Proteoglycan UDP-Galactose:beta -Xylose beta 1,4-Galactosyltransferase I Is Essential for Viability in Drosophila melanogaster*

Hitoshi TakemaeDagger §, Ryu Ueda||**, Reiko OkuboDagger , Hiroshi NakatoDagger Dagger , Susumu Izumi§, Kaoru Saigo§§, and Shoko NishiharaDagger **¶¶

From the Dagger  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 Dagger Dagger  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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Heparan and chondroitin sulfates play essential roles in growth factor signaling during development and share a common linkage tetrasaccharide structure, GlcAbeta 1,3Galbeta 1,3Galbeta 1,4Xylbeta 1-O-Ser. In the present study, we identified the Drosophila proteoglycan UDP-galactose:beta -xylose beta 1,4-galactosyltransferase I (dbeta 4GalTI), and determined its substrate specificity. The enzyme transferred a Gal to the -beta -xylose (Xyl) residue, confirming it to be the Drosophila ortholog of human proteoglycan UDP-galactose:beta -xylose beta 1,4-galactosyltransferase I. Then we established UAS-dbeta 4GalTI-IR fly lines containing an inverted repeat of dbeta 4GalTI ligated to the upstream activating sequence (UAS) promoter, a target of GAL4, and observed the F1 generation of the cross between the UAS-dbeta 4GalTI-IR fly and the Act5C-GAL4 fly. In the F1, double-stranded RNA of dbeta 4GalTI is expressed ubiquitously under the control of a cytoplasmic actin promoter to induce the silencing of the dbeta 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 beta 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

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, GlcAbeta 1,3Galbeta 1, 3Galbeta 1,4Xylbeta 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 beta 1,4-galactosyltransferase I (beta 1,4-galactosyltransferase 7) (beta 4GalTI) (6, 7), one proteoglycan beta 1,3-galactosyltransferase II (beta 1,3-galactosyltransferase 6) (beta 3GalTII) (8), and one glucuronosyltransferase I (GlcATI) (9). In nematodes, two glycosyltransferases, beta 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 beta 4GalT works as proteoglycan beta 4GalTI.

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 beta 4GalTI (dbeta 4GalTI) and performed a biochemical characterization. The protein transferred a Gal to the -beta -Xyl residue, confirming it to be the Drosophila ortholog of human proteoglycan beta 4GalTI (hbeta 4GalTI). After that, we produced an inducible dbeta 4GalTI RNAi fly using the GAL4-UAS system as a first step toward clarifying the biological role of dbeta 4GalTI. The dbeta 4GalTI mRNA was reduced specifically by RNAi, and the severity of the phenotype showed the correlation with the reduction in dbeta 4GalTI mRNA. The death of the flies proved that dbeta 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

Materials-- The Drosophila expressed sequence tag clone CK02622 was obtained from Research Genetics, Inc. (Huntsville, AL). UDP-Gal, p-nitrophenyl-alpha -xylopyranoside (Xylalpha -pNph), Xylbeta -pNph, o-nitrophenyl-beta -xylopyranoside (Xylbeta -oNph), p-nitrophenyl-N-acetyl-1-thio-beta -glucosaminide (GlcNAcbeta -S-pNph), p-nitrophenyl-alpha -glucopyranoside (Glcalpha -pNph), Glcbeta -pNph, p-nitrophenyl-alpha -galactopyranoside (Galalpha -pNph), Galbeta -pNph, p-nitrophenyl-N-acetyl-alpha -galactosaminide (GalNAcalpha -pNph), and GalNAcbeta -pNph were purchased from Sigma. The GlcNAcalpha -pNph, GlcNAcbeta -pNph, and p-nitrophenyl-alpha -mannopyranoside (Manalpha -pNph) were purchased from Calbiochem. Galbeta 1,4Xylbeta 1-p-methoxyphenyl (Galbeta 1,4Xylbeta 1-pMph) was provided by Seikagaku Corp. Uridine diphosphate-[14C]galactose (UDP-[14C]Gal) (325 mCi/mmol) was supplied by PerkinElmer Life Sciences.

Identification of the Drosophila Proteoglycan beta 1,4-Galactosyltransferase I-- We performed a BLAST search of all Drosophila databases and identified one Drosophila proteoglycan beta 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).

Construction and Purification of dbeta 4GalTI and hbeta 4GalTI Proteins Fused with FLAG Peptide-- The putative catalytic domain of dbeta 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-dbeta 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 kappa  (MHFQVQIFSFLLISASVIMSRG), the FLAG peptide (DYKDDDDK), and a conversion site for the GATEWAY system. pVL1393-FLAG-hbeta 4GalTI was also prepared by the same procedure using the two primers, 5'-AAAAAGCAGGCTGGGCAGTCAGGGGACAAG-3' and 5'-AGAAAGCTGGGTCACTGTCCAT- CCAGCTCA-3'.

pVL1393-FLAG-dbeta 4GalTI and pVL1393-FLAG-hbeta 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 beta 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).

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, Xylalpha -pNph, Xylbeta -pNph, Xylbeta -oNph, GlcNAcalpha -pNph, GlcNAcbeta -pNph, GlcNAcbeta -S-pNph, Glcalpha -pNph, Glcbeta -pNph, Galalpha -pNph, Galbeta -pNph, GalNAcalpha -pNph, GalNAcbeta -pNph, Manalpha -pNph, and Galbeta 1,4Xylbeta 1-pMph were utilized as acceptor substrates. With 10 nmol of each acceptor, the beta 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 dbeta 4GalTI and hbeta 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).

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 dbeta 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

The transformation of Drosophila embryos was carried out according to Spradling (22) with w1118 mutant stock as a host to make 23 UAS-dbeta 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.

Quantitative Analysis of dbeta 4GalTI mRNA by Competitive RT-PCR-- Total RNA was extracted from Act5C-GAL4/UAS-dbeta 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 dbeta 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 dbeta 4GalTI, dbeta 4GalTA, dbeta 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 lambda 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 dbeta 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.


                              
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Table I
Gene-specific primers used for competitive RT-PCR


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of the Drosophila Proteoglycan beta 1,4-Galactosyltransferase I-- When human hbeta 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 dbeta 4GalTI. The complete cDNA of the dbeta 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 beta 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.

When the other members of the human beta 1,4-galactosyltransferase family, hbeta 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 beta 1,4-galactosyltransferase family, dbeta 4GalTA and dbeta 4GalTB, respectively. dbeta 4GalTA and dbeta 4GalTB showed much lower homology of amino acids to hbeta 4GalTI (33 and 29%, respectively) than dbeta 4GalTI to hbeta 4GalTI (48%).

The ClustalW alignment of the human and Drosophila beta 4GalT families showed that the three beta 4GalT motifs found in the human family, including the DXD motif, a metal binding site, were also conserved in the Drosophila beta 4GalT family (Fig. 2A) (24, 25). A phylogenetic tree of the three Drosophila (dbeta 4GalTI, dbeta 4GalTA, and dbeta 4GalTB) and seven human beta 4GalTs (hbeta 4GalT1 to -6 and hbeta 4GalTI) was generated based on the amino acid sequences (Fig. 2B). dbeta 4GalTI was confirmed to be the Drosophila ortholog of hbeta 4GalTI. Both dbeta 4GalTA and d4beta GalTB showed higher homology to hbeta 4GalT1 to -6 than hbeta 4GalTI.


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Fig. 2.   Comparison of Drosophila beta 4GalTs and human beta 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 beta 4GalT motifs, including the DXD motif, a metal binding site, are boxed. B, phylogenetic tree of three Drosophila (dbeta 4GalTI, dbeta 4GalTA, and dbeta 4GalTB) and seven human beta 4GalTs (hbeta 4GalT1 to -6 and hbeta 4GalTI). dbeta 4GalTI is the Drosophila ortholog of hbeta 4GalTI, which is human proteoglycan beta 1,4-galactosyltransferase I, namely human beta 4GalT7. The branch lengths indicate amino acid substitution per site.

Characterization of the Galactosyltransferase Activity of dbeta 4GalTI-- The FLAG-tagged recombinant dbeta 4GalTI was expressed in insect cells to determine whether or not dbeta 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 Igkappa 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 hbeta 4GalTI was also prepared by the same procedure.

The purified enzymes were used for a galactosyltransferase assay with various acceptor substrates (Table II). The determined amounts of dbeta 4GalTI and hbeta 4GalTI and the same amount of each substrate were used for the enzyme reactions, so we could determine relative activities that were comparable. hbeta 4GalTI had strong activity toward the -beta -Xyl residue, whereas it had only slight activity toward -alpha -Xyl and no activity toward -beta -GlcNAc, -beta -Glc, -beta -Gal, and -beta -GalNAc as reported previously (6, 7). dbeta 4GalTI also showed strong activity toward the -beta -Xyl residue; only slight activity toward -alpha -Xyl; and no activity toward -beta -GlcNAc, -beta -Glc, -beta -Gal, and -beta -GalNAc. These results demonstrated that dbeta 4GalTI was the Drosophila ortholog of hbeta 4GalTI in view of their activities. But the beta 4GalT activity of dbeta 4GalTI toward the -beta -Xyl residue was almost half that of hbeta 4GalTI at both 25 and 37 °C.


                              
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Table II
Acceptor substrate specificities of purified recombinant dbeta 4GalTI and hbeta 4GalTI expressed in the baculovirus system

Viability of Inducible dbeta GalTI RNAi Flies-- Proteoglycan beta 4GalTI contributes to the synthesis of the common carbohydrate-protein linkage structure, GlcAbeta 1,3Galbeta 1,3Galbeta 1,4Xylbeta 1-O-Ser, of proteoglycan including heparin/HS and CS/DS. If proteoglycan beta 4GalTI is inactivated, every proteoglycan lacks GAG, and a severe biological phenotype is expected. To test this hypothesis, we tried to make inducible dbeta 4GalTI RNAi flies according to the method described under "Experimental Procedures."

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 dbeta 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-dbeta 4GalTI-IR fly stocks having a transgene containing two types of the IR of dbeta 4GalTI ligated to the UAS promoter, a target of GAL4, were established (Table III). The IR of dbeta 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-dbeta 4GalTI-IR fly, dsRNA of dbeta 4GalTI is expressed ubiquitously under the control of the cytoplasmic actin promoter to induce dbeta 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.


                              
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Table III
Viability of inducible dbeta 4GalTI RNAi fly

The phenotypes of the F1 of each UAS-dbeta 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 dbeta 4GalTI dsRNA by crossing the N13 line carrying two copies of UAS-dbeta 4GalTI-IR to the Act5C-GAL4 fly also led to lethality at the pupal stages. These results clearly demonstrated that proteoglycan beta 4GalTI is essential for the viability of flies.

Quantitative Analysis of dbeta 4GalT mRNA in Each Inducible dbeta 4GalTI RNAi Fly by Competitive RT-PCR-- To test the efficiency and specificity of RNAi in this system, the mRNA levels of all Drosophila beta 4GalTs (dbeta 4GalTI, dbeta 4GalTA, and dbeta 4GalTB) were determined in each dbeta 4GalTI RNAi fly by competitive RT-PCR (Fig. 4). The relative amount of each beta 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-dbeta 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 dbeta 4GalTI mRNA in each inducible dbeta 4GalTI RNAi fly by competitive RT-PCR. The mRNA levels of all Drosophila beta 4GalTs (dbeta 4GalTI, dbeta 4GalTA, and dbeta 4GalTB) in each dbeta 4GalTI RNAi fly were determined by competitive RT-PCR. The actual amount of each beta 4GalT mRNA was divided by that of RpL32 mRNA for normalization. The relative amount of each beta 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 beta 4GalTs and RpL32, respectively. The cDNAs for each beta 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 beta 4GalTs in the third instar larvae of the F1 progeny of each N line of UAS-dbeta 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 dbeta 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 beta 4GalTs in the third instar larvae of the F1 progeny of each C line of UAS-dbeta 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 dbeta 4GalTI. Act5C-GAL4/C5 and Act5C-GAL4/C6 were pupal lethal. C, the mRNA level of dbeta 4GalTI in the prepupae of Act5C-GAL4/N4 and Act5C-GAL4/N13.

N lines have a transgene including the IR of the sequence encoding the N-terminal region of dbeta 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 dbeta 4GalTs mRNA in the third instar larvae of these four RNAi flies and the wild-type fly, Act5C-GAL4/+. The ratios of reduction in dbeta 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 dbeta 4GalTI mRNA than those of the N2 line and N4 line, which were crossed to make the N13 line. Reductions in dbeta 4GalTA mRNA and dbeta 4GalTB mRNA were not observed in all RNAi flies. It was clearly demonstrated that the dbeta 4GalTI mRNA was disrupted specifically, and the ratio of degraded dbeta 4GalTI mRNA was well correlated with the severity of the phenotype.

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 dbeta 4GalTI mRNAs in the third instar larvae were also interfered with specifically, and the ratios of degraded dbeta 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.

We also determined the amount of dbeta 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.

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.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We identified the Drosophila proteoglycan beta 4GalTI by molecular and biochemical analyses and then made the RNAi fly to investigate dbeta 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 dbeta 4GalTI mRNA caused lethality, indicating an essential function of dbeta 4GalTI for viability. This is the first example of a reverse genetics approach to the systematic study of Drosophila glycosyltransferase.

Drosophila has three members of the beta 4GalT family, dbeta 4GalTI, dbeta 4GalTA, and dbeta 4GalTB (Fig. 2A). The phylogenetic tree (Fig. 2B) and acceptor substrate specificities (Table II) of the beta 4GalT family clearly demonstrated that dbeta 4GalTI is the Drosophila ortholog of hbeta 4GalTI. dbeta 4GalTA and dbeta 4GalTB showed higher homology to hbeta 4GalT1 to -6 than to hbeta 4GalTI, but the Drosophila ortholog of the six hbeta 4GalTs could not be identified. So there is a possibility that dbeta 4GalTA and dbeta 4GalTB share acceptor substrates, to which hbeta 4GalT1 to -6 can transfer Gal. Recently, hbeta 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 dbeta 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 dbeta 4GalTs.

The three members of the dbeta 4GalT family also conserved the three beta 4GalT motifs found in the hbeta 4GalT family (Fig. 2A) (25). The crystal structure of the bovine beta 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.

dbeta 4GalTI showed roughly half the activity of hbeta 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, hbeta 4GalTI performed a more efficient reaction than dbeta 4GalTI. Very recently, two papers about dbeta 4GalTI have been published (29, 30) reporting similar enzymatic activity to ours.

We made 24 UAS-dbeta 4GalTI-IR fly lines and observed the F1 generation of each UAS-dbeta 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 dbeta 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 dbeta 4GalTI.

We analyzed the amounts of three dbeta 4GalT mRNAs (dbeta 4GalTI, dbeta 4GalTA, and dbeta 4GalTB) to estimate the specificity and efficiency of RNAi in our Drosophila-inducible RNAi system. The RNAi occurred only on dbeta 4GalTI and had no effect on the other members of the dbeta 4GalT family, dbeta 4GalTA and dbeta 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 dbeta 4GalTI with the sequences of dbeta 4GalTA and dbeta 4GalTB, we could not find any identical regions longer than 21 nucleotides. This must be the reason why the RNAi of dbeta 4GalTI was specific with no cross-effect for dbeta 4GalTA and dbeta 4GalTB.

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 dbeta 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.

dbeta 4GalTI contributes to the synthesis of the common linkage structure of heparin/HS and CS/DS. When dbeta 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 hbeta 4GalTI gene of patients with Ehlers-Danlos syndrome has mutations that reduce beta 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 dbeta 4GalTI should serve as a model of this kind of disease.

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 beta 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.

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.

    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.

    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.

    ABBREVIATIONS

The abbreviations used are: GAGs, glycosaminoglycans; beta 4GalT, beta 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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