Tài liệu Báo cáo khoa học: Molecular defect of isovaleryl-CoA dehydrogenase in the skunk mutant of silkworm, Bombyx mori ppt

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Tài liệu Báo cáo khoa học: Molecular defect of isovaleryl-CoA dehydrogenase in the skunk mutant of silkworm, Bombyx mori ppt

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Molecular defect of isovaleryl-CoA dehydrogenase in the skunk mutant of silkworm, Bombyx mori Kei Urano 1 , Takaaki Daimon 1 , Yutaka Banno 2 , Kazuei Mita 3 , Tohru Terada 4 , Kentaro Shimizu 4,5 , Susumu Katsuma 1 and Toru Shimada 1,4 1 Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan 2 Institute of Genetic Resources, Graduate School of Bioresource and Bioenvironmental Science, Kyushu University, Fukuoka, Japan 3 Division of Insect Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan 4 Agricultural Bioinformatics Research Unit, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan 5 Department of Biotechnology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan Introduction Isovaleryl-CoA dehydrogenase (IVD; EC 1.3.99.10) is a tetrameric, mitochondrial flavoenzyme that catalyses the third step of leucine degradation in which isovale- ryl-CoA is converted to 3-methylcrotonyl-CoA. IVD is a member of the acyl-CoA dehydrogenase (ACAD) family of enzymes, all of which share significant sequences and employ a similar enzyme mechanism for the a,b-dehydrogenation of acyl-CoA substrates [1]. Keywords Bombyx mori; branched-chain amino acid; isovaleric acidemia; isovaleryl-CoA dehydrogenase; responsible gene Correspondence T. Shimada, Laboratory of Insect Genetics and Bioscience, Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan Fax: +81 3 5841 8011 Tel: +81 3 5841 8124 E-mail: shimada@ss.ab.a.u-tokyo.ac.jp (Received 2 March 2010, revised 1 August 2010, accepted 25 August 2010) doi:10.1111/j.1742-4658.2010.07832.x The isovaleric acid-emanating silkworm mutant skunk ( sku) was first stud- ied over 30 years ago because of its unusual odour and prepupal lethality. Here, we report the identification and characterization of the gene responsi- ble for the sku mutant. Because of its specific features and symptoms simi- lar to human isovaleryl-CoA dehydrogenase (IVD) deficiency, also known as isovaleric acidaemia, IVD dysfunction in silkworms was predicted to be responsible for the phenotype of the sku mutant. Linkage analysis revealed that the silkworm IVD gene (BmIVD) was closely linked to the odorous phenotype as expected, and a single amino acid substitution (G376V) was found in BmIVD of the sku mutant. To investigate the effect of the G376V substitution on BmIVD function, wild-type and sku-type recombinants were constructed with a baculovirus expression system and the subsequent enzyme activity of sku-type BmIVD was shown to be significantly reduced compared with that of wild-type BmIVD. Molecular modelling suggested that this reduction in the enzyme activity may be due to negative effects of G376V mutation on FAD-binding or on monomer–monomer interactions. These observations strongly suggest that BmIVD is responsible for the sku locus and that the molecular defect in BmIVD causes the characteristic smell and prepupal lethality of the sku mutant. To our knowledge, this is, aside from humans, the first characterization of IVD deficiency in metazoa. Considering that IVD acts in the third step of leucine degradation and the sku mutant accumulates branched-chain amino acids in haemolymph, this mutant may be useful in the investigation of unique branched-chain amino acid catabolism in insects. Abbreviations ACAD, acyl-CoA dehydrogenase BmIVD, Bombyx mori isovaleryl-CoA dehydrogenase; EST, expressed sequence tag; IVD, isovaleryl-CoA dehydrogenase; PMS, phenazinemethosulfate; SNP, single nucleotide polymorphism. 4452 FEBS Journal 277 (2010) 4452–4463 ª 2010 The Authors Journal compilation ª 2010 FEBS IVD dysfunction is well characterized in humans as the first recognized organic acidaemia (isovaleric acida- emia), which causes recurrent episodes of vomiting, lethargy, developmental delay and sometimes acute neonatal death [2,3]. Isovaleric acid, one of the deriva- tives of isovaleryl-CoA, is abnormally excreted in blood and causes a characteristic sweaty feet odour in patients. The isovaleric acid-emanating silkworm mutant skunk (sku) was first described over 30 years ago as an ‘odorous silkworm’ [4,5]. The sku gene is an autosomal recessive lethal gene and the sku mutant exhibits prepupal lethality (Fig. 1). However, the gene responsi- ble for the sku mutant has not yet been identified. The sku mutant has two physiological characteristics. First, the distinctive odour of the mutant is caused by the accumulation of isovaleric acid in the larval excrement. Second, the mutant exhibits abnormally high accumu- lations of branched-chain amino acids, including leucine, in haemolymph [6]. From the second feature, it can be assumed that the branched-chain amino acid degradation mechanism might be dysfunctional in the sku mutant, whereas it is apparent that the first symptom resembles isovaleric acidaemia in humans. Because the isovaleric acid-accumulating mechanism in animals is restricted to isovaleryl-CoA decomposition disorder (Kyoto Encyclopedia of Genes and Genomes; http://www.kegg.jp/en/), it can be expected that IVD deficiency in silkworm may account for the odorous phenotype, similar to the IVD deficiency observed in human isovaleric acidaemia. In this study, the IVD gene of the silkworm Bomb- yx mori was identified and a single nucleotide substitu- tion in the highly conserved site (G376V) in BmIVD of the sku allele was determined. Genetic and biochemical analyses indicated that this substitution caused the sku phenotype. Because G376V was a novel mutation in IVD, the effect of the mutation was further investi- gated by molecular modelling. Together with the previ- ously reported traits of the sku mutant, the molecular and physiological effects of IVD dysfunction in silk- worms are compared with those observed in humans. Results Identification of the B. mori isovaleryl-CoA dehydrogenase (BmIVD) gene as a candidate for the sku mutant A search of the silkworm expressed sequence tag (EST) database revealed the existence of several puta- tive acyl-CoA dehydrogenase genes in silkworm. Among them, one EST clone, fdpeP14_F_F20, exhib- ited the highest homology (69%) to human IVD. After analysis of the full-length sequence of this EST clone, it was apparent that two key residues distinguishing IVD from other ACAD family members are both con- served. One is the catalytic base E254, which abstracts sku / + sku sku / sku sku / + sku sku / sku B A Fig. 1. Phenotypes of control silkworm (sku ⁄ + sku ) and the skunk mutant (sku ⁄ sku) at (A) day 2 of fifth instar and (B) 10 days after spinning. Until spinning, the skunk mutant larva develops normally (A). After spinning, the mutant dies without pupation, whereas control larva successfully moults to pupa (B). Scale bar, 10 mm. K. Urano et al. Odorous silkworm mutant FEBS Journal 277 (2010) 4452–4463 ª 2010 The Authors Journal compilation ª 2010 FEBS 4453 the a-hydrogen of the substrate, and the other is G374, which forms the binding pocket for the branched-chain acyl moiety of substrate isovaleryl- CoA in human IVD [7,8]. As represented in Fig. 2C, both residues were conserved in silkworm proteins (E276 and G396, respectively). Thus, the EST clone was named BmIVD in this study. A homology search of BmIVD on the silkworm revealed that this gene was mapped on the 22nd linkage group [9]. Because sku is also mapped on the same chromosome [10], the genetic loci of sku and BmIVD were compared. In the single nucleotide polymorphism (SNP) linkage map, the genetic distance between BmIVD and ptth genes was estimated to be 10.5 cM [11]. This value strongly agreed with the distance between the sku and ptth loci in the genetic linkage map (13.5 cM), encouraging further investigation of BmIVD as a candidate gene for sku. Comparison of BmIVDs from wild-type (wt) and sku strains RT-PCR analysis revealed that BmIVD mRNA was expressed in both wild-type and sku mutant silkworms with the same molecular size and expression levels (Fig. 2A). Determination of full-length cDNA sequences using the RACE method in both strains revealed the presence of a single point mutation in 1337 nucleotides of BmIVD. In the sku mutant, the 1127th guanine from the start codon was substituted by thymine and none of the other sites were altered (Fig. 2B). A 1127G>T mutation is missense, changing 2.3 2.0 1.1 (kb) B C A wt sku Skunk-RT2 gPCRsku-R2 T poly(A) Start codon Stop codon Probe skunk-RT1 +1 +1127 +1251 +1300–37 G poly(A) (nt) rp49 BmIVD * G376V Fig. 2. Cloning of the BmIVD gene. (A) RT- PCR analysis revealed the BmIVD band amplified from whole-body RNA of standard strains p50T and c108T as well as the sku mutant. Migrations of the molecular mass marker and control gene rp49 are indicated. (B) Full-length mRNA of wild-type and sku mutant BmIVD are represented. Grey boxes depict the open reading frame (ORF) with blank arrowheads indicating the start and stop codons at the edge. The nucleotide length of each part is also shown. The sin- gle nucleotide substitution 1127G>T in the sku mutant is represented as a dashed line. The position of the primers used for RT-PCR and that of the probe used for northern blot- ting are indicated with black arrowheads and a black arrow, respectively. (C) Amino acid sequence alignment of IVDs from Pseu- domonas aeruginosa PAO1 (bacteria, NP_250705), Arabidopsis thaliana (arabidop- sis, NP_190116), Homo sapiens (human, NP_002216), Caenorhabditis elegans (nema- tode, NP_500720) and B. mori (silkworm, AB458683). Alignment was generated using CLUSTAL W algorithm v1.83 and shaded using the BOXSHADE program. The arrow indicates the position of the 376th glycine residue (G) of BmIVD, which is replaced by a valine (V) in the sku mutant. The catalytic base (D), IVD-specific residue in the acyl binding pocket (*) and major functional domains (thick underlines) of IVD are also indicated. Odorous silkworm mutant K. Urano et al. 4454 FEBS Journal 277 (2010) 4452–4463 ª 2010 The Authors Journal compilation ª 2010 FEBS the codon for glycine residue at position 376 to that for valine (G376V) in BmIVD. An investigation of the nucleotide sequence at the 1127th position in eight + sku ⁄ + sku silkworm strains revealed that all strains conserved the canonical guanine at this site (data not shown). To examine other possible variations, genome sequences of BmIVD were determined and 11 SNPs between BmIVDs of wild-type and sku were found, in addition to 1127G>T. However, all were located on the introns of BmIVD (data not shown). Thus, these 11 SNPs appeared to have no functional influence on this gene. Alignment of the amino acid sequences of IVDs (Fig. 2C) exhibited that BmIVD is highly homologous to other IVDs throughout the entire region. Notably, the glycine residue corresponding to G376 of BmIVD, which was substituted to valine in the sku mutant, is highly conserved from bacterial to mammalian IVDs, indicating the importance of this residue. Linkage analysis between sku and BmIVD genes To determine the consistency between the sku pheno- type and the BmIVD genotype, linkage analysis between the wild-type and sku strains was performed. For this, crossing of the strain a85, in which sku locus is marked with or, a recessive ‘oily’ gene that causes translucent epidermis, was performed. As represented in Fig. 3, four genotypes from F 1 progenies were obtained and SNPs from a total of 133 individuals were sequenced for BmIVD at nucleotide 1127 (Table 1). As expected, all the odorous individuals were homozygous for T ⁄ T at nucleotide 1127. How- ever, none of the nonodorous individuals had the T ⁄ T genotype at this site (G ⁄ GorG⁄ T) (Fig. 3), suggesting no recombination between the sku locus and BmIVD gene. Expression pattern of the BmIVD gene Northern blot analysis was performed to investigate the expression profile of the BmIVD gene. For both wild-type and sku strain, a single band of 1.35 kb, cor- responding to the predicted molecular size of BmIVD mRNA, was detected in all the tissues tested (Fig. 4A). The spatial expression pattern of BmIVD mRNA was similar between wild-type and sku and densitometric analysis of three independent experiments showed that relative expression levels of BmIVD (normalized to Actin3) in each tissue were not statistically significant between wild-type and sku strain (P > 0.05, t-test) (data not shown). Therefore, it is likely that the differ- ence in the regulation of BmIVD expression between + sku and sku alleles is not responsible for the odorous phenotype. To further characterize the spatial expres- sion of BmIVD in the wild-type strain, 15 tissues were investigated using RT-PCR analysis. The result showed that BmIVD is expressed in various tissues, ranging from digestive organs such as midgut to reproductive organs such as ovary and testis or the respiratory organ trachea (Fig. 4B). Among these tissues, fat body and midgut showed higher expression levels than other tissues. It is noteworthy that both tissues play essential roles in nutrient turnover in insects. Namely, nutrients are digested and absorbed in the midgut and stored and metabolized in the fat body which is equivalent to liver in mammals. Thus, it is likely that BmIVD may G/G G/T T/T 1127 | P F 1 1127 | 1127 | or + ++ or sku ++ or + or sku or sku or sku or sku or + or sku ++ Non-odorous Odorous SNP of BmIVD Oily Non-oily Oily G/T 1127 | Phenotypes Fig. 3. Linkage analysis between sku and BmIVD. Recessive gene or linked to sku on the 22nd linkage group was utilized to distin- guish sku heterozygous mutants. The upper part indicates that sku heterozygous mutants were crossed and three kinds (nonodorous- non-oily, nonodorous-oily and odorous-oily) of F 1 generation were distinguished by combination of or and sku phenotypes. The lower part indicates the representative results of genomic DNA direct sequencing of PCR products harbouring the SNP 1127G>T of BmIVD from each of the three phenotypes. Table 1. Results of linkage analysis between sku and BmIVD.A single nucleotide polymorphism at the 1127th base pair of BmIVD ORF was analysed from 133 individuals obtained from an F 1 inter- cross (for details, see Fig. 3). Phenotype (genotype) Number of larvae screened 1127th base pair of BmIVD ORF G ⁄ GG⁄ TT⁄ T Normal (+ ⁄ +, sku ⁄ +)30 12180 Oily and nonodorous (sku ⁄ +) 60 0 60 0 Oily and odorous (sku ⁄ sku)43 0 043 K. Urano et al. Odorous silkworm mutant FEBS Journal 277 (2010) 4452–4463 ª 2010 The Authors Journal compilation ª 2010 FEBS 4455 participate in amino acid catabolism for energy pro- duction in the silkworm. Expression of recombinant BmIVD by the baculovirus expression system To evaluate the effect of the G376V amino acid substi- tution in the sku mutant on the IVD activity and sub- strate specificity, we first carried out overexpression and purification of wild-type and sku-type (G376V) BmIVDs. Because the expression level in IVD-recom- binant Escherichia coli was previously reported to be extremely low and 5¢-end alteration to mimic codon usage of E. coli is necessary for improved expression levels [12], a baculovirus expression system was employed to overexpress the BmIVD. Sf9 cells were infected with a recombinant baculovirus that expresses the full-length BmIVD with His-tagged sequences at the C-terminus. As indicated in Fig. 5A, the expression level of recombinant BmIVD was sufficiently high that a putative BmIVD band could be observed in Coomas- sie Brilliant Blue staining. Western blot analysis revealed that the molecular mass of the expressed BmIVD is apparently lower than that of the predicted full-length recombinant protein (46.5 kDa). However, a size difference between wild-type and sku-type BmIVDs was not observed (Fig. 5B). This suggests that the point mutation at Gly376 does not have an effect on the processing of mitochondrial leader pep- tide common in IVDs [13,14]. The recombinant protein was successfully purified to homogeneity by a single- step, nickel-chelating chromatography procedure (Fig. 5C, D) and used for enzymological studies. Enzymatic activity and substrate specificity of wild-type and sku-type recombinant BmIVDs The enzymatic activity of purified recombinant BmIVD was measured with a variety of acyl-CoA sub- strates using a dye-reduction assay (Fig. 6). When iso- valeryl-CoA was used as a substrate, significant activity (614 nmol of 2,6-dichloroindophenol reduced mg protein )1 Æmin )1 ) was observed in wild-type BmIVD. Meanwhile, when substrates for other ACADs such as isobutyryl-CoA for isobutyryl-CoA dehydrogenase and hexanoyl-CoA for medium-chain acyl-CoA dehydrogenase were used, wild-type BmIVD exhibited residual but much lower activities against these substrates compared with isovaleryl-CoA. This confirms that BmIVD specifically functions in isovale- ryl-CoA dehydrogenation, similar to IVDs observed in other species [15]. Next, sku-type BmIVD (G376V) was examined to determine if it retained enzymatic activities. As indicated in Fig. 6, sku-type BmIVD exhibited only faint ACAD activities against all the substrates investigated. This 1.3 kb FB MG MT EP wt FB MG MT EP sku Actin3 BmIVD rp49 BmIVD B A Fig. 4. Expression profiles of BmIVD. (A) Northern blotting compares the expression levels of BmIVD from several tissues obtained from fifth instar larvae at day 2 of wild-type (p50T) and mutant (sku) strains. Total RNA (5 lg) prepared from fat body (FB), midgut (MG), Malpi- ghian tubule (MT) and epidermis (EP) were blotted and hybridized with the digoxigenin (DIG)-labelled probe. The arrowhead indicates the positive signal. Silkworm Actin3 is represented as a control. (B) RT-PCR analysis using cDNAs from 15 tissues of wild-type p50T strain are indicated. Lane 1, brain (BR); lane 2, prothoracic gland (PG); lane 3, salivary gland (SaG); lane 4, central nervous system (CNS); lane 5, tra- chea (TR); lane 6, fat body (FB); lane 7, ovary (OV); lane 8, testis (TES); lane 9, anterior silk gland (ASG); lane 10, middle silk gland (MSG); lane 11, posterior silk gland (PSG); lane 12, midgut (MG); lane 13, hindgut (HG); lane 14, Malpighian tubule (MT); lane 15, epidermis (EP). Silkworm rp49 was the control. Odorous silkworm mutant K. Urano et al. 4456 FEBS Journal 277 (2010) 4452–4463 ª 2010 The Authors Journal compilation ª 2010 FEBS result demonstrates that the point mutation at Gly376 in BmIVD almost totally disrupts the function of BmIVD as an enzyme, indicating BmIVD dysfunction in sku mutants. Sequence alignment and position of the mutation in the 3D structure The results of the sequence alignment within the ACAD family revealed that the mutated residue in the sku mutant was strictly conserved and positioned within the loop structure connecting helices I and J (Fig. 7A). The loop region consists of 10 amino acids, seven of which are conserved within the species (Fig. 2C) and two of which are conserved within all ACAD family of enzymes, forming a highly conserved motif of Gly–Gly–X–Gly. The second glycine of this motif (Gly374) is known to make a hydrogen bond with the pyrophosphate moiety of the FAD of the next monomer in the tetramer [16]. However, the third gly- cine (Gly376), which is conserved within all ACAD family members and is substituted to valine in the sku mutant, has not been functionally clarified. To investi- gate how the protein loses its enzymatic activity with the G376V mutation (Fig. 6), comparative models of wild-type and mutant BmIVDs were constructed by using the crystal structure of human IVD (PDB ID: 1IVH) as a template [7]. The model of wild-type BmIVD indicated that all the side chains in the loop connecting helices I and J are exposed on the surface of the monomer, pointing toward FAD or its neigh- bouring monomer in the tetramer. By contrast, the side chain of the mutated residue (Val376) points toward the inside of the helix–loop–helix structure. Consequently, the side-chain atoms of Val376 overlap with those of Ile369, Leu372 and Thr383 with inter- atomic distances of < 3 A ˚ in the mutant structure (Fig. 7B). To avoid these overlaps, the mutant proba- bly has a different structure in this region. Discussion In this study, a candidate gene approach was utilized to discover the gene responsible for the odorous silk- worm mutant sku. The candidate gene BmIVD was identified and a single nucleotide substitution was found in the codon of a highly conserved residue, not only in the species, but also in all enzyme family mem- bers (Figs 2C and 7A). It was demonstrated that this substitution perfectly cosegregated with the sku loci (Fig. 3 and Table 1) and dramatically decreased the enzymatic activity (Fig. 6). These genetic and biochem- ical data, along with previous observations that the sku mutant accumulates isovaleric acid and branched- chain amino acids, strongly indicate that a single amino acid substitution (G376V) in BmIVD is respon- sible for the sku mutant. In the sku mutant, dysfunc- tion of BmIVD would cause hydrolytic degradation of 75 50 37 25 (kDa) 1 2 3 4 5 6 7 8 9 10 AB C D 75 50 37 25 (kDa) 50 37 Control Control Fig. 5. Expression and purification of His-tagged BmIVD protein. Protein samples were electrophoresed by SDS ⁄ PAGE and analysed by Coo- massie Brilliant Blue staining (A,C) or western blotting with the anti-His IgG (B,D). The molecular mass markers are indicated on the left. (A,B) Confirmation of baculovirus-expressed recombinant BmIVD. Three kinds of whole cells, Sf9 cells infected with parental AcMNPV (control), wild-type BmIVD (wt) and sku-type BmIVD (sku) under the polyhedrin promoter, were electrophoresed. Arrows indicate the BmIVD band around 40 kDa. (C,D) Recombinant His-tagged BmIVD was purified from virus-infected cells by nickel chromatography. Arrows indicate the position of the recombinant BmIVD. Lane 1, cell lysate; lane 2, proteins not binding to the column; lanes 3, 4 and 5, wash fraction (5, 20 and 40 m M imidazole, respectively) and lanes 6–10, eluate fraction (500 mM imidazole). K. Urano et al. Odorous silkworm mutant FEBS Journal 277 (2010) 4452–4463 ª 2010 The Authors Journal compilation ª 2010 FEBS 4457 isovaleryl-CoA to isovaleric acid, instead of the dehy- drogenation of the substrate, resulting in the character- istic odour of the mutant. To our knowledge, this is the first report of IVD deficiency in animals aside from humans. IVD is a member of the ACAD family of enzymes, all of which employ a similar enzyme mechanism for a,b-dehydrogenation of acyl-CoA substrates [1]. Con- servation of the substituted glycine residue in all ACAD members (Fig. 7A) and significant reduction of ACAD activities in G376V protein (Fig. 6) suggest that Gly376 (corresponding to position 354 in human IVD) is essential for common mechanisms in ACADs. ACAD is a homotetrameric or homodimeric flavopro- tein with each monomer containing one molecule of FAD [17]. FAD not only serves as a catalyst, but also bridges the monomers; it is located at the interface between the monomers and forms many hydrogen bonds with both [16,18]. Molecular modelling revealed that the G376V mutation causes the side chain of Val376 to overlap with other residues, possibly result- ing in changes in the loop structure. Although the loop has only been characterized to make a hydrogen bond with FAD [16], the model structures indicated that it is also involved in interactions with the neigh- bouring monomer. As shown in Fig. 7B, Tyr377 and Asn379 in the loop region interact with Leu236 and Asp234, respectively, of the neighbouring monomer via a hydrogen bond. These results suggest that the G376V mutation alters the structure of the loop region and affects the interactions between monomers and with FAD. Imperfect FAD-binding or tetramer formation would lead to disappearance of the enzy- matic activity (Fig. 6). Recent clinical mutation stud- ies about ACAD deficiency in humans support this prediction [17]. An identical substitution at the homologous position (G371V) in human short-chain acyl-CoA dehydrogenase has also been reported and, though this protein’s enzymatic activity was not men- tioned, in vitro import studies revealed that this muta- tion led to a temperature-dependent inability to form tetramers [19]. The sku larvae begin emanating isovaleric acid odour from the first day after hatching, but do not show any signs of developmental abnormality until the onset of spinning (Fig. 1A). The mutants start spinning after the normal duration of the final instar (6–8 days) but stop after a short time and develop a very thin cocoon. They eventually die without becom- ing pupae in about a week after spinning (Fig. 1B). Isovaleric acid seems to be the cause of prepupal lethality because injection of isovaleric acid into normal spinning larva induces a phenocopy of the pupation defect observed in the sku mutant [5]. Because the silkworm larvae cannot excrete after the onset of spinning, highly accumulated isovaleric acid in sku prepupae may have toxic effects and cause prepupal lethality. In humans, patients with isovaleric acidaemia suffer from recurrent episodes of vomiting, lethargy, developmental delay and sometimes acute neonatal death [2,3]. These symptoms are also thought to be caused by isovaleric acid, but the underlying mechanisms are largely unknown. Because a narcotic effect of short chain fatty acids has been known [20,21] and isovaleric acid is also toxic to silkworm [5], there may be common mechanisms between silkworms and humans in how isovaleric acid causes severe symp- toms in addition to the characteristic odour. One of the most intriguing features of the sku mutant is that the mature larva accumulates branched-chain amino a cids, leucine, isoleucine and valine, in haemolymph at levels  4 times higher in females and  7–12 times wt 120 100 80 60 40 20 0 sku Relative activity (%) * * ** IV-CoA IB-CoA HX-CoA Fig. 6. Relative enzymatic activities of wild-type (wt) and sku-type BmIVD (sku). Enzymatic activities of isovaleryl-CoA (IV-CoA), isobu- tyryl-CoA (IB-CoA) and hexanoyl-CoA (HX-CoA) were assayed by the 2,6-dichloroindophenol ⁄ PMS dye-reduction method. The data show means ± SD of pooled data from two independent experi- ments each performed in triplicate. (**P < 0.0001, *P < 0.05, one- tailed, Student’s t-test). Odorous silkworm mutant K. Urano et al. 4458 FEBS Journal 277 (2010) 4452–4463 ª 2010 The Authors Journal compilation ª 2010 FEBS higher in males than in normal silkworms [6]. In human cases, however, patients with isovaleric acidaemia do not exhibit such accumulation of amino acids [2] because an enzyme reaction one step before IVD dehy- drogenation, in which a-ketoisocaproate is catalysed by branched-chain a-keto acid dehydrogenase, is irrevers- ible in humans [22]. This indicates that, in the silk- worm, there might be an additional mechanism bypassing branched-chain a-keto acid dehydrogenase irreversibility, which enables indirect accumulation of upstream leucine in the sku mutant. It is interesting that isoleucine and valine also accumulate to the same degree as leucine [6]. This phenomenon suggests that leucine catabolism plays an important role in regu- lating all three branched-chain amino acid levels in haemolymph. Thus far, little is known about amino acid catabolism in insects. It is hoped that the sku mutant will give insights into the unique catabolism of branched-chain amino acids in insects. Using recently released B. mori genome data [9] (also see KAIKObase, http:// sgp.dna.affrc.go.jp/KAIKObase/), several genes respon- sible for amino acid turnover in silkworm mutants have been identified [23,24]. The genetic resources of the silk- worm, together with its advantageous large body size for physiological study, will facilitate the further study of amino acid catabolism in insects. Materials and methods Materials B. mori strains p50T and c108T were used as wild-type silk- worms, which are maintained at the University of Tokyo. Odorous silkworm-segregating strain a85, which is main- tained at Kyushu University, was also used. To identify homozygous sku mutants, larvae were individually reared in Petri dishes and the odour was determined by sniffing. wt sku A G376V B G376 V376 I369 I369 BmIVD IVD IBD SBCAD GCD SCAD MCAD LCAD VLCAD ACAD9 ACAD10 398 376 399 415 415 369 377 413 423 427 938 755 J T383 T383 L372 L372 Y377 L236 D234 N379 G374 G374 L236 N379 Y377 D234 I Loop region Fig. 7. Sequence alignment and predicted BmIVD structure. (A) Alignment of BmIVD with all 11 acyl-CoA dehydrogenase family members found in humans. The arrow indicates the substituted residue in the sku mutant and the thick line represents conserved helices I and J. The loop region is also shown. IVD (isovaleryl-CoA dehydrogenase, NP_002216); IBD (isobutyryl-CoA dehydrogenase, NP_055199); SBCAD (short ⁄ branched-chain acyl-CoA dehydrogenase, NP_001600); GCD (glutaryl-CoA dehydrogenase, NP_000150); SCAD (short-chain acyl-CoA dehydrogenase, NP_000008); MCAD (medium-chain acyl-CoA dehydrogenase, NP_000007); LCAD (long-chain acyl-CoA dehydrogenase, NP_001599); VLCAD (very long-chain acyl-CoA dehydrogenase, NP_000009); ACAD9 (acyl-CoA dehydrogenase 9, NP_054768); ACAD10 (acyl-CoA dehydrogenase 10, NP_001130010) and ACAD11 (acyl-CoA dehydrogenase 11, NP_115545). (B) Close-up view of residue 376 to comparative models of wild-type (wt) and sku-type (sku) BmIVD based on an X-ray structure of human IVD. The main chains are represented by ribbons and the atoms of key residues and FAD are shown with a stick model. Helices I and J are coloured gray and the loop between the two helices is coloured yellow, except for mutation site 376 which is coloured pink. FAD of the neighbouring monomer is coloured orange and main chain of neighbouring monomer is coloured blue. Two-headed arrows indicate distances from the side chain of residue 376 to side chains of other residues that are < 3 A ˚ apart. Hydrogen bonds between Gly374 and FAD, Tyr377 and Leu236, and Asn379 and Asp 234 are represented as a dashed line. K. Urano et al. Odorous silkworm mutant FEBS Journal 277 (2010) 4452–4463 ª 2010 The Authors Journal compilation ª 2010 FEBS 4459 The silkworms were reared on fresh mulberry leaves in an insect rearing chamber under standard conditions (25 °C, 12L : 12D photoperiod). Sf9 cells were cultured at 27 °Cin TC-100 insect medium (SAFC Biosciences, Lenexa, KS, USA) supplemented with 10% fetal bovine serum. Autogra- pha californica multiple nucleopolyhedrovirus (AcMNPV) was propagated in the Sf9 cells as described previously [25]. All tissues and cells to be examined were washed twice in NaCl ⁄ P i (137 mm NaCl, 2.7 mm KCl, 8.1 mm Na 2 HPO 4 , 1.5 mm KH 2 PO 4 ), immediately frozen in liquid nitrogen and stored at )80 °C. PCRs were performed using the ExTaq Kit (Takara Bio, Shiga, Japan), unless otherwise mentioned. Isolation of B. mori cDNA encoding the IVD-like gene To identify the Bombyx gene which is homologous to the IVD gene, the EST database was screened [26]. The cDNA clone fdpeP14_F_F20 exhibited the highest homology to human IVD and was subjected to further analysis. Assess- ment of the genetic loci of the EST clone was performed using KAIKObase (http://sgp.dna.affrc.go.jp/KAIKObase/). The nucleotide sequence was determined using the ABI PRISM BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and the ABI Prism 3130 DNA Sequencer (Applied Biosystems). Sequence data were analysed using the program package genetyx-mac version 12.0 (Genetyx Corporation, Tokyo, Japan). PCR primers used in this study are listed in Table S1. RT-PCR of BmIVD Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA). One lg of total RNA was reverse transcribed using the RNA PCR Kit (Takara Bio). PCR was performed using skunkRT1 and skunkRT2 primers (Table S1). Temperature cycling consisted of 40 cycles of denaturing at 94 °C for 30 s, annealing at 54 °C for 30 s and extension at 72 °C for 90 s. 5¢- and 3¢-rapid amplification of cDNA ends (RACE) In order to examine differences in the full-length cDNA nucleotide sequences between odorous and normal silk- worm BmIVD,5¢- and 3¢-RACE was performed using the GeneRacer Kit (Invitrogen). Five micrograms of total RNA was used to dephosphorylate, remove the 5¢ cap, ligate the RNA Oligo and reverse-transcribe the nucleotide sequences. The PCR primers used in this experiment are listed in Table S1. PCRs were carried out according to the manufacturer’s instructions. PCR products were subcloned into the pGEM-T Easy vector (Promega, Madison, WI, USA). The nucleotide sequences were determined as described above. Preparation and sequencing of the BmIVD genomic clone Genomic DNA was extracted from the silk glands of fifth instar larvae according to standard methods [27]. Because the genomic structure of BmIVD is long ( 11 kb), the gen- ome sequence was divided into two parts and sequenced separately. The genomic sequence of BmIVD was PCR- amplified using the TaKaRa LA Taq Kit (Takara Bio). Amplified PCR fragments were subcloned and sequenced as described above. Full-length cDNA and genomic sequences of wild-type BmIVD were deposited into the GenBank ⁄ EMBL ⁄ DDBJ data bank with accession numbers AB458683 for cDNA and AB462483 for genomic DNA. Linkage analysis between sku and BmIVD The heterozygous mutant of sku can be identified using the sku-linked recessive oily gene or. Crossing was performed as indicated in Fig. 3. Thirty normal larvae, 60 oily but nonodorous larvae and 43 oily and odorous larvae were screened at fifth instar. To extract genomic DNA, caudal portions of the larvae were cut and homogenized with a pestle and DNeasy Blood and Tissue Kit (Qiagen, Venlo, The Netherlands) was used. The genomic DNA was ampli- fied by PCR with primers PCRseqF and gPCRsku-R1, which were designed to amplify the fragment that contains the substitution site in BmIVD. The PCR product was then cleaned using the QIAquick PCR Purification Kit (Qiagen) and directly sequenced as described above. Northern blot analysis Total RNA from the fat body, midgut, Malpighian tubule and epidermis of day 2 fifth instar larvae was prepared using Trizol reagent (Invitrogen). Probes for BmIVD mRNA were amplified by PCR using the DIG probe syn- thesis Kit (Roche, Basel, Switzerland) with primers skunkRT1 and gPCRsku-R2. The vector synthesized in the protein expression experiment was used as the template. Northern blot analysis was performed according to proce- dures described previously [28,29]. Production of recombinant baculoviruses Recombinant AcMNPVs were constructed using the Bac-to-Bac Baculovirus Expression System (Invitrogen). Two recombinant viruses were constructed, one expressing wild-type BmIVD and the other expressing sku-type BmIVD. For this, the coding region of BmIVD was Odorous silkworm mutant K. Urano et al. 4460 FEBS Journal 277 (2010) 4452–4463 ª 2010 The Authors Journal compilation ª 2010 FEBS PCR-amplified using either the cDNA from p50T or sku mutant as a template with primers recombi-IVD-F2 and recombi-IVD-R2. In this procedure, a high-fidelity DNA polymerase, KOD-Plus (TOYOBO Life Science, Osaka, Japan), was used for PCR. The PCR products were digested with EcoRI and XbaI and ligated into the corre- sponding site of the pFastBac1 vector (Invitrogen). The construction and propagation of recombinant AcMNPVs were performed according to the manufacturer’s instruc- tions (Invitrogen). Expression and purification of recombinant BmIVD protein Monolayers of Sf9 cells in a 150-mm dish were infected with BmIVD-recombinant AcMNPV. After 72 h, the culture med- ium was discarded and cells were suspended in 10 mL NaCl ⁄ P i . The suspension was centrifuged at 3000 g for 10 min and the cell pellet was washed and stored at )80 °C until use. To purify recombinant proteins, harvested cells were resuspended in 5 mL of 50 mm potassium phosphate buffer (pH 8.0) per dish, together with protease inhibitor cocktail tablets (Roche). The cells were lysed by sonication for 1 min in the Branson Sonifier 250 (Branson, Danbury, CT, USA) and added with the same volume of binding buffer (5 mm imidazole, 20 mm sodium phosphate, 500 mm NaCl, pH 7.4). After centrifugation at 14 000 g for 10 min, the resulting supernatants were loaded onto a HisGraviTrap col- umn (GE Healthcare Bioscience, Little Chalfont, UK). The eluate was dialysed twice in 100 mm potassium phosphate (pH 8.0) and 100 mm NaCl using the Slide-A-Lyser Dialysis Cassette (Pierce, Rockford, IL, USA). The protein concentra- tion was determined using the Coomassie Plus Protein Assay Reagent (Pierce) with bovine serum albumin as the standard. Expression and purification of recombinant protein was confirmed by SDS ⁄ PAGE [30] and western blot as described previously [31]. Enzyme assays The isovaleryl-CoA dehydrogenase activity was assayed spectrophotometrically by the dye-reduction method using 2,6-dichloroindophenol as an electron acceptor and phenaz- inemethosulfate (PMS) as an intermediate electron carrier as described previously [32,33], with slight modifications. The incubation medium was composed of 50 mm potassium phosphate buffer (pH 8.0), 1.5 mm PMS, 0.05 mm 2,6-di- chloroindophenol, 0.1 mm FAD and 0.1 mm acyl-CoA sub- strate. The final volume was 100 lL. The enzyme reaction was carried out at 25 °C and the reaction was started with the addition of the acyl-CoA substrate. A reduction rate of 600 nm absorbancy, resulting from bleaching of 2,6-dichlo- roindophenol, was measured for 2 min using a Beckman DU 640 spectrophotometer (Beckman Coulter, Brea, CA, USA). Enzymatic activity was calculated by subtracting the reduction rate of the enzyme-excluded solution from that of the enzyme-containing solution and was expressed as nmols of 2,6-dichloroindophenol reduced per mg of protein per min. The extinction coefficient of 2,6-dichloroindophenol (21 000 MÆcm )1 ) at 600 nm was used to compute the amount of 2,6-dichloroindophenol reduced. FAD, PMS and 2,6-dichloroindophenol were obtained from Wako Pure Chemical Industries (Osaka, Japan) and isovaleryl-CoA, isobutyryl-CoA and hexanoyl-CoA substrates were obtained from Sigma-Aldrich (St. Louis, MO, USA). Comparative modelling of BmIVD structure Comparative models of wild-type and mutant BmIVDs were generated based on the crystal structure of human IVD (PDB ID: 1VH) [7]. The primary sequence of BmIVD was aligned with that of human IVD using blast [34]. The model structures were generated to have the same tetra- meric structure as the human IVD protein in the crystal structure. FAD and a substrate in the crystal structure were also included in the model. modeller 9v3 was used to gen- erate the models [35]. Conformations of the side chains were refined with SCWRL 3.0 [36] and the quality of the models was evaluated with Verify3D [37]. Acknowledgements This work was supported by grants from MEXT (Nos. 17018007 to T.S.), JSPS (21248006 to TD and TS), MAFF-NIAS (Agrigenome Research Program) and JST (Professional Program for Agricultural Bioinfor- matics), Japan. The silkworm strains and DNA clones were provided by the National Bioresource Project (NBRP), Japan. References 1 Thorpe C & Kim JJ (1995) Structure and mechanism of action of the acyl-CoA dehydrogenases. FASEB J 9, 718–725. 2 Tanaka K, Budd MA, Efron ML & Isselbacher KJ (1966) Isovaleric acidemia: a new genetic defect of leu- cine metabolism. Proc Natl Acad Sci USA 56, 236–242. 3 Vockley J & Ensenauer R (2006) Isovaleric acidemia: new aspects of genetic and phenotypic heterogeneity. Am J Med Genet C Semin Med Genet 142, 95–103. 4 Yoshitake N, Kobayashi M & Miyashita T (1978) On the ‘skunk’ mutant in the silkworm. 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One of the most intriguing features of the sku mutant is that the mature larva accumulates branched-chain amino a cids, leucine, isoleucine and valine, in. effect of the mutation was further investi- gated by molecular modelling. Together with the previ- ously reported traits of the sku mutant, the molecular and

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