Recent progress in molecular genetic studies on the carotenoid transport system using cocoon-color mutants of the silkworm.

The existence of tissue-specific delivery for certain carotenoids is supported by genetic evidence from the silkworm Bombyx mori and the identification of cocoon color mutant genes, such as Yellow blood (Y), Yellow cocoon (C), and Flesh cocoon (F). Mutants with white cocoons are defective in one of the steps involved in transporting carotenoids from the midgut lumen to the middle silk gland via the hemolymph lipoprotein, lipophorin, and the different colored cocoons are caused by the accumulation of specific carotenoids into the middle silk gland. The Y gene encodes carotenoid-binding protein (CBP), which is expected to function as the cytosolic transporter of carotenoids across the enterocyte and epithelium of the middle silk gland. The C and F genes encode the C locus-associated membrane protein, which is homologous to a mammalian high-density lipoprotein receptor-2 (Cameo2) and scavenger receptor class B member 15 (SCRB15), respectively; these membrane proteins are expected to function as non-internalizing lipophorin receptors in the middle silk gland. Cameo2 and SCRB15 belong to the cluster determinant 36 (CD36) family, with Cameo2 exhibiting specificity not only for lutein, but also for zeaxanthin and astaxanthin, while SCRB15 seems to have specificity toward carotene substrates such as α-carotene and ß-carotene. These findings suggest that Cameo2 and SCRB15 can discriminate the chemical structure of lutein and ß-carotene from circulating lipophorin during uptake. These data provide the first evidence that CD36 family proteins can discriminate individual carotenoid molecules in lipophorin.

Lipid transfer particle from the silkworm, Bombyx mori, is a novel member of the apoB/large lipid transfer protein family.

Lipid transfer particle (LTP) is a high-molecular-weight, very high-density lipoprotein known to catalyze the transfer of lipids between a variety of lipoproteins, including both insects and vertebrates. Studying the biosynthesis and regulation pathways of LTP in detail has not been possible due to a lack of information regarding the apoproteins. Here, we sequenced the cDNA and deduced amino acid sequences for three apoproteins of LTP from the silkworm (Bombyx mori). The three subunit proteins of the LTP are coded by two genes, apoLTP-II/I and apoLTP-III. ApoLTP-I and apoLTP-II are predicted to be generated by posttranslational cleavage of the precursor protein, apoLTP-II/I. Clusters of amphipathic secondary structure within apoLTP-II/I are similar to Homo sapiens apolipoprotein B (apoB) and insect lipophorins. The apoLTP-II/I gene is a novel member of the apoB/large lipid transfer protein gene family. ApoLTP-III has a putative conserved juvenile hormone-binding protein superfamily domain. Expression of apoLTP-II/I and apoLTP-III genes was synchronized and both genes were primarily expressed in the fat body at the stage corresponding to increased lipid transport needs. We are now in a position to study in detail the physiological role of LTP and its biosynthesis and assembly.

CD36 homolog divergence is responsible for the selectivity of carotenoid species migration to the silk gland of the silkworm Bombyx mori.

Dietary carotenoids are absorbed in the intestine and delivered to various tissues by circulating lipoproteins; however, the mechanism underlying selective delivery of different carotenoid species to individual tissues remains elusive. The products of the Yellow cocoon (C) gene and the Flesh (F) gene of the silkworm Bombyx mori determine the selectivity for transport of lutein and ß-carotene, respectively, to the silk gland. We previously showed that the C gene encodes Cameo2, a CD36 family member, which is thought to function as a transmembrane lipoprotein receptor. Here, we elucidated the molecular identity of the F gene product by positional cloning, as SCRB15, a paralog of Cameo2 with 26% amino acid identity. In the F mutant, SCRB15 mRNA structure was severely disrupted, due to a 1.4 kb genomic insertion in a coding exon. Transgenic expression of SCRB15 in the middle silk gland using the binary GAL4-UAS expression system enhanced selective ß-carotene uptake by the middle silk gland, while transgenic expression of Cameo2 enhanced selective lutein uptake under the same GAL4 driver. Our findings indicate that divergence of genes in the CD36 family determines the selectivity of carotenoid species uptake by silk gland tissue and that CD36-homologous proteins can discriminate among carotenoid species.

A possible overestimation of the effect of acetylation on lysine residues in KQ mutant analysis.

Acetylation of lysine residues, one of the most common protein post-transcriptional modifications, is thought to regulate protein affinity with other proteins or nucleotides. Experimentally, the effects of acetylation have been studied using recombinant mutants in which lysine residues (K) are substituted with glutamine (Q) as a mimic of acetyl lysine (KQ mutant), or with arginine (R) as a mimic of nonacetylated lysine (KR mutant). These substitutions, however, have not been properly validated. The effects lysine acetylation on Ku, a multifunctional protein that has been primarily implicated in DNA repair and cell survival, are characterized herein using a series of computer simulations. The binding free energy was reduced in the KQ mutant, while the KR mutant had no effect, which is consistent with previous experimental results. Unexpectedly, the binding energy between Ku and DNA was maintained at almost the same level as in the wild type protein despite full acetylation of the lysine residues. These results suggest that the effects of acetylation may be overestimated when the KQ mutant is used as a mimic of the acetylated protein.

A CD36-related transmembrane protein is coordinated with an intracellular lipid-binding protein in selective carotenoid transport for cocoon coloration.

The transport pathway of specific dietary carotenoids from the midgut lumen to the silk gland in the silkworm, Bombyx mori, is a model system for selective carotenoid transport because several genetic mutants with defects in parts of this pathway have been identified that manifest altered cocoon pigmentation. In the wild-type silkworm, which has both genes, Yellow blood (Y) and Yellow cocoon (C), lutein is transferred selectively from the hemolymph lipoprotein to the silk gland cells where it is accumulated into the cocoon. The Y gene encodes an intracellular carotenoid-binding protein (CBP) containing a lipid-binding domain known as the steroidogenic acute regulatory protein-related lipid transfer domain. Positional cloning and transgenic rescue experiments revealed that the C gene encodes Cameo2, a transmembrane protein gene belonging to the CD36 family genes, some of which, such as the mammalian SR-BI and the fruit fly ninaD, are reported as lipoprotein receptors or implicated in carotenoid transport for visual system. In C mutant larvae, Cameo2 expression was strongly repressed in the silk gland in a specific manner, resulting in colorless silk glands and white cocoons. The developmental profile of Cameo2 expression, CBP expression, and lutein pigmentation in the silk gland of the yellow cocoon strain were correlated. We hypothesize that selective delivery of lutein to specific tissue requires the combination of two components: 1) CBP as a carotenoid transporter in cytosol and 2) Cameo2 as a transmembrane receptor on the surface of the cells.

Purification and partial characterization of a lutein-binding protein from human retina.

Dietary intake of lutein and zeaxanthin appears to be advantageous for protecting human retinal and macular tissues from degenerative disorders such as age-related macular degeneration. Selective concentration of just two of the many dietary carotenoids suggests that uptake and transport of these xanthophyll carotenoids into the human foveal region are mediated by specific xanthophyll-binding proteins such as GSTP1 which has previously been identified as the zeaxanthin-binding protein of the primate macula. Here, a membrane-associated human retinal lutein-binding protein (HR-LBP) was purified from human peripheral retina using ion-exchange chromatography followed by size-exclusion chromatography. After attaining 83-fold enrichment of HR-LBP, this protein exhibited a significant bathochromic shift of approximately 90 nm in association with lutein, and equilibrium binding studies demonstrated saturable, specific binding toward lutein with a K(D) of 0.45 muM. Examination for cross-reactivity with antibodies raised against known lutein-binding proteins from other organisms revealed consistent labeling of a major protein band of purified HR-LBP at approximately 29 kDa with an antibody raised against silkworm (Bombyx mori) carotenoid-binding protein (CBP), a member of steroidogenic acute regulatory (StAR) protein family with significant homology to many human StAR proteins. Immunolocalization with antibodies directed against either CBP or GSTP1 showed specific labeling of rod and cone inner segments, especially in the mitochondria-rich ellipsoid region. There was also strong labeling of the outer plexiform (Henle fiber) layer with anti-GSTP1. Such localizations compare favorably with the distribution of macular carotenoids as revealed by resonance Raman microscopy. Our results suggest that HR-LBP may facilitate lutein's localization to a region of the cell subject to considerable oxidative stress.

Integration of the 5' end of the retrotransposon, R2Bm, can be complemented by homologous recombination.

R2Bm is a non-long-terminal-repeat (non-LTR) retrotransposon that was identified at a specific target site in the 28S rRNA genes of the silkworm, Bombyx mori. Although in vitro analysis has revealed that the 3' end of R2Bm is integrated into the target site by means of target-primed reverse transcription (TPRT), the mechanism of the 5' end integration is not well understood. We established a novel in vivo system to assay the insertion mechanism of R2Bm using a cultured cell line, C65, and a baculovirus, AcNPV, as host and vector, respectively. The 3' end of R2Bm integrated at the target site in the rRNA genes of C65 cells when an AcNPV containing both the full-length 3' UTR and the entire open reading frame (ORF) of R2Bm was introduced while the 5' end integration was incorrect. The 5' end of R2Bm was integrated, however, when the 28S gene sequence upstream of the R2Bm target site was added to the R2Bm sequence. Thus, in our assay, homologous sequences were likely essential for the successful integration of the entire R2Bm into the host cell genome. We also demonstrated that the failure to integrate caused by a frame-shifted ORF was rescued by co-infection with a helper virus that contained only the R2Bm ORF. This indicates that R2 retrotransposition can be complemented in trans. These findings suggest that the host's mechanism for DNA repair may be necessary for the integration of the 5' end of R2Bm and that R2Bm protein may only have the ability to integrate the 3' end of the element by TPRT.

A carotenoid-binding protein (CBP) plays a crucial role in cocoon pigmentation of silkworm (Bombyx mori) larvae.

We examined the role of carotenoid-binding protein (CBP) in yellow cocoon pigmentation. First, using yellow or white cocoon races, we investigated the linkage between the yellow pigmentation and CBP expression. CBP was expressed only in the silk gland of the yellow cocoon races, which utilize carotenoids for cocoon pigmentation. Furthermore, CBP expression in the silk glands of day 1-7 fifth instar larvae matched the period of carotenoid uptake into the silk gland. Finally, we gave double-stranded CBP RNA to Bombyx mori (B. mori) larvae to induce RNA interference. The significantly reduced expression of CBP in the silk gland of fifth instar larva was confirmed on day 4 and a decrease in yellow pigmentation was observed in the cocoon. We showed that CBP plays a key role in the yellow cocoon pigmentation caused by carotenoids.

The basis for colorless hemolymph and cocoons in the Y-gene recessive Bombyx mori mutants: a defect in the cellular uptake of carotenoids.

Bombyx mori is an excellent model for the study of carotenoid-binding proteins (CBP). In previous papers, we identified and molecularly characterized a CBP from the Y-gene dominant mutants. In the present study, we attempted to correlate and establish lipid metabolism and distribution in these mutants. When [3H]-triolein was fed to the mutants, typical patterns of uptake of labeled fatty acids from midgut to hemolymph and subsequent delivery to fat body and silk glands were obtained in all mutants. Further analysis of lipid and carotenoid profiles revealed that the yellow coloration in the hemolymph associated with lipophorin is not attributed to a difference in lipophorin concentrations among the mutants, nor to its lipid composition, but rather to its carotenoid content. Lipophorin of the Y+I mutant exhibited the highest concentration of total carotenoids of 55.8 microg/mg lipophorin compared to 3.1 microg/mg in the +Y+I mutant, 1.2 microg/mg in the YI mutant and 0.5 microg/mg in the +YI mutant. Characteristic retention time in HPLC of the different classes of carotenoids of lipophorin identified the presence of lutein as the major chromophore (62-77%), followed by beta-carotenes (22-38%). Although lutein and beta-carotene content of mutants' lipophorin differed significantly, the ratio of lutein to beta-carotene of 3:1 was not different among mutants. Similarly, lipid compositions of mutant silk glands were not significantly different, but carotenoid contents were. The significantly high concentration of lutein in the Y+I mutant silk gland represented more than 160-fold increase compared to +Y+I mutant (p<0.001). In this report, we conclude that lipid metabolism in the mutants is not defected and that the molecular basis for colorless hemolymph and cocoons is a defect in the cellular uptake of lutein associated with the Y-gene recessive mutants.

Characterization of the carotenoid-binding protein of the Y-gene dominant mutants of Bombyx mori.

Carotenoids play important and diverse roles in insects. Recently, we purified and partially characterized a carotenoid-binding protein (CBP) from the wild type of Bombyx mori. In this report, we utilized immunoblotting, ELISA and immunocytochemistry to further characterize and localize the expression of CBP in the larval midgut and silk gland obtained from the wild type and four naturally occurring mutants linked to carotenoids transport. CBP was expressed throughout the 5th stadium, with highest expressions on days 4-5 in the silk gland and days 3-5 in the midgut. Immunoblotting analyses demonstrated the presence of CBP along the middle part of the midgut. Microscopic immunocytochemistry demonstrated that the 33 kDa CBP was uniformly expressed along the brush border of columnar cells in the epithelium of the midgut typifying its function in aiding absorption of dietary carotenoids. Similarly, CBP was highly expressed along the distal membrane of the middle part of the silk gland demonstrating its function in uptake of carotenoids from lipophorin. When the middle silk glands and midguts of the four mutants were incubated with rabbit anti-CBP antibody, only proteins of the Y-gene dominant mutants cross reacted with the antibody further accentuating the hypothesis that the CBP is a Y-gene dependent protein.

Diversity in copy number and structure of a silkworm morphogenetic gene as a result of domestication.

The carotenoid-binding protein (CBP) of the domesticated silkworm, Bombyx mori, a major determinant of cocoon color, is likely to have been substantially influenced by domestication of this species. We analyzed the structure of the CBP gene in multiple strains of B. mori, in multiple individuals of the wild silkworm, B. mandarina (the putative wild ancestor of B. mori), and in a number of other lepidopterans. We found the CBP gene copy number in genomic DNA to vary widely among B. mori strains, ranging from 1 to 20. The copies of CBP are of several types, based on the presence of a retrotransposon or partial deletion of the coding sequence. In contrast to B. mori, B. mandarina was found to possess a single copy of CBP without the retrotransposon insertion, regardless of habitat. Several other lepidopterans were found to contain sequences homologous to CBP, revealing that this gene is evolutionarily conserved in the lepidopteran lineage. Thus, domestication can generate significant diversity of gene copy number and structure over a relatively short evolutionary time.

Apolipophorin-III expression and low density lipophorin formation during embryonic development of the silkworm, Bombyx mori.

We examined the expression of apolipophorin-III (apoLp-III) during embryonic development of the silkworm Bombyx mori. ApoLp-III mRNA was first expressed 24h after oviposition, which corresponds to the time of germ band formation. The amount of apoLp-III in the eggs increased from day 2, peaked on day 4, and then gradually decreased until hatching (on day 9.5). ApoLp-III was apparently synthesized during early embryogenesis, as radioactive amino acids were incorporated into newly synthesized apoLp-III in three-day-old eggs. Moreover, radioactive apoLp-III was found only in the embryo and not in the extraembryonic tissue. KBr density gradient ultracentrifugation of egg homogenates showed that apoLp-III was associated with low-density lipophorin (LDLp). These results suggest that LDLp is required for the delivery of lipids for organogenesis during embryogenesis.

Purification and expression analysis of imaginal disc growth factor in the silkworm, Bombyx mori.

In the present study, we purified and sequenced a homolog of the Drosophila imaginal disc growth factor (IDGF) from the hemolymph of Bombyx mori (BmIDGF). Antibodies against BmIDGF were produced and subsequently used in immunoblotting analyses. The immunoblotting analyses demonstrated an extremely high level of BmIDGF in the hemolymph throughout the period of rapid growth of the organs of B. mori. The results of RT-PCR showed that BmIDGF was predominantly expressed in fat bodies. Real-time RT-PCR revealed that BmIDGF transcripts in fat bodies were highly expressed during the feeding stage but significantly suppressed during the molting, wandering, and pupal stages. Starvation brought about a significant decline of BmIDGF mRNAs in the fat bodies and BmIDGF proteins in the hemolymph. After re-feeding, the BmIDGF transcripts in fat bodies and BmIDGF proteins in the hemolymph increased again. In addition, an immunocytochemical study revealed BmIDGF proteins on the surface of wing discs. The present findings suggest that the level of BmIDGF in the hemolymph was modulated by the fat body in response to nutritional conditions and that BmIDGF was transported to target organs through the hemolymph.

Carotenoid silk coloration is controlled by a carotenoid-binding protein, a product of the Yellow blood gene.

Mechanisms for the uptake and transport of carotenoids, essential nutrients for humans, are not well understood in any animal system. The Y (Yellow blood) gene, a critical cocoon color determinant in the silkworm Bombyx mori, controls the uptake of carotenoids into the intestinal mucosa and the silk gland. Here we provide evidence that the Y gene corresponds to the intracellular carotenoid-binding protein (CBP) gene. In the Y recessive strain, the absence of an exon, likely due to an incorrect mRNA splicing caused by a transposon-associated genomic deletion, generates a nonfunctional CBP mRNA, resulting in colorless hemolymph and white cocoons. Enhancement of carotenoid uptake and coloration of the white cocoon was achieved by germ-line transformation with the CBP gene. This study demonstrates the existence of a genetically facilitated intracellular process beyond passive diffusion for carotenoid uptake in the animal phyla, and paves the way for modulating silk color and lipid content through genetic engineering.

Lipophorin receptor of Bombyx mori: cDNA cloning, genomic structure, alternative splicing, and isolation of a new isoform.

The cDNA and genomic structure of a putative lipophorin receptor from the silkworm, Bombyx mori (BmLpR), indicated the presence of four isoforms, designated LpR1, LpR2, LpR3, and LpR4. The deduced amino acid sequence of each isoform showed five functional domains that are homologous to vertebrate very low density lipoprotein receptor (VLDLR). All four isoforms seem to have originated from a single gene by alternative splicing and were differentially expressed in a tissue- and stage-specific manner. BmLpR1 harbored an additional 27 amino acids in the O-linked sugar domain, resulting in an extra exon. The silkworm BmLpR gene consisted of 16 exons separated by 15 introns spanning >122 kb and was at least three times larger than the human VLDLR gene. Surprisingly, one of the isoforms, LpR4, was expressed specifically in the brain and central nervous system. Additionally, it had a unique cytoplasmic tail, leading to the proposition that it represents a new candidate LpR for possible brain-related function(s). This is the first report on the genomic characterization of an arthropod lipoprotein receptor gene and the identification of a brain-specific receptor variant from a core member of the low density lipoprotein receptor family in invertebrates.

Proteome analysis of silk gland proteins from the silkworm, Bombyx mori.

The silk gland of Bombyx mori is an organ specialized for the synthesis and secretion of silk proteins. We report here the resolution of silk gland proteins by 2-DE and the identification of many of those proteins. This was accomplished by dissecting the glands into several sections, with each exhibiting more than 400 protein spots by 2-DE, of which 100 spots were excised and characterized by in-gel digestion followed by PMF. Ninety-three proteins were tentatively identified. These were then categorized into groups involved in silk protein secretion, transport, lipid metabolism, defense, etc. Western blotting of a 2-DE gel using an antibody of the carotenoid binding protein confirmed the presence of this protein in the silk gland. Proteins including fibroin L-chain and P25 were found as multiple isoforms, some of which contained differential amounts of phosphate residues as analyzed by on-probe dephosphorylation. The current analysis contributes to our understanding of proteins expressed by the silk gland not only of the model lepidopteran B. mori, but also to proteins from other silk-producing insects such as Philosamia cynthia ricini.

BmStart1, a novel carotenoid-binding protein isoform from Bombyx mori, is orthologous to MLN64, a mammalian cholesterol transporter.

Carotenoid-binding protein (CBP) from the silkworm Bombyx mori is an essential molecule for carotenoid dependent cocoon pigmentation. We identified a novel isoform of CBP, Start1 of B. mori (BmStart1). BmStart1 contains a membrane-spanning MENTAL domain in its N-terminus and a lipid-binding START domain in its C-terminus. This domain architecture is identical to the mammalian MLN64 and Start1 of Drosophila melanogaster (DmStart1), both of which have been implicated to function in cholesterol transport and regulation of steroidogenesis. BmStart1 is expressed in both white and yellow cocoon strains of B. mori, while CBP is only detected in the yellow cocoon strain. BmStart1 mRNA abundance in the prothoracic gland, the main ecdysteroidogenic tissue, positively correlates with changes in the hemolymph ecdysteroid level. Genomic sequence analysis revealed that BmStart1 and CBP are generated from the same gene locus by alternative splicing. Splice site comparison and homology search indicate that BmStart1 is orthologous to both MLN64 and DmStart1. This study implies that alternative splicing of the BmStart1/CBP gene generates unique protein isoforms whose endogenous ligands, sterol or carotenoid, are structurally different.

Isolation, characterization, and cDNA sequence of a carotenoid binding protein from the silk gland of Bombyx mori larvae.

A carotenoid binding protein (CBP) has been isolated from the silk glands of Bombyx mori larvae. The protein has an apparent molecular mass of 33 kDa and binds carotenoids in a 1:1 molar ratio. Lutein accounts for 90% of the bound carotenoids, whereas alpha-carotene and beta-carotene are minor components. Immunological analysis demonstrated the presence of CBP only in the yellow-colored tissues of the silk gland, midgut, testis, and ovary. Several phenotypes of B. mori mutants linked to carotenoid transport have been utilized to characterize CBP. The Y (yellow hemolymph) gene controls uptake of carotenoids from the midgut lumen into the midgut epithelium, and larvae with the +(Y) gene lack this property. Immunoblotting analysis confirmed the presence of CBP in mutants with the dominant Y gene only. Immunohistochemistry verified the localization of CBP in the villi of the midgut epithelium, indicating that CBP might be involved in absorption of carotenoids. A cDNA clone for CBP encoding a protein of 297 amino acids has been isolated from the B. mori silk gland cDNA library. The deduced amino acid sequence revealed that CBP is a novel member of the steroidogenic acute regulatory (StAR) protein family with its unique structural feature of a StAR-related lipid transfer domain, known to aid in lipid transfer and recognition. Lutein-binding capacity of the recombinant CBP (rCBP) determined by incubating rCBP with lutein followed by immunoprecipitation using anti-CBP IgG conjugated to protein A-Sepharose, demonstrated the formation of a lutein-rCBP complex. Sequence analyses coupled with binding specificity suggest that CBP is a new member of the StAR protein family that binds carotenoids rather than cholesterol.