Keywords Bombyx mori; branched-chain amino acid; isovaleric acidemia; isovaleryl-CoA dehydrogenase; responsible gene Correspondence T.. Linkage analysis revealed that the silkworm IVD ge
Trang 1skunk mutant of silkworm, Bombyx mori
Kei Urano1, Takaaki Daimon1, Yutaka Banno2, Kazuei Mita3, Tohru Terada4, Kentaro Shimizu4,5, Susumu Katsuma1and Toru Shimada1,4
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.
Trang 2IVD 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
skumutant 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 moriwas 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.
Trang 3the 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)
Probe
skunk-RT1
–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
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.
Trang 4the 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 genopheno-type, 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 F1 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⁄ G or G ⁄ 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
+skuand 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
1127 |
P
F1
1127 | 1127 |
or + + +
or sku
+ +
or +
or sku
or sku
or sku
or sku
or +
or sku
+ +
Non-odorous Odorous
SNP of BmIVD
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 F1generation 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 F1 inter-cross (for details, see Fig 3).
Phenotype (genotype)
Number of larvae screened
1127th base pair
of BmIVD ORF
G ⁄ G G ⁄ T T ⁄ T
Trang 5participate 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)1Æmin)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.
Trang 6result 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 degradadysfunc-tion of
75
50
37
25
(kDa)
1 2 3 4 5 6 7 8 9 10
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 m M imidazole).
Trang 7isovaleryl-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 spinnbecom-ing (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 acids, 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
*
*
**
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).
Trang 8higher 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 moristrains 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
B
G376
V376 I369
I369
BmIVD IVD SBCAD GCD SCAD LCAD VLCAD ACAD10
398 399 415 377 423 938
J
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.
Trang 9The silkworms were reared on fresh mulberry leaves in an
insect rearing chamber under standard conditions (25C,
12L : 12D photoperiod) Sf9 cells were cultured at 27C in
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⁄ Pi (137 mm NaCl, 2.7 mm KCl, 8.1 mm Na2HPO4,
1.5 mm KH2PO4), 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
IVDgene, 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 94C for 30 s, annealing at 54 C for 30 s
and extension at 72C 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
Trang 10PCR-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⁄ Pi 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 25C 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
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