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J. Biol. Chem., Vol. 279, Issue 25, 26469-26474, June 18, 2004
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¶||||**¶¶¶¶
From the
Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, Japan, the Department of Surgery, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan, **Seikagaku Corporation, Central Research Laboratories, 3-1253 Tateno, Higashiyamato, Tokyo 207-0021, Japan, Mitsui Knowledge Industry Co., Ltd., 1-32-2 Honcho, Nakano-ku, Tokyo 164-8721, Japan, the Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Umezono, Tsukuba, Ibaraki, 305-8586 Japan, and ¶Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology Corporation (JST), Kawaguchi Center Building, 4-1-8, Hon-cho, Kawaguchi, Saitama 332-0012, Japan
Received for publication, October 15, 2003 , and in revised form, April 9, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Recent studies have demonstrated that some NSTs are implicated in growth factor signaling during development through the regulation of proteoglycan synthesis. In Caenorhabditis elegans, sqv-7 mutants show a defect in vulva formation, with epithelial invaginations, during ontogeny (6). SQV-7 has been identified as a transporter of UDP-glucuronic acid (UDP-GlcUA), UDP-galacose (UDP-Gal), and UDP-N-acetylgalactosamine (UDP-GalNAc) (7). Biochemical analysis of sqv-7 mutants has demonstrated defects in both chondroitin and heparan sulfate biosynthesis in vivo (8). Furthermore, Selva et al. (9) and Goto et al. (10) reported that the Drosophila melanogaster gene of fringe connection (frc)is involved in embryonic Wingless/Hedgehog and fibroblast growth factor signaling (9, 10). Frc encodes a multisubstrate-specific NST that transfers UDP-sugars into the Golgi apparatus (9, 10). UDP-GlcUA and UDP-N-acetylglucosamine (UDP-GlcNAc) act as substrates for the synthesis of heparan sulfate. Embryos with a mutation of frc display severe segment polarity phenotypes (9).
Although sqv-7 and frc are considered to be orthologous to each other, it remains obscure which NSTs of humans have corresponding functions. In humans, the UGTrel7 protein has a similar multisubstrate specificity to those of FRC and SQV-7; namely, UDP-GlcUA/UDP-GalNAc (11). However, localization of UGTrel7 to the ER, not to the Golgi apparatus (11), leaves open the possibility that other transporters may be involved in proteoglycan synthesis in the Golgi apparatus.
The structural conservation present among NSTs enables the identification of many putative NST sequences from data bases. By a data base search, we identified a putative NST gene that is closely related to sqv-7, frc, and UGTrel7. HFRC1 had a multisubstrate specificity for UDP-GlcNAc/UDP-glucose (UDP-Glc)/GDP-mannose (GDP-Man) in the Golgi fraction of yeast cells expressing this gene. Furthermore, alteration in the expression of hfrc1 affected the heparan sulfate on the cell surface of mammalian cells. Thus, the present study raises the possibility that HFRC1 takes part in heparan sulfate synthesis by supplying UDP-GlcNAc.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Isolation of a Novel Human NST cDNA and Construction of Expression Plasmids—A novel putative NST gene was cloned using the same procedures as previously described (12). Briefly, a TBLASTN search was performed for the amino acid sequence of the open reading frame (ORF) of the GDP-fucose (GDP-Fuc) transporter gene (13). We identified a cDNA sequence encoding a full-length ORF (GenBankTM accession number XM_047286). 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'-aaaaagcaggcttcccgcaggagatgacgg-3') and a reverse template-specific primer with attB2 (5'-agaaagctgggtcgctcttcaaatccaaaca-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 pDONRTM 201 vector to create an entry clone for the subsequent subcloning were performed according to the instruction manual. The ORF of the UGTrel7 cDNA was obtained by the same procedures as mentioned above using the forward template-specific primer 5'-aaaaagcaggcttcgcagccatggcggaagt-3' and the reverse template-specific primer 5'-agaaagctgggtccactgctcctttcccctt-3'.
Expression vectors were inserted with three copies of the 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 Microscopy—Transient transfection and immunofluorescence microscopy was performed as previously described (12). HCT116 cells were subcultured onto a 4-well Lab-Tek chamber slide (Nalge Nunc International, New York) in Dulbecco's modified Eagle's/Ham's medium containing 10% fetal bovine serum. After 24 h, cells were transfected with 0.25 µg/well of pCXN2 (14) or pCXN2 inserted with HA-tagged hfrc1 using LipofectAMINE 2000 reagent (Invitrogen) according to the manufacturer's protocol. After 72 h, cells were fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde for 30 min at 4 °C, then permeabilized in permeabilizing buffer (PBS 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 (4GalT1) mAb (15) or anti-ERp57 mAb 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, then stained with fluorescein isothiocyanate-conjugated anti-HA mAb for 90 min at room temperature. Finally, cells were washed and mounted with PermaFluor (Thermo Shandon, Pittsburgh, PA). The fluorescence was observed under a fluorescence microscope.
Subcellular Fractionation of Yeast and Transport Assay—Yeast of the Saccharomyces cerevisiae strain W303-1a (MATa, ade2-1, ura3-1, his3-11,15, trp1-1, leu2-3,112, and can1-100) was transformed by the lithium acetate procedure (16) using a yeast expression vector, YEp352GAP-II (17). 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. (18). The cells were harvested, washed with ice-cold 10 mM NaN3, then converted into spheroplasts by incubation at 37 °C for 30 min in spheroplast buffer (1.4 M sorbitol, 50 mM potassium phosphate, pH 7.5, 10 mM NaN3, 40 mM 2-mercaptoethanol, and 1 mg of Zymolyase 100T per g of cells). Spheroplasts were pelleted in a refrigerated centrifuge and washed twice with 1.0 M ice-cold sorbitol to remove Zymolyase. The cells were suspended in ice-cold lysis buffer (0.8 M sorbitol in 10 mM triethanolamine, pH 7.2, 5 µg/ml pepstatin A, and 1 mM phenylmethylsulfonyl fluoride), then homogenized using a Dounce homogenizer. The lysate was centrifuged at 1,000 x g for 10 min to remove unlysed cells and cell-wall debris. The supernatant was then centrifuged at 10,000 x g for 15 min at 4 °C (Beckman XL-90 ultracentrifuge with 90.1 Ti rotor), which yielded a pellet of the P10 membrane fraction. The supernatant was further centrifuged at 100,000 x g yielding a pellet of the P100 membrane fraction and a supernatant of the S100 fraction. Then, 200 µg of protein of each fraction were incubated in 100 µl of reaction buffer (20 mM Tris-HCl, pH 7.5, 0.25 M sucrose, 5.0 mM MgCl2, 1.0 mM MnCl2, and 10 mM 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 from the background value obtained from the time 0 assay for each sample.
Western Blot Analysis—To determine the expression status of the HA-tagged protein, subcellular fractions of yeast cells were analyzed by Western blot analysis. Fifty micrograms of protein of each sample were added to 3x SDS sample buffer (New England Biolabs Inc.), then incubated at room temperature for 2 h. The samples were fractionated on a 5–20% gradient SDS-polyacrylamide gel (Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan). The separated proteins were electrotransferred onto polyvinyldene difluoride membrane. HA-tagged proteins were immunostained with anti-HA mouse mAb and horseradish peroxidase-conjugated anti-mouse IgG mAb. Bound horseradish peroxidase was detected using ECL+plus (Amersham Biosciences) according to the manufacturer's directions.
Quantitative Analysis of hfrc1 and UGTrel7 Transcripts in Human Tissues by Real Time PCR—The amount of hfrc1 and UGTrel7 transcripts in human tissues was determined by real time PCR. Total RNA was extracted from human tissues by the method of Chomczynski and Sacchi (19). First-strand cDNA was synthesized using a SuperScript II first-strand synthesis kit (Invitrogen) according to the manufacturer's instructions. Real time PCR was performed using a qPCR Mastermix QuickGoldStar (EUROGENTEC) and ABI PRISM 7700 Sequence Detection System (Applied Biosystems). Multiplex real time PCR was used to compare the amounts of hfrc1 and UGTrel7 transcripts in normal human tissues. The sequences of the PCR primer pairs and TaqMan probes used for each gene were as follows. For the quantitation of hfrc1, the forward primer 5'-cctgacgacagcagtggtt-3', reverse primer 5'-ctccaccgattaatatcccaat-3', and probe 5'-agccatcaagaatgtatccgttgcct-3' were used. The probe was labeled at the 5'-end with the reporter dye FAM, and at the 3'-end with the quencher dye TAMRA. For the detection of UGTrel7, the forward primer 5'-cattgcgtatttcacaggagatg-3', reverse primer 5'-ctgcagaagaaagagggtgtca-3', and probe 5'-cccagccttcaaactccacagccttt-3' were used. The probe was labeled at the 5'-end with Yakima Yellow and at the 3'-end with Darquencher. The relative amounts of hfrc1 and UGTrel7 transcripts were normalized with respect to the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts in the same cDNA.
Stable Transfection—HCT116 cells were subcultured onto 6-cm dishes in Dulbecco's modified Eagle's/Ham's (1:1) medium containing 10% fetal bovine serum. After 24 h, the cells were transfected with 8 µg of pCXN2 vector or pCXN2 inserted with HA-tagged hfrc1 using Lipo-fectAMINE 2000 reagent according to the manufacturer's protocol. After 48 h, transfectants were selected by the addition of 600 µg/ml Geneticin (Invitrogen) to the medium, and cultured for 1 month.
Flow Cytometric Analysis—After 16 h of subculture, cells were harvested with PBS containing 1 mM EDTA, and washed with PBS containing 0.1% bovine serum albumin. Then, 100 µl of cell suspensions (1 x 106 cells) were incubated with anti-heparan sulfate mAb or antichondroitin 4-sulfate mAb for 30 min on ice, and washed three times with 1.5 ml of PBS containing 1% bovine serum albumin. Then, cells were resuspended in 100 µl of fluorescein isothiocyanate-conjugated goat anti-mouse IgM and incubated for 30 min on ice. Flow cytometric analysis was performed using EPICS ELITE flow cytometer (Beckman-Coulter). As the negative control, cells were treated with 10 milliunits/ml of heparitinase I or 100 milliunits/ml of chondroitinase ABC for 90 min at 37 °C before immunoblotting. Data were analyzed using WinMDI 2.8 software (The Scripps Research Institute Cytometry software page).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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4GalT1 mAb or anti-ERp57 mAb, and the immunofluorescence was observed microscopically. As shown in Fig. 2, HFRC1-HA was co-localized with 4GalT1, which is a typical protein of trans-Golgi localization (15). In contrast, UGTrel7-HA was co-localized with ERp57, which is a typical protein of the ER (Fig. 2). These results indicate that HFRC1 is localized to the Golgi apparatus, unlike UGTrel7.
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The gene expression profiles of these genes in various human tissues are shown in Fig. 3. All transcript levels are shown relative to that of GAPDH. Hfrc1 and UGTrel7 displayed similar tissue distributions, each showing high levels of expression in the colon, stomach, lung, and leukocyte. However, the expression level of hfrc1 transcripts was 10 times that of UGTrel7 (note the different scales in the two panels in Fig. 3).
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Hfrc1 Has a Role in Heparan Sulfate Synthesis—Heparan sulfate molecules are found on the cell surface as glucosaminoglycan chains covalently attached to a core protein. Heparan sulfate has a repeat of GlcUA and GlcNAc, whereas chondroitin sulfate has a repeat of GlcUA and GalNAc in the glucosaminoglycan chains. Because HFRC1 can transport UDP-GlcNAc, the possibility needed to be considered that HFRC1 might contribute to the elongation of heparan sulfate by supplying the donor substrate. To investigate whether HFRC1 is involved in the synthesis of heparan sulfate, HCT116 cells stably expressing HA-tagged hfrc1 or mock vector were immunostained with an antibody against heparan sulfate, and analyzed by flow cytometry. As shown in Fig. 5A, the cell surface expression levels of heparan sulfate on hfrc1 transfectant cells were increased compared with that on mock transfectant cells. On the other hand, the cell surface expression levels of chondroitin sulfate were not different between the hfrc1 and mock transfectants. These results suggest that hfrc1 is involved in the synthesis of heparan sulfate by supplying UDP-GlcNAc, a donor substrate for the heparan sulfate synthases.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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As shown in Fig. 5, an alteration in the expression of hfrc1 affected heparan sulfate on the cell surface of mammalian cells. UDP-GlcNAc, a transport substrate for HFRC1, can be utilized by the enzymes that catalyze the elongation of heparan sulfate. Because the elongation of proteoglycan takes place within the Golgi apparatus (2, 21–23), the localization of HFRC1 to the Golgi apparatus would allow it to regulate the donor-substrate level for the enzymes. Heparan sulfate is known to be involved in a variety of biological phenomena, such as morphogenesis, development, angiogenesis, blood coagulation, cell adhesion, and lipid metabolism (23, 24). It is also involved in a variety of signaling pathways, in particular those of fibroblast growth factor (25), Wnt/Wingless (26–28), Decapentaplegic (27), and Hedgehog (29). Selva et al. (9) reported that frc mutant embryos display defects in Wingless, Hedgehog, and fibroblast growth factor signaling (9). Hfrc1 is expected to be involved in growth factor signaling through the regulation of heparan sulfate synthesis in a way similar to that observed for frc.
The substrate specificity of HFRC1 also suggests the possibility that HFRC1 may be able to modulate Notch activity in humans by supplying UDP-GlcNAc. The interaction of the Notch receptor with its ligands, Serrate and Delta, is modulated by the addition of GlcNAc to an O-linked Fuc on the Notch by FRINGE enzyme (30–33). In Drosophila, mutation of frc leads to a defect in Notch maturation (10). Here, HFRC1 exhibited an apparent Km value for UDP-GlcNAc comparable with that of FRC (8 versus 7.8 µM, respectively). It would be interesting to investigate whether hfrc1 can rescue the defects in glycosylation and signaling caused by frc mutations.
We also observed that HFRC1 has a UDP-Glc transport activity. HFRC1 is the first UDP-Glc transporter to be reported in a mammal, although the physiological significance of UDP-Glc transport in the Golgi apparatus is obscure. Possibly, it is involved in some glycosylation process in the Golgi apparatus, such as the addition of O-linked glucose (34) or the glucosyl-transferase reaction by 4GalT1 in the presence of 
-lactalbumin (35). On the other hand, we found that HFRC1 transported GDP-Man alone in the yeast, not in the mammalian cells. This difference is perhaps because of the abundance of antiport molecules for GDP-Man within the yeast Golgi apparatus. It has been suggested that GDP-Man is transported into the lumen of the Golgi apparatus of yeast and Leishmania, but not into that of mammals (3). Whether or not HFRC1 can act as a real GDP-Man transporter in mammalian cells needs to be evaluated in future investigations.
In humans, another UDP-GlcNAc transporter exists within the Golgi apparatus (36). We did not investigate whether HFRC1 and this UDP-GlcNAc transporter operate in a complementary fashion or in different ways within the Golgi apparatus. Identification of the glycoconjugates that are affected by these genes may hold the key to a clarification of their functions. Analysis using RNA interference of each gene may also help to elucidate the significance of these transporters in glycosylation.
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FOOTNOTES |
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* This work was supported by the New Energy and Industrial Technology Development Organization (NEDO) as part of the R&D Project of Industrial Science and Technology Frontier Program, and by Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Corporation (JST). 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. 
|| Both authors contributed equally to this article.
¶¶ To whom correspondence should be addressed: Laboratory of Cell Biology, Dept. of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, Japan. Tel.: 81-426-91-8140; Fax: 81-426-91-9315; E-mail: shoko{at}t.soka.ac.jp.
1 The abbreviations used are: ER, endoplasmic reticulum; 4GalT1, 1,4-galactosyltransferase 1; CMP-Sia, CMP-sialic acid; HA, influenza hemagglutinin epitope; frc, fringe connection; mAb, monoclonal antibody; NST, nucleotide-sugar transporter; GDP-Fuc, GDP-fucose; GDP-Man, GDP-mannose; sqv, squashed vulva; UDP-Gal, UDP-galactose; UDP-GalNAc, UDP-N-acetyl-D-galactosamine; UDP-Glc, UDP-glucose; UDP-GlcNAc, UDP-N-acetyl-D-glucosamine; UDP-GlcUA, UDP-glucuronic acid; ORF, open reading frame; PBS, phosphate-buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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0.5). Hydropathy analysis and predictions of transmembrane helices of the amino acid sequence were carried out using the SOSui program developed by Mitsui Knowledge Industry Co., Ltd. The putative transmembrane domains are underlined. Possible N-glycosylation site in the HFRC1 sequence is double underlined. C, the genomic structures of the hfrc1 (upper) and UGTrel7 (lower) genes were constructed from the genomic and cDNA sequences. Solid boxes indicate exons, and open boxes indicate untranslated sequences.
