular basis of the blindness of the Drosophila ninaD mutant is a nonsense mutation in a gene encoding a lipid class B type scavenger receptor, leading to a defect in carotenoid utilization for the synthesis of visual chromophore. The identity of the ninaD gene with this class B scavenger receptor was confirmed by P element-mediated rescue with a ninaD(wt) scavenger receptor allele. Its heat shock-induced expression restored carotenoid uptake and visual pigments in the ninaDP245 mutant. Furthermore, ectopic UAS-ninaD(wt) expression under control of a ninaE-GAL4 driver specifically led to an elevation of the carotenoid content in the fly's head. Thus, these analyses provide genetic and functional evidence that class B scavenger receptors are causally involved in the cellular uptake of carotenoids into cells of target tissues.

ninaD belongs to a small family of Drosophila class B scavenger receptor-like proteins. Two other representatives, encoded by croquemort and emp, have so far been characterized (27, 28). For croquemort, expressed in embryonic macrophages, a crucial role in the recognition and phagocytosis of apoptotic cells during Drosophila embryonic development has been recently demonstrated (27). Comparable functions of scavenger receptors have also been reported in mammals (31). The physiological functions of the emp scavenger receptor have yet not been addressed in detail.

In the past few years, there has been growing evidence that class B scavenger receptors, in particular SR-BI, are substantially involved in lipid metabolism, especially the cholesterol homeostasis in mammals (32, 33). It could be shown that these receptors mediate the bidirectional flux of unesterified cholesterol between target cells and the circulating lipoproteins (34, 35). Yet, a direct involvement of class B scavenger receptors in insect lipid metabolism has not been reported. In insects, carotenoids are transported in the lipophorins of the hemolymph (36). These lipophorins are structurally related to the mammalian lipoprotein particles, also being transport vehicles for carotenoids (6). Insect in vitro systems provided evidence that the cellular uptake of lipids occurs by a flux between lipophorins and target cells. Like cholesterol exchange in mammals, the lipophorin particles are not necessarily internalized by receptor-mediated phagocytosis (37, 38). Based on our analyses, we speculate that the ninaD scavenger receptor mediates the carotenoid transport from lipophorins in a mechanistically similar manner.

ninaD mRNA levels were particularly high in pupae when lipids acquired during the larval stage are mobilized and redistributed for metamorphogenesis. The high mRNA levels during this developmental stage may indicate that NinaD plays a specific role for the redistribution of xanthophylls, the pupal storage form of carotenoids (14). This mobilization is necessary to deliver carotenoids from adipose tissues to the developing eyes for the synthesis of the visual chromophores. Because ninaD flies develop normally and possess, besides reduced carotenoids, no significant alterations in their lipid contents, the loss of this scavenger receptor seems not to interfere with the fly's lipid metabolism in general. This situation may be explained by the specificity of the NinaD function for carotenoids or by a bypass of the lack of NinaD function by different, yet to be identified receptor types.

With the molecular identification of the Drosophila NinaD and NinaB functions, the two crucial molecular players involved in the synthesis of the visual chromophore from dietary carotenoid precursors are now molecularly identified in this model organism. As for mammals, we already showed that the ortholog of NinaB, a β,β-carotene-15,15′-oxygenase, catalyzes β-carotene conversion to vitamin A (10). In mammals, the NinaB ortholog is expressed in a variety of different tissues, including the eyes and reproductive organs (10, 12, 13), so vitamin A-dependent physiological processes like vision and reproduction may be directly influenced by carotenoids, which then are tissue specifically acquired from lipoproteins of the circulation. It remains to be shown whether the cellular uptake of carotenoids necessary for their conversion to vitamin A is mediated via the mammalian NinaD receptor counterparts. Vitamin A also plays an important role in the mammalian immune response (39) and a role of scavenger receptors in host defense has been well established (40). Therefore, a putative role of class B scavenger receptors in provitamin A uptake may interlink some aspects of vitamin A and class B scavenger receptor functions in the immune system. Furthermore, it has recently been reported that in SR-BI-deficient mice, vitamin E metabolism is impaired, resulting in an elevated plasma concentration of this fat-soluble vitamin (41). This finding, together with our results from Drosophila, indicates a more general role of class B scavenger receptors in the metabolism of fat-soluble vitamins belonging to the isoprenoid substance class. No defects related to carotenoid utilization have as yet been reported in mice deficient in the class B scavenger receptors, SR-BI or CD36. However, it should be noted that under laboratory conditions mice are usually kept on a diet rich in preformed vitamin A. Therefore, an impaired carotenoid utilization would not necessarily result in vitamin A deficiency accompanied by phenotypic alterations such as blindness.

Besides vitamin A formation, carotenoids are of importance for various physiological processes. For example, lutein and zeaxanthin are accumulated as macular pigments in the primate and human eye (42). Additionally, beneficial effects of carotenoids mainly due to their antioxidant properties have been discussed in the context of several diseases (43). The identification of the scavenger receptor ninaD as being essential for carotenoid uptake in Drosophila delivers molecular insight into how dietary carotenoids may be distributed to their site of action in the body and promises to elucidate new aspects of class B scavenger receptor functions.

Acknowledgments

We thank W. L. Pak (Purdue University, West Lafayette, IN) for the gift of the fly stock ninaDP245 and H. Huber (University of Karlsruhe, Gemany) for the gift of the Rh1 antiserum. We also thank Randall Cassada, who corrected the English version of the manuscript. This work was supported by Grant LI 956 of the German Research Foundation (to D.F.G.).

Abbreviations

  • wt, wild type
  • RT, reverse transcription
  • hs, heat shock
  • RACE, rapid amplification of cDNA ends

Notes

This paper was submitted directly (Track II) to the PNAS office.

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