From the Division of Cell Biology, Soka
University, Hachioji, Tokyo 192-8577, Japan, § Department of
Biology, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan, ¶ Invertebrate Genetics Laboratory, National Institute of
Genetics, Mishima, Shizuoka 441-8540, Japan,
Genetic
Networks Research Group, Mitsubishi Kagaku Institute of Life Science,
Machida, Tokyo 194-8511, Japan, ** Core Research for
Evolutional Science and Technology of Japan Science and Technology
Corporation, Kawaguchi, Saitama 332-0012, Japan, the
Department of Molecular and Cellular Biology and
Division of Neurobiology, University of Arizona, Tucson, Arizona 85721, and the §§ Department of Biophysics and
Biochemistry, Graduate School of Science, University of Tokyo,
Bunkyo-ku, Tokyo 113-0033, Japan
Received for publication, August 27, 2002, and in revised form, February 3, 2003
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ABSTRACT |
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Heparan and chondroitin sulfates play essential
roles in growth factor signaling during development and share a
common linkage tetrasaccharide structure,
GlcA Proteoglycans are widely expressed on the cell surface
and in the extracellular matrix of various tissues and play important roles in the control of growth and differentiation. Proteoglycans consist of a core protein and negatively charged glycosaminoglycans (GAGs)1 that interact with
growth factors, the components of extracellular matrix, morphogens, and
cytokines (1-3). GAGs are classified into two categories,
heparin/heparan sulfate (HS) and chondroitin sulfate (CS)/dermatan
sulfate (DS). The biosynthesis of GAG is initiated by the formation of
the linkage tetrasaccharide structure, GlcA RNA interference (RNAi) was first recognized in C. elegans
as a biological response to exogeneous double-stranded RNA (dsRNA), which induces sequence-specific gene silencing. RNAi is an
evolutionarily conserved phenomenon and a multistep process involving
the generation of active small interfering RNA (siRNA) in
vivo through a reaction with an RNase III endonuclease, Dicer
(14-16). The resulting 21-23-nucleotide siRNA mediates degeneration
of the complementary homologous RNA (17). RNAi has recently emerged as
a powerful reverse genetics tool to study gene function in many model
organisms, including plants, C. elegans and D. melanogaster in which large dsRNAs efficiently induce
gene-specific silencing (18, 19). Only recently, DNA vector-based siRNA
has been reported to suppress the expression of the corresponding gene
in mammalian cells (20, 21).
In the present study, we identified the Drosophila
proteoglycan Materials--
The Drosophila expressed
sequence tag clone CK02622 was obtained from Research Genetics, Inc.
(Huntsville, AL). UDP-Gal, p-nitrophenyl- Identification of the Drosophila Proteoglycan
Construction and Purification of d
pVL1393-FLAG-d Western Blot Analysis--
The enzymes purified above were
subjected to 12.5% SDS-polyacrylamide gel electrophoresis, followed by
Western blot analysis. The separated proteins were transferred to a
Hybond-P membrane (Amersham Biosciences). The membrane was probed with
anti-FLAG M2-peroxidase conjugate (Sigma) and stained with Konica
Immunostaining HRP-1000 (Konica, Tokyo, Japan). The intensity of
positive bands on Western blotting was measured by densitometer to
determine the amount of the purified enzyme using FLAG-BAP Control
Protein (Sigma).
Assay of Galactosyltransferase Activity--
To determine the
galactosyltransferase activity, Xyl RNAi Fly--
A cDNA fragment encoding the C-terminal region
(nucleotide 685-969 of coding sequence) or the amino-terminal
(N-terminal) region (nucleotide 1 to 506 of coding sequence) of
d
The transformation of Drosophila embryos was carried
out according to Spradling (22) with w1118 mutant
stock as a host to make 23 UAS-d Quantitative Analysis of d Identification of the Drosophila Proteoglycan
When the other members of the human
The ClustalW alignment of the human and Drosophila Characterization of the Galactosyltransferase Activity of
d
The purified enzymes were used for a galactosyltransferase assay with
various acceptor substrates (Table II).
The determined amounts of d Viability of Inducible d
A scheme of the heritable and inducible RNAi system is shown in Fig.
3. In this report, we used
Act5C-GAL4 as a GAL4 driver to induce
d
The phenotypes of the F1 of each
UAS-d Quantitative Analysis of d
N lines have a transgene including the IR of the sequence
encoding the N-terminal region of d
Similar analyses were performed for the F1 progeny of C
lines crossed with Act5C-GAL4 fly,
Act5C-GAL4/C5, and
Act5C-GAL4/C6. Both RNAi flies showed lethality
at the pupal stages (Table III). The d
We also determined the amount of d
The above results clearly demonstrated that the expression of the
target gene was specifically reduced by RNAi in this
Drosophila RNAi system to induce the phenotype.
We identified the Drosophila proteoglycan Drosophila has three members of the The three members of the d d We made 24 UAS-d We analyzed the amounts of three d The efficiency of RNAi largely did not depend on the target sequences
using the constructions of IR (Fig. 4, A and B),
and the ratio of degraded d d D. melanogaster is well established as a model for genetic
analysis. Recently, fly mutants with severe phenotypes related to
developmental processes have begun to be used to address the defects of
glycosyltransferases. As one example, the Drosophila fringe
gene has been demonstrated to encode an O-fucose
In this report, we demonstrated the first systematic reverse genetics
approach using RNAi of Drosophila glycosyltransferase and
showed that RNAi worked well in the case of a glycosyltransferase. We
found almost 70 Drosophila glycosyltransferases by
performing a TBLASTN search of the Drosophila data bases
using mammalian glycosyltransferases as the query sequence. It is
possible to make a RNAi fly for each of the 70 glycosyltransferases,
whereas knock-out mice cannot be made. The inducible
glycosyltransferase RNAi fly obtained using the GAL4-UAS system will
open the way for the analysis of the biological role of glycans.
1,3Gal
1,3Gal
1,4Xyl
1-O-Ser. In the
present study, we identified the Drosophila
proteoglycan UDP-galactose:
-xylose
1,4-galactosyltransferase I (d
4GalTI), and determined its
substrate specificity. The enzyme transferred a Gal to the -
-xylose
(Xyl) residue, confirming it to be the Drosophila ortholog
of human proteoglycan UDP-galactose:
-xylose
1,4-galactosyltransferase I. Then we established
UAS-d
4GalTI-IR fly lines containing an
inverted repeat of d
4GalTI
ligated to the upstream activating sequence (UAS) promoter, a
target of GAL4, and observed the F1 generation of the cross
between the UAS-d
4GalTI-IR fly and the
Act5C-GAL4 fly. In the F1, double-stranded RNA
of d
4GalTI is expressed ubiquitously under
the control of a cytoplasmic actin promoter to induce the silencing of
the d
4GalTI gene. The expression of the
target gene was disrupted specifically, and the degree of interference
was correlated with phenotype. The lethality among the progeny proved
that
4GalTI is essential for viability. This study is the first to
use reverse genetics, RNA interference, to study the
Drosophila glycosyltransferase systematically.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,3Gal
1,
3Gal
1,4Xyl
1-O-Ser, which is common to heparin/HS and CS/DS. In humans, all four kinds of
glycosyltransferases related to the synthesis of the linkage
tetrasaccharide structure have been cloned: two peptide
O-xylosyltransferases (O-XylT) (4, 5), one
proteoglycan
1,4-galactosyltransferase I
(
1,4-galactosyltransferase 7) (
4GalTI) (6, 7), one
proteoglycan
1,3-galactosyltransferase II
(
1,3-galactosyltransferase 6) (
3GalTII) (8), and one
glucuronosyltransferase I (GlcATI) (9). In nematodes, two
glycosyltransferases,
4GalTI and GlcATI, have
already been cloned and characterized (10). Biochemical analysis of
GAGs has demonstrated that both Caenorhabditis elegans and
Drosophila melanogaster have HS and CS (11, 12). Recently,
Drosophila peptide O-XylT has been reported to
transfer xylose (Xyl) to the syndecan peptide (13). But it is still
unknown which Drosophila
4GalT works as proteoglycan
4GalTI.
4GalTI (d
4GalTI) and performed
a biochemical characterization. The protein transferred a Gal to the
-
-Xyl residue, confirming it to be the Drosophila
ortholog of human proteoglycan
4GalTI (h
4GalTI). After that, we
produced an inducible d
4GalTI RNAi fly using
the GAL4-UAS system as a first step toward clarifying the
biological role of d
4GalTI. The d
4GalTI
mRNA was reduced specifically by RNAi, and the severity of the
phenotype showed the correlation with the reduction in
d
4GalTI mRNA. The death of the flies
proved that d
4GalTI is essential for viability. This is the first
example of the use of reverse genetics to study Drosophila
glycosyltransferase systematically.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-xylopyranoside (Xyl
-pNph), Xyl
-pNph,
o-nitrophenyl-
-xylopyranoside (Xyl
-oNph), p-nitrophenyl-N-acetyl-1-thio-
-glucosaminide
(GlcNAc
-S-pNph), p-nitrophenyl-
-glucopyranoside
(Glc
-pNph), Glc
-pNph,
p-nitrophenyl-
-galactopyranoside (Gal
-pNph), Gal
-pNph,
p-nitrophenyl-N-acetyl-
-galactosaminide (GalNAc
-pNph), and GalNAc
-pNph were
purchased from Sigma. The GlcNAc
-pNph,
GlcNAc
-pNph, and
p-nitrophenyl-
-mannopyranoside (Man
-pNph)
were purchased from Calbiochem.
Gal
1,4Xyl
1-p-methoxyphenyl (Gal
1,4Xyl
1-pMph) was provided by Seikagaku Corp.
Uridine diphosphate-[14C]galactose
(UDP-[14C]Gal) (325 mCi/mmol) was supplied by PerkinElmer
Life Sciences.
1,4-Galactosyltransferase I--
We performed a BLAST search of all
Drosophila databases and identified one
Drosophila proteoglycan
1,4-galactosyltransferase I gene,
CG11780. The Drosophila expressed sequence tag
clone CK02622 including CG11780 was obtained. The plasmid
DNA was prepared from CK02622 and sequenced using an ABI PRISM
BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit
(ABI, Foster City, CA).
4GalTI and h
4GalTI
Proteins Fused with FLAG Peptide--
The putative catalytic domain of
d
4GalTI (amino acids 36-322) was expressed as a secreted protein
fused with a FLAG peptide in insect cells according to the instruction
manual of GATEWAYTM Cloning Technology (Invitrogen). An ~0.9-kb DNA
fragment was amplified by two-step PCR. The first PCR used the plasmid
DNA from expressed sequence tag clone CK02622 as a template, the
forward primer 5'-AAAAAGCAGGCTTGTGCCCGCTGTCCAATCCGCTG-3', and
the reverse primer 5'-AGAAAGCTGGGTACCCATCAGGTTTGTACCGC-3'. The second
PCR used the first PCR product as a template, the forward primer
5'-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3' and the reverse primer 5'-GGGGACCACTTTGTACAAGAAAGCTGGGT-3'. The forward and reverse primers were flanked with attB1 and attB2 sequences,
respectively, to create the recombination sites. The amplified fragment
was recombined between the attP1 and attP2 sites
of the pDONRTM201 vector using the BP CLONASE Enzyme Mix
(Invitrogen). Then the insert was transferred between the
attR1 and attR2 sites of pVL1393-FLAG to yield
pVL1393-FLAG-d
4GalTI. pVL1393-FLAG is an expression
vector derived from pVL1393 (Pharmingen, San Diego, CA) and
contains a fragment encoding the signal peptide of human immunoglobulin
(MHFQVQIFSFLLISASVIMSRG), the FLAG peptide (DYKDDDDK), and a
conversion site for the GATEWAY system.
pVL1393-FLAG-h
4GalTI was also prepared by the
same procedure using the two primers, 5'-AAAAAGCAGGCTGGGCAGTCAGGGGACAAG-3' and
5'-AGAAAGCTGGGTCACTGTCCAT- CCAGCTCA-3'.
4GalTI and
pVL1393-FLAG-h
4GalTI were cotransfected with
BaculoGold viral DNA (Pharmingen, San Diego, CA) into Sf21 insect cells according to the manufacturer's
instructions and incubated for 3 days at 27 °C to produce
recombinant viruses. Sf21 cells were infected with
each recombinant virus at a multiplicity of infection of 5 and
incubated for 72 h to yield conditioned media containing
recombinant
4GalTI proteins fused with FLAG peptide. A 5-ml volume
of culture medium was mixed with 100 µl of anti-FLAG M1 AFFINITY GEL
(Sigma). The protein-gel mixture was washed twice with 50 mM Tris-buffered saline (50 mM Tris-HCl, pH
7.4, and 150 mM NaCl) containing 1 mM
CaCl2 and eluted with 100 µl of 100 µg/ml FLAG peptide
in 10 mM Tris-buffered saline (Sigma).
-pNph,
Xyl
-pNph, Xyl
-oNph,
GlcNAc
-pNph, GlcNAc
-pNph,
GlcNAc
-S-pNph, Glc
-pNph,
Glc
-pNph, Gal
-pNph, Gal
-pNph,
GalNAc
-pNph, GalNAc
-pNph, Man
-pNph, and Gal
1,4Xyl
1-pMph were
utilized as acceptor substrates. With 10 nmol of each acceptor, the
4GalT activity reaction was performed at both 25 and 37 °C for
2 h in 20 µl of a reaction mixture containing 14 mM
Hepes buffer (pH 7.4), 0.5% Triton X-100, 11 mM
MnCl2, 3 µM UDP-[14C]Gal (325 mCi/mmol), 250 µM UDP-Gal, and 1.15 and 0.44 pmol of purified d
4GalTI and h
4GalTI, respectively. The enzyme reaction was terminated by the addition of 400 µl of water. After
centrifugation of the reaction mixture, the supernatant was applied to
a Sep-PakC18 column (Millipore Corp.) equilibrated with water. The
unreacted UDP-Gal was washed out with water, and the products were
eluted with methanol. The eluates were dried with an N2
evaporator and dissolved in 30 µl of methanol. Then 10 µl of the
product was applied to an HPTLC plate (Merck) and developed in
chloroform/methanol/0.2% CaCl2 (55:45:10). The bands of
reaction products incorporating radioactivity were detected with a
BAS2000 Imaging Analyzer system (Fuji, Tokyo, Japan).
4GalTI was amplified by PCR and inserted as an inverted repeat (IR)
in a modified Bluescript vector, pSC1, which possesses an IR formation site consisting of paired CpoI and SfiI
restriction sites. In all cases, the IR was in a head-to-head
orientation. IR-containing fragments were cut out by NotI
and subcloned into pUAST, a transformation vector. The cloning
procedures will be described
elsewhere.2
4GalTI-IR fly lines. Then
we made one line, N13, which has two copies of the IR, by crossing two
lines, N2 and N4, in which the IR was on different chromosomes. Each
line was mated with the Act5C-GAL4 fly line, and
F1 progeny was raised at 28 °C to observe phenotypes.
4GalTI mRNA by
Competitive RT-PCR--
Total RNA was extracted from
Act5C-GAL4/UAS-d
4GalTI-IR and
Act5C-GAL4/+ third instar larvae and prepupae by the methods
of Chomczynski and Sacchi (23). Poly(A)+ RNA was isolated
from total RNA using
OligotexTM-dT30<super>
(Takara Bio Inc.) according to the manufacturer's instructions.
First-strand cDNA was synthesized in 50 µl of a reaction mixture
containing 300 ng of mRNA, 5 mM MgCl2, 10 mM dithiothreitol, 0.5 µg of oligo(dT)12-18,
0.5 mM each dNTP, 40 units of RNasin, and 50 units of
Superscript II RT (Invitrogen). After incubation at 50 °C for 50 min, the reaction was terminated by heating at 70 °C for 15 min,
followed by rapid chilling on ice. Competitive RT-PCR of
d
4GalTI was carried out for the region except
for the sequences using the IR construction for RNAi. The gene-specific
primers used for amplification of d
4GalTI,
d
4GalTA, d
4GalTB, and
ribosomal protein L32 (RpL32) genes are listed in Table I.
The sense and antisense primers for construction of the DNA competitor
were prepared by flanking the sequence for amplification of
DNA at
the 3' terminus of each sense and antisense primer for amplification of
the target cDNA. The DNA competitors were generated using reagents
supplied in the Competitive DNA construction kit (Takara Bio Inc.).
Competitive RT-PCRs were performed using 1 µl of the first-strand
cDNA mixture with a serial dilution of DNA competitor in 50-µl
reaction mixtures containing a 0.2 µM concentration of
each of the relevant primers (listed in Table I), 0.2 mM each dNTP, and 2.5 units of TaKaRa Ex Taq. To normalize the efficiency of
cDNA preparation among individual samples, measurement of
RpL32 mRNA in each cDNA (0.5 µl) was carried out
using the same competitive RT-PCR method as for
d
4GalTI mRNA. Amplifications involved 30 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. PCR products were subjected to electrophoresis in a 3-4% NuSieve 3:1 agarose gel (Cambrex, Corp., East Rutherford, NJ)
and stained with ethidium bromide. ImageMaster VDS-CL (Amersham Biosciences) was used to generate digital images of the agarose gel.
The intensities of amplified fragments were quantified using ImageMaster analysis software. The amount of target mRNA was
estimated from the ratio of the intensity of the competitor band and
the target band.
Gene-specific primers used for competitive RT-PCR
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,4-Galactosyltransferase I--
When human h
4GalTI was used as
the query sequence for a TBLASTN search of the Berkeley
Drosophila Genome Project, one highly homologous gene,
CG11780, was obtained as d
4GalTI. The complete cDNA of the d
4GalTI gene and the predicted amino acid
sequence are shown in Fig. 1A.
The putative protein, consisting of 323 amino acids, was a type II
transmembrane protein with a hydrophobic domain in the N-terminal
region (Fig. 1B).
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Fig. 1.
cDNA and predicted amino acid sequence of
D. melanogaster proteoglycan
1,4-galactosyltransferase I. A, the
nucleotide sequence and the predicted amino acid sequence. The putative
transmembrane domain is underlined. B, the
hydrophobicity plot estimated by the method of Kyte and Doolittle with
a window of 7 amino acid residues.
1,4-galactosyltransferase
family, h
4GalT1 to -6, were used as query sequences for the TBLASTN
search, two highly homologous genes, CG8536 and
CG14517, were also obtained as members of the
Drosophila
1,4-galactosyltransferase family,
d
4GalTA and d
4GalTB, respectively.
d
4GalTA and d
4GalTB showed much lower homology of amino acids to
h
4GalTI (33 and 29%, respectively) than d
4GalTI to h
4GalTI
(48%).
4GalT
families showed that the three
4GalT motifs found in the human family, including the DXD motif, a metal binding site, were
also conserved in the Drosophila
4GalT family (Fig.
2A) (24, 25). A phylogenetic
tree of the three Drosophila (d
4GalTI, d
4GalTA, and
d
4GalTB) and seven human
4GalTs (h
4GalT1 to -6 and h
4GalTI) was generated based on the amino acid sequences (Fig. 2B).
d
4GalTI was confirmed to be the Drosophila ortholog of
h
4GalTI. Both d
4GalTA and d4
GalTB showed higher homology to
h
4GalT1 to -6 than h
4GalTI.
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Fig. 2.
Comparison of Drosophila
4GalTs and human
4GalTs. A, ClustalW alignment of
the predicted amino acid sequences. Introduced gaps are indicated by
hyphens. The asterisks indicate the amino acids
identical among all proteins. Conserved amino acids are shown by
dots. The three
4GalT motifs, including the
DXD motif, a metal binding site, are boxed.
B, phylogenetic tree of three Drosophila
(d
4GalTI, d
4GalTA, and d
4GalTB) and seven human
4GalTs
(h
4GalT1 to -6 and h
4GalTI). d
4GalTI is the
Drosophila ortholog of h
4GalTI, which is human
proteoglycan
1,4-galactosyltransferase I, namely human
4GalT7.
The branch lengths indicate amino acid substitution per site.
4GalTI--
The FLAG-tagged recombinant d
4GalTI was expressed
in insect cells to determine whether or not d
4GalTI has
galactosyltransferase activity. The soluble form was prepared by
replacing the N-terminal region including the cytoplasmic and
transmembrane domains, amino acids 1-35, with an Ig
signal sequence
and FLAG peptide sequence. The secreted enzyme was purified with
anti-FLAG M1 gel and quantitated by Western blotting analysis using
Anti-FLAG anti-body. FLAG-tagged recombinant h
4GalTI was also
prepared by the same procedure.
4GalTI and h
4GalTI and the same amount
of each substrate were used for the enzyme reactions, so we could
determine relative activities that were comparable. h
4GalTI had
strong activity toward the -
-Xyl residue, whereas it had only slight
activity toward -
-Xyl and no activity toward -
-GlcNAc, -
-Glc,
-
-Gal, and -
-GalNAc as reported previously (6, 7). d
4GalTI
also showed strong activity toward the -
-Xyl residue; only slight activity toward -
-Xyl; and no activity toward -
-GlcNAc, -
-Glc, -
-Gal, and -
-GalNAc. These results demonstrated that d
4GalTI was the Drosophila ortholog of h
4GalTI in view of their
activities. But the
4GalT activity of d
4GalTI toward the -
-Xyl
residue was almost half that of h
4GalTI at both 25 and 37 °C.
Acceptor substrate specificities of purified recombinant d4GalTI and
h
4GalTI expressed in the baculovirus system
GalTI RNAi Flies--
Proteoglycan
4GalTI contributes to the synthesis of the common
carbohydrate-protein linkage structure,
GlcA
1,3Gal
1,3Gal
1,4Xyl
1-O-Ser, of
proteoglycan including heparin/HS and CS/DS. If proteoglycan
4GalTI
is inactivated, every proteoglycan lacks GAG, and a severe biological
phenotype is expected. To test this hypothesis, we tried to make
inducible d
4GalTI RNAi flies according to the method described under "Experimental Procedures."
4GalTI gene silencing in all cells of the
fly. The Act5C-GAL4 fly has a transgene containing yeast
transcriptional factor GAL4, the expression of which is
under the control of the cytoplasmic actin promoter. 24 UAS-d
4GalTI-IR fly stocks having a transgene
containing two types of the IR of d
4GalTI
ligated to the UAS promoter, a target of GAL4, were established (Table III). The IR of
d
4GalTI was separated by an unrelated DNA
sequence that acts as a spacer to give a hairpin loop-shaped RNA. C-1
to C-11 have a transgene containing the IR of the sequence encoding the
C-terminal region of the catalytic domain (amino acids
209-322). N-1 to N-13 have a transgene containing the IR
of the sequence encoding the N-terminal region (amino acids 1-167). In
the F1 generations of the Act5C-GAL4 fly and the
UAS-d
4GalTI-IR fly, dsRNA of
d
4GalTI is expressed ubiquitously under the
control of the cytoplasmic actin promoter to induce
d
4GalTI gene silencing.
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Fig. 3.
Schematic representation of the heritable and
inducible RNAi system. Two transgenic fly stocks,
GAL4-driver and UAS-IR, are used in this system.
The GAL4-driver fly used in this experiment has a transgene
containing yeast transcriptional factor GAL4, the expression
of which is under the control of the cytoplasmic actin promoter. The
UAS-IR fly has a transgene containing the inverted repeat of
the target gene ligated to the UAS promoter, a target of GAL4. In the
F1 progeny of these flies, the dsRNA of the target gene is
expressed ubiquitously in all cells to induce the gene silencing.
Viability of inducible d4GalTI RNAi fly
4GalTI-IR fly crossed with the
Act5C-GAL4 fly are shown in Table III; 65% (15 of 23) of
these crosses caused lethality in the progeny (i.e. the
flies could not develop into adults). The expression of
d
4GalTI dsRNA by crossing the N13 line
carrying two copies of UAS-d
4GalTI-IR to the
Act5C-GAL4 fly also led to lethality at the pupal stages. These results clearly demonstrated that proteoglycan
4GalTI is essential for the viability of flies.
4GalT mRNA in Each Inducible
d
4GalTI RNAi Fly by Competitive RT-PCR--
To test the efficiency
and specificity of RNAi in this system, the mRNA levels of all
Drosophila
4GalTs
(d
4GalTI, d
4GalTA, and d
4GalTB) were determined in each
d
4GalTI RNAi fly by competitive RT-PCR (Fig.
4). The relative amount of
each
4GalT mRNA to RpL32 mRNA in
F1 progeny of w1118 crossed with the
Act5C-GAL4 fly, Actin5C-GAL4/+, which corresponds to the wild type, was presented as 1. The F1 progeny of
each N or C line of the UAS-d
4GalTI-IR fly
crossed with Act5C-GAL4 fly was designated as
Act5C-GAL4/N or
Act5C-GAL4/C, respectively.
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Fig. 4.
Quantitative analysis of d 4GalTI
mRNA in each inducible d
4GalTI RNAi fly by
competitive RT-PCR. The mRNA levels of all
Drosophila
4GalTs
(d
4GalTI, d
4GalTA,
and d
4GalTB) in each d
4GalTI
RNAi fly were determined by competitive RT-PCR. The actual amount of
each
4GalT mRNA was divided by that of
RpL32 mRNA for normalization. The relative amount of
each
4GalT mRNA to RpL32 mRNA in F1 progeny of the
w1118 crossed with Act5C-GAL4 fly,
Act5C-GAL4/+, which corresponds to the wild type,
was presented as 1. Amounts of 1 and 0.5 µl of synthesized cDNA
were used for the quantitation of
4GalTs and
RpL32, respectively. The cDNAs for each
4GalT mRNA and RpL32 mRNA were
amplified together with 0.5-100 × 105 copies and
2.5-10 × 107 copies, respectively, of the
corresponding competitor DNAs. A, the mRNA levels of
three kinds of
4GalTs in the third instar larvae of the
F1 progeny of each N line of
UAS-d
4GalTI-IR fly crossed with
Act5C-GAL4, designated as
Act5C-GAL4/N. Each N line has the IR of the
sequence encoding the N-terminal region of d
4GalTI.
Act5C-GAL4/N2,
Act5C-GAL4/N4, and
Act5C-GAL4/N13 showed lethality at the pupal
stages, whereas Act5C-GAL4/N6 was viable and
morphologically normal. The N13 line has two copies of the IR on
chromosomes 2 and 3. B, the mRNA levels of three kinds
of
4GalTs in the third instar larvae of the
F1 progeny of each C line of
UAS-d
4GalTI-IR fly crossed with
Act5C-GAL4 fly, designated as
Act5C-GAL4/C. Each C line has the IR of the
sequence that encodes the C-terminal region of d
4GalTI.
Act5C-GAL4/C5 and
Act5C-GAL4/C6 were pupal lethal. C,
the mRNA level of d
4GalTI in the prepupae of
Act5C-GAL4/N4 and
Act5C-GAL4/N13.
4GalTI. N13 having two copies of
the IR on chromosomes 2 and 3 was made from the N2 and N4 lines. The
degree of expression of the transgene is known to depend on its sites
of insertion on the chromosome. Act5C-GAL4/N2,
Act5C-GAL4/N4, and
Act5C-GAL4/N13 were pupal lethal, whereas
Act5C-GAL4/N6 was viable and morphologically
normal (Table III). First, we determined the amounts of the three kinds
of d
4GalTs mRNA in the third instar larvae
of these four RNAi flies and the wild-type fly,
Act5C-GAL4/+. The ratios of reduction in
d
4GalTI mRNA of
Act5C-GAL4/N13,
Act5C-GAL4/N4, Act5C-GAL4/N2, and
Act5C-GAL4/N6 were 0.26, 0.32, 0.36, and 0.76, respectively, demonstrating a correlation with the severity of the
phenotype (Fig. 4A). F1 progeny of the N13 line
having two copies of the IR had less d
4GalTI
mRNA than those of the N2 line and N4 line, which were crossed to
make the N13 line. Reductions in d
4GalTA
mRNA and d
4GalTB mRNA were not
observed in all RNAi flies. It was clearly demonstrated that the
d
4GalTI mRNA was disrupted specifically,
and the ratio of degraded d
4GalTI mRNA was
well correlated with the severity of the phenotype.
4GalTI
mRNAs in the third instar larvae were also interfered with
specifically, and the ratios of degraded
d
4GalTI mRNA of Act5C-GAL4/C5 and
Act5C-GAL4/C6 (0.35 and 0.45, respectively) were
almost the same as those of Act5C-GAL4/N2 and
Act5C-GAL4/N4 (Fig. 4B). The
efficiency of RNAi did not depend greatly on the target sequences using
the constructions of IR.
4GalTI mRNAs in
prepupae of Act5C-GAL4/N4 and
Act5C-GAL4/N13 (Fig. 4C). The target
efficiency in the prepupae was almost the same as that in the third
instar larvae.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4GalTI by
molecular and biochemical analyses and then made the RNAi fly to
investigate d
4GalTI function in vivo. The expression of
the target gene was disrupted specifically in the RNAi fly and the
degree of interference was correlated to phenotype. The reduction of
d
4GalTI mRNA caused lethality, indicating
an essential function of d
4GalTI for viability. This is the first
example of a reverse genetics approach to the systematic study of
Drosophila glycosyltransferase.
4GalT family,
d
4GalTI, d
4GalTA, and d
4GalTB (Fig. 2A). The
phylogenetic tree (Fig. 2B) and acceptor substrate
specificities (Table II) of the
4GalT family clearly demonstrated
that d
4GalTI is the Drosophila ortholog of h
4GalTI.
d
4GalTA and d
4GalTB showed higher homology to h
4GalT1 to -6 than to h
4GalTI, but the Drosophila ortholog of the six h
4GalTs could not be identified. So there is a possibility that d
4GalTA and d
4GalTB share acceptor substrates, to which
h
4GalT1 to -6 can transfer Gal. Recently, h
4GalT1 has been
reported to transfer Gal to fringe-modified
O-fucose glycans on the Notch protein and the elongation of
glycans was necessary to modulate Notch signaling. A novel
Drosophila galectin has also been isolated (26). It is still
unknown which of the two d
4GalTs works to elongate
O-fucose glycans on Notch or synthesize the ligands of Drosophila galectin. We are now attempting to determine the
substrate specificity of the two d
4GalTs.
4GalT family also conserved the
three
4GalT motifs found in the h
4GalT family (Fig.
2A) (25). The crystal structure of the bovine
4GalT1 has
already been reported (24, 27, 28). The DXD motif is a
Mn2+-binding site, and the other two motifs expose the
surface of the catalytic pocket. The FNRA motif is involved in UDP-Gal
binding and the negatively charged residues of the
GWGXEDD(D/E) motif contribute to UDP-Gal and UDP-glucose
binding. The three motifs conserved between human and
Drosophila had amino acid sequences that related to the
binding of metal or donor substrates.
4GalTI showed roughly half the activity of h
4GalTI toward
each substrate at both 25 and 37 °C, although the substrate
specificities of the two were similar (Table II). The breeding
temperature of flies and the body temperature of humans are 25 and
37 °C, respectively. Under physiological conditions, h
4GalTI
performed a more efficient reaction than d
4GalTI. Very recently, two
papers about d
4GalTI have been published (29, 30) reporting similar
enzymatic activity to ours.
4GalTI-IR fly lines and
observed the F1 generation of each
UAS-d
4GalTI-IR fly crossed with the
Act5C-GAL4 fly. The severity of the phenotype differed
between the stocks. Approximately 65% of the flies died at the pupal
stages (Table III), but some lived to become adults, similar in
morphology to the wild type. Because the degree of expression of the
transgene is known to depend on its sites of insertion on the
chromosome, it is reasonable that the phenotypes differed. The
reduction in d
4GalTI mRNA was correlated
with the severity of the phenotype (Fig. 4A). The severest
phenotype should be considered to represent the real phenotype of the
mutant. Although we have no data indicating that cell, tissue, or organ
abnormalities caused the death of individual flies, finer analyses of
the phenotype should reveal the in vivo function of
d
4GalTI.
4GalT
mRNAs (d
4GalTI,
d
4GalTA, and d
4GalTB)
to estimate the specificity and efficiency of RNAi in our
Drosophila-inducible RNAi system. The RNAi occurred only on
d
4GalTI and had no effect on the other members
of the d
4GalT family, d
4GalTA and
d
4GalTB (Fig. 4, A and
B). During the process of RNAi, 21-23-nucleotide siRNA
mediates the degeneration of the complementary homologous RNA (17). If
even one nucleotide differs between the siRNA and target mRNA,
siRNA cannot mediate the degeneration of target RNA (17). Comparing the
DNA sequence of d
4GalTI with the sequences of
d
4GalTA and d
4GalTB,
we could not find any identical regions longer than 21 nucleotides.
This must be the reason why the RNAi of
d
4GalTI was specific with no cross-effect for
d
4GalTA and
d
4GalTB.
4GalTI mRNA was
well correlated with the severity of the phenotypes. These findings
demonstrate that our Drosophila inducible RNAi system has
the potential to become a powerful tool for analyses of the biological
roles of glycosyltransferase. However, one might have to make an effort
to increase the efficiency of RNAi. A small amount of maternal RNA
might remain even after the occurrence of RNAi.
4GalTI contributes to the synthesis of the common linkage structure
of heparin/HS and CS/DS. When d
4GalTI is inactivated in the RNAi
fly, levels of both GAGs are reduced. Our results clearly demonstrated
that GAGs on core proteins are important to viability (Table III). It
has been reported that the h
4GalTI gene of
patients with Ehlers-Danlos syndrome has mutations that reduce
4GalTI activity (7, 31). The patients show an aged appearance,
developmental delay, dwarfism, craniofacial disproportion, generalized
osteopenia, and various connective abnormalities. The RNAi fly of
d
4GalTI should serve as a model of this kind of disease.
1,3-N-acetylglucosaminyltransferase that extends the
O-fucose moieties on Notch to modulate Notch activation by
the ligands, Delta and Serrate/Jagged (32, 33). Some flies with
mutations related to proteoglycan, dally (34), sugarless, tout-velu, and sulfateless
(2), have demonstrated defects in signaling of the growth factors
including Wingless, Decapentaplegic, Hedgehog, and fibroblast growth
factors. Recently, one recessive lethal mutant has been reported to
have a missense mutation causing a reduction of
UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase activity (35, 36). Considering the recent progress made in studies of
Drosophila glycans as mentioned above, D. melanogaster will become a powerful tool for analysis of the
biological roles of glycans.
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ACKNOWLEDGEMENTS |
---|
We thank Seikagaku Corporation for providing the substrate. We also thank M. Hisamatsu, N. Hashimoto, K. Ohtsu, M. Yamamoto, W. Awano, and R. Shimamura for technical assistance.
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FOOTNOTES |
---|
* This work was supported in part by Core Research for Evolutional Science and Technology of Japan Science and Technology Corporation. This work was also supported in part by Grant-in-Aid for Scientific Research (C) 12680620 from MEXT Japan (to S. N.) and a Grant-in-Aid for Scientific Research on Priority Areas (C) "Genome Science" from MEXT Japan (to R. U. and K. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶¶ To whom all correspondence should be addressed. Tel.: 81-426-91-8140; Fax: 81-426-91-8140; E-mail: shoko@t.soka.ac.jp.
Published, JBC Papers in Press, February 17, 2003, DOI 10.1074/jbc.M301123200
2 R. Ueda and K. Saigo, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are:
GAGs, glycosaminoglycans;
4GalT,
1,4-galactosyltransferase;
HS, heparan
sulfate;
CS, chondroitin sulfate;
DS, dermatan sulfate;
Xyl, xylose;
RNAi, RNA interference;
dsRNA, double-stranded RNA;
siRNA, small
interfering RNA;
pNph, p-nitrophenyl-;
oNph, o-nitrophenyl-;
pMph, p-methoxyphenyl-;
IR, inverted repeat;
RpL32, ribosomal
protein L32;
RT, reverse transcriptase.
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