From the aDivision of Cell Biology, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, Japan, bSeikagaku Corp., Central Research Laboratories, 3-1253 Tateno, Higashiyamato, Tokyo 207-0021, Japan, the cDepartment of Surgery, Tokyo Medical University, 6-7-1 Nishi-shinjuku, Shinjuku-ku, Tokyo 160-0023, Japan, dCore Research for Evolutional Science and Technology of Japan Science and Technology Corp., Kawaguchi Center Building, 4-1-8, Hon-cho, Kawaguchi, Saitama 332-0012, Japan, eInvertebrate Genetics Laboratory, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 441-8540, Japan, fGenetic Networks Research Group, Mitsubishi Kagaku Institute of Life Science, 11 Minamiooya, Machida, Tokyo 194-8511, Japan, gMitsui Knowledge Industry Co., Ltd., 1-32-2 Honcho, Nakano-ku, Tokyo 164-8721, Japan, hResearch Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, 1-1-1 Umezono, Tsukuba, Ibaraki, 305-8586, Japan, the iDepartment of Bioanalytical Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi, Inage, Chiba 263-8522, Japan, the jDepartment of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan, and the kDepartment of Surgery, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
Received for publication, March 10, 2003 , and in revised form, April 22, 2003.
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ABSTRACT |
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INTRODUCTION |
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Earlier studies had shown a saturable transport activity of PAPS using isolated Golgi vesicles (6) or reconstituted proteoliposome (7). To identify this transporter protein, the proteins responsible for PAPS translocating activity have been purified (810). The characterization of these purified proteins revealed the kinetic behavior of a PAPS-specific transport through an antiport mechanism (810); however, cloning of the transporter has not been reported.
Recently, several nucleotide-sugar transporters (NSTs) have been cloned and characterized in mammals, yeast, protozoa, and plants (1113). Nucleotide-sugars are the donor substrates for glycosylation, which is catalyzed by glycosyltransferases. These NST proteins are highly hydrophobic Type III multitransmembrane proteins localized in the Golgi or endoplasmic reticulum membrane and provide a specific substrate for the glycosylation. The structural conservation among NSTs has contributed to the identification of novel NST-related sequences from existing databases, whereas the levels of the amino acids identified are not indicators of their substrate specificities (14). A large number of putative NST sequences of mammals, Drosophila, Caenorhabditis elegans, plants, and yeast are under investigation for substrate specificity and function.
By a BLAST search of the data base, we identified a putative NST gene homologous to human UGTrel1 (UDP-galactose transporter-related isozyme 1) (15). Unexpectedly, the heterologous expression of PAPST1 in yeast Saccharomyces cerevisiae did not result in any nucleotide-sugar transport activity but revealed PAPS transport activity. Furthermore, the Drosophila melanogaster orthologous gene, slalom (sll), had the same substrate specificity. When double-stranded RNA of sll was expressed ubiquitously under the control of a cytoplasmic actin promoter to induce the silencing of the sll gene, the RNAi fly showed marked lethality. Here we reported the functional properties of these novel PAPS transporters.
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EXPERIMENTAL PROCEDURES |
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Isolation of Human and Drosophila PAPS Transporter cDNAs and Construction of Expression PlasmidsA TBLASTN search was performed for the amino acid sequence of the open reading frame (ORF) of UGTrel1 (15). We succeeded in identifying a candidate cDNA sequence encoding a full-length ORF. To obtain this cDNA and create recombination sites for the GATEWAYTM cloning system (Invitrogen), we used two steps of attB adaptor PCR for the preparation of attB-flanked PCR products. For the first gene-specific amplification, a forward template-specific primer with attB1 (5'-aaaaagcaggcttcgcctggaccatggacgc-3') and a reverse template-specific primer with attB2 (5'-agaaagctgggtcaaccttctgcacaggaga-3') were used. PCR was performed using Platinum® Pfx DNA polymerase (Invitrogen) and a cDNA library derived from human colon tissue. The insertion of a complete attB adaptor and cloning into the pDONRTM201 vector to create an entry clone for the subsequent subcloning were performed according to the instruction manual. A Drosophila cDNA encoding the ORF of slalom (sll) was obtained from expressed sequence tag clone SD04658 (Invitrogen) by PCR using a forward template-specific primer (5'-aaaaagcaggcttccgccacatgtacgcctat-3') and a reverse template-specific primer (5'-agaaagctgggtcgacagccattttcggttt-3'). The rest of the cloning procedure was the same as described above.
Expression vectors were inserted with three copies of HA epitope tags (YPYDVPDYA) at the position corresponding to the C terminus of the expressing protein and converted to a GATEWAY destination vector with a conversion site. Each entry clone was subcloned into appropriate expression vectors using the GATEWAY cloning system according to the instruction manual.
Transient Transfection and Immunofluorescence MicroscopyThe
mammalian expression vector, pCXN2, was kindly provided by Dr. K. Miyazaki
(Tokyo University). SW480 cells (CCL-228; ATCC) were subcultured onto a
four-well Lab-Tek chamber slide (Nalge Nunc International) in Dulbecco's
modified Eagle's medium containing 10% fetal bovine serum. After 24 h, cells
were transfected with 0.25 µg/well of pCXN2 or pCXN2 inserted with
PAPST1 using LipofectAMINE 2000 reagent (Invitrogen) according to the
manufacturer's protocol. After 72 h, cells were fixed in phosphate-buffered
saline containing 4% paraformaldehyde for 30 min at 4 °C and permeabilized
in permeabilizing buffer (phosphate-buffered saline containing 5% bovine serum
albumin and 0.1% Triton X-100) for 1 h at 4 °C. Then cells were stained
with anti-1,4-galactosyltransferase 1 mAb
(16) for 16 h at 4 °C,
washed four times with permeabilizing buffer, and incubated with
rhodamine-labeled anti-IgG mAb for 90 min at room temperature. After
incubation, the cells were washed and stained with fluorescein
isothiocyanate-conjugated anti-HA mAb for 90 min at room temperature. Cells
were washed and mounted with PermaFluor (Thermo Shandon, Pittsburgh, PA). The
fluorescence was observed under a fluorescence microscope.
Stable TransfectionBurkitt's lymphoma Namalwa cells (CRL-1432; ATCC) were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum. First, 4 µg of pAMo vector (17) or pAMo inserted with PAPST1 was transfected into 1 x 107 cells by electroporation using a GenePulser apparatus (Bio-Rad). The transfectants were selected by the addition of 600 µg/ml Geneticin (Invitrogen) to the medium.
Subcellular Fractionation of Yeast and Transport AssayYeast S. cerevisiae strain W3031a (MATa, ade2-1, ura3-1, his3-11,15, trp1-1, leu2-3,112, and can1-100) was transformed by the lithium acetate procedure (18) using a yeast expression vector, YEp352GAP-II, kindly provided by Dr. K. Nakayama (National Institute of Advanced Industrial Science and Technology, Japan). Transformed yeast cells were grown at 30 °C in synthetic defined medium in which uracil was omitted for the selection of transformants. Subcellular fractionation and nucleotide-sugar transport assays were performed as described by Roy et al. (19). Yeast cells were converted into spheroplasts, homogenized, and fractionated to yield a 10,000 x g membrane fraction (P10), a 100,000 x g membrane fraction (P100), and the supernatant of the cytosolic fraction (S100). Then 200 µg of protein of each fraction was incubated in 100 µl of reaction buffer (20 mM Tris-HCl, pH 7.5, 0.25 M sucrose, 5.0 mM MgCl2, 1.0 m 2 phenotype. M MnCl, and 10 m M 2-mercaptoethanol) containing 1 µM radiolabeled substrate at 30 °C for 5 min. After incubation, the radioactivity incorporated in the microsomes was trapped with a 0.45-µm nitrocellulose filter and measured by liquid scintillation. The amount of incorporated substrate was calculated as the difference with the background value obtained from the time 0 assay for each sample.
Western Blot AnalysisThe indicated amounts of protein of samples were added to 3x SDS sample buffer (New England Biolabs Inc., Beverly, MA) and incubated at room temperature for 2 h. The samples were fractionated on a 520% gradient SDS-polyacrylamide gel using the XV PANTELA electrophoresis system (DRC Corp., Tokyo, Japan). The separated proteins were electrotransferred onto polyvinylidene difluoride membrane. Anti-HA mouse mAb and horseradish peroxidase-conjugated anti-mouse IgG mAb were detected by ECL+plus (Amersham Biosciences) according to the manufacturer's directions.
Quantitative Analysis of the PAPST1 Transcript in Human Tissues by Real Time PCRTotal RNA was extracted from human tissues by the methods of Chomczynski and Sacchi (20). First-strand cDNA was synthesized using a SuperscriptII first strand synthesis kit (Invitrogen) according to the manufacturer's instructions. Quantitation of PAPST1 expression in the different tissues was performed by real time PCR using the following primers: forward, 5'-ggcaggccctgaagct-3'; reverse, 5'-tgcgggtcatcactctttc-3'. The probe, which consisted of 5'-ccacagggctccaggtgtcttatctg-3', was labeled at the 5'-end with the reporter dye, 3FAM, and at the 3'-end with the quencher dye TAMRA (Applied Biosystems, Foster City, CA). Real time PCR was performed using a TaqMan Universal PCR Master Mix (Applied Biosystems) and ABI PRISM 7700 Sequence Detection System (Applied Biosystems). The relative amount of PAPST1 transcript was normalized to an endogenous control, human glyceraldehyde-3-phosphate dehydrogenase transcript in the same cDNA.
Construction of sll RNAi Fly and Quantitative Analysis of the TranscriptA 500-bp cDNA fragment of sll was amplified by PCR (forward primer, 5'-acacttcttctcggatctgct-3'; reverse primer, 5'-gacgattggaaaacaccagga-3') and inserted as an inverted repeat with a head-to-head orientation into a modified Bluescript vector, pSC1. The cloning of sll into the transformation vector pUAST was done as previously reported (21). The transformation of Drosophila embryos was carried out according to Spradling (22) with w1118 stock as a host to make four UAS-sll inverted repeat fly lines. Each line was mated with the Act5CGAL4 fly line, and the F1 progeny was raised at 28 °C to determine the phenotype.
The quantitation of the sll transcript in third instar larvae was performed using real time PCR as described above. Sequences of used primers and probe are as follows: forward, 5'-ggcccagttgtgtttacgataat-3'; reverse, 5'-ggtagatgaagcaggagagcataat-3'; probe, 5'-ccaccgcctgacgcagtgtcat-3'. The relative amount of sll transcript was normalized to an endogenous control, ribosomal protein L32 (RpL32) transcript in the same cDNA.
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RESULTS |
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Subcellular Localization of PAPST1 in Mammalian Cells The
subcellular localization of transiently expressed PAPST1 protein in SW480
cells was investigated by immunofluorescence staining. A mammalian expression
vector, pCXN2, was inserted with the ORF of PAPST1 with an HA epitope
tag at the C terminus and transfected transiently into SW480 cells. The cells
were double immunostained with anti-HA mAb and
anti-1,4-galactosyltransferase 1 mAb. The immunofluorescence microscopy
of cells expressing HA-tagged PAPST1 is shown in
Fig. 2A. PAPST1-HA
showed partial co-localization with
1,4-galactosyltransferase 1, which
is a typical protein of trans-Golgi localization
(16). This indicates that
PAPST1 is localized in the Golgi apparatus but not endoplasmic reticulum.
Further support for this observation was derived from Western blotting of
stable transfectants. Namalwa cells stably expressing HA-tagged PAPST1 or mock
vector (vector alone) were fractionated into a 10,000 x g
fraction (P10), a 100,000 x g microsomal fraction (P100), and
the supernatant of the cytosolic fraction (S100). PAPST1 transfectant showed
16.3 times the PAPST1 transcript level of the mock transfectant (4.41
and 71.71 x 103/glyceraldehyde-3-phosphate
dehydrogenase transcript, respectively). As shown in
Fig. 2B, PAPST1
protein was detected mainly in the P100 microsomal membrane fraction with a
small amount in the P10 fraction by Western blotting. HA-tagged PAPST1
proteins migrated as a 48-kDa protein. These proteins were not detected in
cells transfected with mock vector. These results indicate that HA-tagged
PAPST1 protein is predominantly localized on Golgi membrane.
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Substrate Specificities of PAPST1 and SLL Proteins Expressed in Yeast CellsTo investigate the functional properties of PAPST1 and SLL, we used a heterologous yeast expression system. Yeast strain S. cerevisiae is widely used, because the isolated microsomal vesicles have little nucleotide-sugar transport activity except for GDP-mannose. A yeast expression vector, YEp352GAP-II, was inserted with the ORF of PAPST1 or sll and introduced into W3031a yeast. PAPST1 and SLL proteins were expressed in the yeast P100 membrane fraction (Fig. 3A). The substrate specificities of PAPST1 and SLL were examined using radiolabeled substrates. As shown in Fig. 3B, the transport activity of PAPS into the P100 fraction prepared from yeast cells expressing PAPST1 and SLL is significantly higher than that shown for the mock (2.5 and 4.9 times, respectively). No difference was observed among PAPST1, SLL, and mock in the transport of other nucleotide-sugars. This was confirmed in Namalwa cells stably transfected with PAPST1. The P100 fraction of the PAPST1 transfectant showed 4.3 times the PAPS transport activity of the mock transfectant (1.34 ± 0.13 and 5.72 ± 0.06 pmol/5 min/mg of protein, respectively). The substrate concentration dependences of PAPS transport by PAPST1 and SLL are shown in Fig. 3C. The apparent Km values of PAPST1 and SLL were estimated to be 0.8 and 1.2 µM, respectively.
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Tissue Distribution of PAPST1 Transcripts in Human TissuesThe gene expression of PAPST1 in human tissues was analyzed using real time PCR. The distribution of PAPST1 transcripts in human tissues is shown in Fig. 4. The placenta had the most PAPST1 among the tissues tested. Relatively high levels of PAPST1 expression in the pancreas, mammary gland, and skeletal muscle were also observed. PAPST1 transcripts were hardly detectable at all in colon, heart, and prostate. All transcript levels are shown relative to that of glyceraldehyde-3-phosphate dehydrogenase.
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Lethality of Inducible sll RNAi FliesProteoglycans, including heparan sulfate, chondroitin sulfate, and dermatan sulfate, are sulfated at various positions along their glycosaminoglycan chains. Since PAPS is the sole substrate for sulfation, the down-regulation of PAPS transport into Golgi lumen may display an abnormal biological phenotype. To elucidate the importance of the PAPS transporter to the viability of D. melanogaster, we made an inducible sll RNAi fly using the GAL4-UAS system (21, 23). First we made four UAS-sll inverted repeat fly lines, and then we used Act5C-GAL4 as a GAL4 driver to induce sll gene knock-down in all cells of the fly. In the F1 generations of the Act5C-GAL4 fly and the UAS-sll inverted repeat fly, double-stranded RNA of sll was expressed ubiquitously under the control of the cytoplasmic actin promoter to induce sll gene silencing.
The amount of sll transcript in the third instar larvae of each F1 is shown in Table I. All transcripts were analyzed by real time PCR and are shown as relative amounts to that of RpL32. The relative amount of sll transcript in the F1 of the UAS-sll inverted repeat fly crossed with the Act5C-GAL4 fly is reduced to approximately one-fifth of that in the F of w11181 crossed with Act5C-GAL4, Act5C-GAL4/+, which corresponds to the wild type. All four lines of the F1 of the UAS-sll inverted repeat fly crossed with Act5C-GAL4 exhibited pupal lethality, and no fly developed into an adult (Table I). These results clearly demonstrated that sll is essential for the viability of flies.
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DISCUSSION |
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To isolate these novel transporters, we used the cloning strategy of
searching databases for cDNAs homologous to human UGTrel1. UGTrel1 is
a gene of unknown function that has similarity with human UDP-galactose
transporter genes (15). In
humans, Muraoka et al.
(24) identified a gene related
to UGTrel1 and reported that the product transports both
UDP-glucuronic acid and UDP-N-acetylgalactosamine. The phylogenetic
tree of these transporters indicated that PAPST1 is more closely
related to UGTrel1 (Fig.
1B). PAPST1 was defined in GenBankTM as a
nuclear factor of a light polypeptide gene enhancer in B-cell
inhibitor epsilon (NF
BIE), although no evidence of this has been
provided. Two distinct NF
BIE proteins were reported in 1997
independently by two laboratories
(25,
26). In some data bases, there
has been confusion over PAPST1 and two other NF
BIEs about the sequence
and gene locus. Although we did not assess the effect of PAPST1 on nuclear
factor
B DNA binding activity, we failed to find any ankyrin repeat
motif in PAPST1, which is essential for interactions with nuclear factor
B. Furthermore, PAPST1 is multitransmembrane protein, whereas other
NF
BIEs are toplasmic proteins. These findings strongly indicate that
PAPST1 is not an NF
BIE.
Mandon et al. (8) purified rat liver Golgi membrane transporter to a 75-kDa protein. The PAPS transport activity was characterized using phosphatidylcholine liposome and sessed to have an apparent Km of 1.7 µM. Independently, Ozeran et al. (9, 10) purified and characterized a 230-kDa liver Golgi membrane translocase protein. From its kinetic properties, they characterized it as a specific transporter PAPS, which acts through an antiport mechanism with adenosine 3',5'-bisphosphate as the returning ligand. The kinetic behavior of PAPST1 resembles that of a rat protein (a saturable transport of PAPS with an apparent Km of 0.8 µM (Fig. 4B)); however, PAPST1 is different from these proteins in regard its apparent molecular mass. The HA-tagged PAPST1 protein expressed in Namalwa cells showed a band of 48-kDa on Western blot analysis (Fig. 3). It is not clear whether these proteins are homo- or heteropolymeric forms of PAPST1 or distinct PAPS transporters. Whether PAPST1 is the sole PAPS transporter or not should be evaluated in further investigations.
As shown in Table I, the
RNAi fly of sll induced with GAL4-UAS system confirmed that
sll is essential for viability. Recent studies on Drosophila
demonstrated that the mutation of some genes required for proteoglycan
biosynthesis, dally
(27,
28), sugarless
(2932),
and tout-velu (32,
33), resulted in defective
signaling during development. A number of reports have suggested that heparan
sulfate proteoglycans are involved in variety of signaling pathways, in
particular those of fibroblast growth factor
(34), Wnt/Wingless
(35), Decapentaplegic
(27), and Hedgehog
(36). Heparan sulfate
proteoglycans are thought to be required for stabilizing the complex between a
ligand and its receptors or restricting the extracellular diffusion of ligands
(35). It is known that the
developmental signaling functions cell surface heparan sulfate proteoglycans
are dependent their sulfation states
(35,
37). In Drosophila,
the mutation of gene encoding
N-deacetylase/N-sulfotransferase (sulfateless)
caused defects in Wingless
(2731)
and fibroblast growth factor signaling
(38). Indeed, sll in
the flybase is involved in signal transduction by growth factors of Wingless
and Hedgehog during patterning and morphogenesis
(39). We reported the
quirement of Drosophila 1,4-galactosyltransferase I, which
contributes to the synthesis of the linkage structure of proteoglycans, for
viability (21). However, we
did not investigate whether the lethality of the sll RNAi fly is
caused by a reduction in the sulfation of proteoglycans or other sulfated
glycoconjugates, such as sulfatides
(40) and HNK-1 epitope
(41). shown in
Fig. 2A, PAPST1 was
only partially co-localized with trans-Golgi
1,4-galactosyltransferase
1. Thus, it might involved in not only proteoglycan synthesis but also
sulfatide synthesis, which occurs in the early Golgi compartment. Further
clarification is necessary regarding the role of the PAPS transporter in
proteoglycan synthesis and the signaling pathway.
Mutations of some genes related to PAPS synthesis are known to be responsible for human inherited disorders. The diastrophic dysplasia sulfate transporter gene was identified as being responsible for diastrophic dysplasia by Hästbacka et al. (42). The diastrophic dysplasia sulfate transporter plays an important role in providing sulfate ion from the extracellular milieu to the cytosol. Mutations in the diastrophic dysplasia sulfate transporter gene impair sulfate uptake across the plasma membrane and the sulfation of proteoglycans in cartilage matrix. Achondrogenesis type IB, atelosteogenesis type II, dysplasia epiphysealis multiplex, and other diastrophic dysplasia variant disorders are caused by allelic mutations in the same gene, although the molecular basis of the majority of osteochondrodysplasias is still unknown. In addition, Kurima et al. found that the PAPS synthase 2 gene is responsible for mouse brachymorphism characterized by a dome-shaped skull, short thick tail, and shortened limbs (43). Missense and nonsense mutations of PAPS synthase 2 were demonstrated in the human inherited disorder, spondylo-epimetaphyseal dysplasia (44). On the other hand, no genetic disorder has been associated with the subsequent PAPS transport pathway.
We also identified the Drosophila ortholog, sll. D. melanogaster is a well established model for genetic analysis. The analysis of the RNAi fly of sll may help to elucidate the biological function of PAPS transport and its role in post-translational sulfation.
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FOOTNOTES |
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* This work has been supported by Core Research for Evolutional Science and
Technology of Japan Science and Technology Corp. and by the New Energy and
Industrial Technology Development Organization as part of the research and
development project of the Industrial Science and Technology Frontier Program.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
l To whom all correspondence should be addressed. Tel.: 81-426-91-8140 Fax: 81-426-91-8140; E-mail: shoko{at}t.soka.ac.jp.
1 The abbreviations used are: PAPS, 3'-phosphoadenosine
5'-phosphosulfate; HA, influenza hemagglutinin epitope; NST,
nucleotide-sugar transporter; NFBIE, nuclear factor of
light
polypeptide gene enhancer in B-cells inhibitor epsilon; RNAi, RNA
interference; UGTrel1, UDP-galactose transporter-related isozyme 1; mAb,
monoclonal antibody; ORF, open reading frame.
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ACKNOWLEDGMENTS |
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REFERENCES |
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