Molecular Cloning and Characterization of a Novel 3′-Phosphoadenosine 5′-Phosphosulfate Transporter, PAPST2
2006; Elsevier BV; Volume: 281; Issue: 16 Linguagem: Inglês
10.1074/jbc.m508991200
ISSN1083-351X
AutoresS. Kamiyama, Norihiko Sasaki, Emi Goda, Kumiko Ui‐Tei, Kaoru Saigo, Hisashi Narimatsu, Yoshifumi Jigami, Reiji Kannagi, Tatsuro Irimura, Shoko Nishihara,
Tópico(s)Chronic Myeloid Leukemia Treatments
ResumoSulfation is an important posttranslational modification associated with a variety of molecules. It requires the involvement of the high energy form of the universal sulfate donor, 3′-phosphoadenosine 5′-phosphosulfate (PAPS). Recently, we identified a PAPS transporter gene in both humans and Drosophila. Although human colonic epithelial tissues express many sulfated glycoconjugates, PAPST1 expression in the colon is trace. In the present study, we identified a novel human PAPS transporter gene that is closely related to human PAPST1. This gene, called PAPST2, is predominantly expressed in human colon tissues. The PAPST2 protein is localized on the Golgi apparatus in a manner similar to the PAPST1 protein. By using yeast expression studies, PAPST2 protein was shown to have PAPS transport activity with an apparent Km value of 2.2 μm, which is comparable with that of PAPST1 (0.8 μm). Overexpression of either the PAPST1 or PAPST2 gene increased PAPS transport activity in human colon cancer HCT116 cells. The RNA interference of the PAPST2 gene in the HCT116 cells significantly reduced the reactivity of G72 antibody directed against the sialyl 6-sulfo N-acetyllactosamine epitope and total sulfate incorporation into cellular proteins. These findings indicate that PAPST2 is a PAPS transporter gene involved in the synthesis of sulfated glycoconjugates in the colon. Sulfation is an important posttranslational modification associated with a variety of molecules. It requires the involvement of the high energy form of the universal sulfate donor, 3′-phosphoadenosine 5′-phosphosulfate (PAPS). Recently, we identified a PAPS transporter gene in both humans and Drosophila. Although human colonic epithelial tissues express many sulfated glycoconjugates, PAPST1 expression in the colon is trace. In the present study, we identified a novel human PAPS transporter gene that is closely related to human PAPST1. This gene, called PAPST2, is predominantly expressed in human colon tissues. The PAPST2 protein is localized on the Golgi apparatus in a manner similar to the PAPST1 protein. By using yeast expression studies, PAPST2 protein was shown to have PAPS transport activity with an apparent Km value of 2.2 μm, which is comparable with that of PAPST1 (0.8 μm). Overexpression of either the PAPST1 or PAPST2 gene increased PAPS transport activity in human colon cancer HCT116 cells. The RNA interference of the PAPST2 gene in the HCT116 cells significantly reduced the reactivity of G72 antibody directed against the sialyl 6-sulfo N-acetyllactosamine epitope and total sulfate incorporation into cellular proteins. These findings indicate that PAPST2 is a PAPS transporter gene involved in the synthesis of sulfated glycoconjugates in the colon. Sulfation of a variety of molecules, including glycoproteins, proteoglycans, and glycolipids, is an important posttranslational modification. The process requires the involvement of the high energy form of the universal sulfate donor, namely, 3′-phosphoadenosine 5′-phosphosulfate (PAPS). 2The abbreviations used are: PAPS, 3′-phosphoadenosine 5′-phosphosulfate; FITC, fluorescein isothiocyanate; Gal, galactose; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GlcNAc, UDP-N-acetyl d-glucosamine; HA, influenza hemagglutinin epitope; Lex, Lewis x antigen, galactose β1,4 (fucose α1,3) N-acetyl d-glucosamine; Lea, Lewis a antigen, galactose β1,3 (fucose α1,4) N-acetyl d-glucosamine; mAb, monoclonal antibody; PBS, phosphate-buffered saline; RNAi, RNA interference; siRNA, small interfering RNA; sll, slalom. In higher organisms, PAPS is synthesized in the cytosol or nucleus by PAPS synthetases (1Li H. Deyrup A. Mensch Jr., J.R. Domowicz M. Konstantinidis A.K. Schwartz N.B. J. Biol. Chem. 1995; 270: 29453-29459Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 2Kurima K. Warman M.L. Krishnan S. Domowicz M. Krueger Jr., R.C. Deyrup A. Schwartz N.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8681-8685Crossref PubMed Scopus (129) Google Scholar) and is subsequently translocated into the Golgi lumen via the PAPS transporter(s). Because most of the sulfation of glycoconjugates occurs in the Golgi apparatus, the translocation of PAPS is considered to be an essential process. Recently, we identified and characterized a PAPS transporter in both humans and Drosophila (3Kamiyama S. Suda T. Ueda R. Suzuki M. Okubo R. Kikuchi N. Chiba Y. Goto S. Toyoda H. Saigo K. Watanabe M. Narimatsu H. Jigami Y. Nishihara S. J. Biol. Chem. 2003; 278: 25958-25963Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Human PAPST1 and the Drosophila ortholog SLALOM (SLL) are Golgi-localized proteins that exhibit PAPS-specific transport activity. Analysis of the sll gene by using the RNA interference (RNAi) fly demonstrated that the PAPS transporter is essential for viability in vivo (3Kamiyama S. Suda T. Ueda R. Suzuki M. Okubo R. Kikuchi N. Chiba Y. Goto S. Toyoda H. Saigo K. Watanabe M. Narimatsu H. Jigami Y. Nishihara S. J. Biol. Chem. 2003; 278: 25958-25963Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Furthermore, Lüders et al. (4Lüders F. Segawa H. Stein D. Selva E.M. Perrimon N. Turco S.J. Häcker U. EMBO J. 2003; 22: 3635-3644Crossref PubMed Scopus (67) Google Scholar) demonstrated that sll is involved in growth factor signaling pathways during patterning and morphogenesis. Heparan sulfate proteoglycans (HSPGs) possess glycosaminoglycan chains that contain diversely sulfated uronic acid and glucosamine residues. Cell surface HSPGs are involved in a variety of developmental signaling pathways, and the functions of HSPGs are dependent on their sulfation state (5Reichsman F. Smith L. Cumberledge S. J. Cell Biol. 1996; 135: 819-827Crossref PubMed Scopus (261) Google Scholar, 6Zioncheck T.F. Richardson L. Liu J. Chang L. King K.L. Bennett G.L. Fugedi P. Chamow S.M. Schwall R.H. Stack R.J. J. Biol. Chem. 1995; 270: 16871-16878Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 7Olwin B.B. Rapraeger A. J. Cell Biol. 1992; 118: 631-639Crossref PubMed Scopus (210) Google Scholar, 8Yayon A. Klagsbrun M. Esko J.D. Leder P. Ornitz D.M. Cell. 1991; 64: 841-848Abstract Full Text PDF PubMed Scopus (2094) Google Scholar, 9Rapraeger A.C. Krufka A. Olwin B.B. Science. 1991; 252: 1705-1708Crossref PubMed Scopus (1292) Google Scholar, 10Aviezer D. Yayon A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12173-12177Crossref PubMed Scopus (126) Google Scholar). A mutation in the sll gene caused defects in multiple signaling pathways, including Wingless and Hedgehog signaling, probably because of the lack of HSPG sulfation (4Lüders F. Segawa H. Stein D. Selva E.M. Perrimon N. Turco S.J. Häcker U. EMBO J. 2003; 22: 3635-3644Crossref PubMed Scopus (67) Google Scholar). Despite the low expression of the PAPST1 gene in the colon (3Kamiyama S. Suda T. Ueda R. Suzuki M. Okubo R. Kikuchi N. Chiba Y. Goto S. Toyoda H. Saigo K. Watanabe M. Narimatsu H. Jigami Y. Nishihara S. J. Biol. Chem. 2003; 278: 25958-25963Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), human colonic tissues highly express many sulfated glycoconjugates such as proteoglycans and sulfomucins. For example, the 3′-sulfo Lewis a epitope (3′-sulfo Lea: Gal β1,3 (fucose α1,4) GlcNAc that is sulfated at the C-3 position of Gal) is strongly expressed in the normal colonic epithelium but diminishes considerably in primary colon carcinomas (11Irimura T. Wynn D.M. Hager L.G. Cleary K.R. Ota D.M. Cancer Res. 1991; 51: 5728-5735PubMed Google Scholar, 12Yamori T. Ota D.M. Cleary K.R. Hoff S. Hager L.G. Irimura T. Cancer Res. 1989; 49: 887-894PubMed Google Scholar, 13Matsushita Y. Yamamoto N. Shirahama H. Tanaka S. Yonezawa S. Yamori T. Irimura T. Sato E. Jpn. J. Cancer Res. 1995; 86: 1060-1067Crossref PubMed Scopus (35) Google Scholar). The sialyl 6-sulfo Lewis x epitope (sialyl 6-sulfo Lex: Sia α2,3 Gal β1,4 (fucose α1,3) GlcNAc that is sulfated at the C-6 position of GlcNAc) is also expressed in normal human colonic tissues but not in cancerous tissues (14Izawa M. Kumamoto K. Mitsuoka C. Kanamori C. Kanamori A. Ohmori K. Ishida H. Nakamura S. Kurata-Miura K. Sasaki K. Nishi T. Kannagi R. Cancer Res. 2000; 60: 1410-1416PubMed Google Scholar). These sulfated glycoconjugate epitopes are believed to regulate many biological processes in the colon (11Irimura T. Wynn D.M. Hager L.G. Cleary K.R. Ota D.M. Cancer Res. 1991; 51: 5728-5735PubMed Google Scholar, 12Yamori T. Ota D.M. Cleary K.R. Hoff S. Hager L.G. Irimura T. Cancer Res. 1989; 49: 887-894PubMed Google Scholar, 13Matsushita Y. Yamamoto N. Shirahama H. Tanaka S. Yonezawa S. Yamori T. Irimura T. Sato E. Jpn. J. Cancer Res. 1995; 86: 1060-1067Crossref PubMed Scopus (35) Google Scholar, 14Izawa M. Kumamoto K. Mitsuoka C. Kanamori C. Kanamori A. Ohmori K. Ishida H. Nakamura S. Kurata-Miura K. Sasaki K. Nishi T. Kannagi R. Cancer Res. 2000; 60: 1410-1416PubMed Google Scholar, 15Nakayama T. Watanabe M. Katsumata T. Teramoto T. Kitajima M. Cancer Res. 1995; 75: 2051-2056Google Scholar, 16Nakamori S. Kameyama M. Imaoka S. Furukawa H. Ishikawa O. Sasaki Y. Kabuto T. Iwanaga T. Matsushita Y. Irimura T. Cancer Res. 1993; 53: 3632-3637PubMed Google Scholar, 17Yamachika T. Nakanishi H. Inada K. Kitoh K. Kato T. Irimura T. Tatematsu M. Virchows Arch. 1997; 431: 25-30Crossref PubMed Scopus (19) Google Scholar). In the present study, we attempted to identify the PAPS transporter that is responsible for the sulfation of glycoconjugates in the colon tissue. We found a gene that is closely related to the human PAPST1 gene by performing a BLAST search of data bases. This gene, called PAPST2, is preferentially expressed in human colon tissues. The PAPST2 protein exhibited PAPS transport activity similar to that of the PAPST1 protein. Here, we report the functional properties of this novel PAPS transporter. Materials—GDP-[2-3H]mannose (15 Ci/mmol), UDP-[1-3H]glucose (15 Ci/mmol), UDP-N-acetyl [6-3H]d-galactosamine (15 Ci/mmol), UDP-[14C(U)]glucuronic acid (15 Ci/mmol), and carrier-free [35S]H2SO4 (100 m Ci/ml) were purchased from American Radiolabeled Chemicals Inc. (St. Louis, MO). GDP-[1-3H]fucose (6.95 Ci/mmol), UDP-[4,5-3H]galactose (48.3 Ci/mmol), CMP-[9-3H]sialic acid (33.6 Ci/mmol), UDP-N-acetyl [6-3H(N)]d-glucosamine (39.7 Ci/mmol), and [35S]PAPS (1.82 Ci/mmol) were purchased from Perkin Elmer Life Sciences Inc. Zymolyase 100T was obtained from Seikagaku Kogyo Co. Ltd. (Tokyo, Japan). Fluorescein isothiocyanate (FITC)-conjugated anti-c-Myc monoclonal antibody (mAb) (9E10) and rhodamine-conjugated anti-influenza hemagglutinin epitope (HA) mAb (HA-probe, F-7) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All the other reagents used in the study were of the highest purity grade available commercially. Isolation of Human PAPS Transporter cDNA and Construction of Expression Plasmids—The human PAPST2 gene was identified and cloned using the same procedures as described previously (3Kamiyama S. Suda T. Ueda R. Suzuki M. Okubo R. Kikuchi N. Chiba Y. Goto S. Toyoda H. Saigo K. Watanabe M. Narimatsu H. Jigami Y. Nishihara S. J. Biol. Chem. 2003; 278: 25958-25963Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The amino acid sequence of the open reading frame of UGTrel1 (18Ishida N. Miura N. Yoshioka S. Kawakita M. J. Biochem. 1996; 120: 1074-1078Crossref PubMed Scopus (92) Google Scholar) was used as a query sequence for the TBLASTN search that was performed to detect novel genes. To obtain the cDNA of NM_015948, a human gene that was identified in this study, and to create recombination sites for the GATEWAY™ cloning system (Invitrogen), we used two steps of attB adaptor PCR and prepared attB-flanked PCR products. For the first gene-specific amplification, a forward template-specific primer with attB1, 5′-AAAAAGCAGGCTTCCATAATGGCATGGACTTG-3′, and a reverse template-specific primer with attB2, 5′-AGAAAGCTGGGTCTACAGTCTGTGCCAGCGT-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 pDONR™201 vector were performed in accordance with the manufacturer's protocol to create an entry clone for use during the subsequent subcloning steps. The entry clone was subcloned into the appropriate expression vectors by using the GATEWAY™ cloning system in accordance with the manufacturer's protocol. A 3 × HA epitope tag or a c-Myc tag was inserted into the expression vectors at the position corresponding to the C terminus of the expressing protein. Transient Transfection and Immunofluorescence Microscopy—Transient transfection and immunofluorescence microscopy were performed by using one of two procedures. The first procedure is similar to one described previously (3Kamiyama S. Suda T. Ueda R. Suzuki M. Okubo R. Kikuchi N. Chiba Y. Goto S. Toyoda H. Saigo K. Watanabe M. Narimatsu H. Jigami Y. Nishihara S. J. Biol. Chem. 2003; 278: 25958-25963Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 19Suda T. Kamiyama S. Suzuki M. Kikuchi N. Nakayama K. Narimatsu H. Jigami Y. Aoki T. Nishihara S. J. Biol. Chem. 2004; 279: 26469-26474Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Briefly, HCT116 cells were subcultured onto a 4-well Lab-Tek chamber slide (Nalge Nunc International) in Dulbecco's modified Eagle's medium/Ham's nutrient mixture F-12 (1:1) containing 10% fetal bovine serum. After 24 h of subculturing, the HCT116 cells were transfected with 0.25 μg/well of pCXN2 (20Niwa H. Yamamura K. Miyazaki J. Gene. 1991; 108: 193-199Crossref PubMed Scopus (4619) Google Scholar), pCXN2 inserted with HA-tagged PAPST1, or pCXN2 inserted with HA-tagged PAPST2 by using Lipofectamine 2000 reagent (Invitrogen) in accordance with the manufacturer's protocol. After 72 h, the cells were fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde for 30 min at 4 °C, and they were then permeabilized in a permeabilizing buffer (PBS containing 0.1% Triton X-100) for 1 h at 4 °C. The cells were subsequently double immunostained with rhodamine-conjugated anti-HA mAb and anti-β1,4-galactosyltransferase 1 mAb (21Uemura M. Sakaguchi T. Uejima T. Nozawa S. Narimatsu H. Cancer Res. 1992; 52: 6153-6157PubMed Google Scholar) as described previously (3Kamiyama S. Suda T. Ueda R. Suzuki M. Okubo R. Kikuchi N. Chiba Y. Goto S. Toyoda H. Saigo K. Watanabe M. Narimatsu H. Jigami Y. Nishihara S. J. Biol. Chem. 2003; 278: 25958-25963Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 19Suda T. Kamiyama S. Suzuki M. Kikuchi N. Nakayama K. Narimatsu H. Jigami Y. Aoki T. Nishihara S. J. Biol. Chem. 2004; 279: 26469-26474Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Finally, the cells were washed four times and mounted with PermaFluor (Thermo Shandon, Pittsburgh, PA). The fluorescence was observed using a confocal laser scanning microscope, LSM5 Pascal (Carl Zeiss, Goettingen, Germany). In the second procedure, HA-tagged PAPST2 and c-Myc-tagged PAPST1 were expressed simultaneously in the HCT116 cells. After 24 h of subculturing, the cells were transfected with 0.25 μg/well of pCXN2 or pCXN2 inserted with HA-tagged PAPST2 and pCXN2 inserted with c-Myc-tagged PAPST1 by using Lipofectamine 2000 reagent. The cells were fixed, permeabilized, and immunostained with FITC-conjugated anti-c-Myc mAb and rhodamine-conjugated anti-HA mAb for 30 min at 37 °C after 72 h. The remainder of the procedure was the same as that described above. Stable Transfection and Subcellular Fractionation—HCT116 cells were subcultured onto 6-cm dishes in Dulbecco's modified Eagle's medium/Ham's nutrient mixture F-12 (1:1) containing 10% fetal bovine serum. After 24 h, the cells were transfected with 8 μg of pCXN2 vector, pCXN2 inserted with HA-tagged PAPST1, or pCXN2 inserted with HA-tagged PAPST2 by using Lipofectamine 2000 reagent in accordance with the manufacturer's protocol. The transfectants were selected by the addition of 600 μg/ml of geneticin (Invitrogen) to the medium and cultured for 1 month after 48 h. Subcellular fractionation was performed as described previously (3Kamiyama S. Suda T. Ueda R. Suzuki M. Okubo R. Kikuchi N. Chiba Y. Goto S. Toyoda H. Saigo K. Watanabe M. Narimatsu H. Jigami Y. Nishihara S. J. Biol. Chem. 2003; 278: 25958-25963Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 19Suda T. Kamiyama S. Suzuki M. Kikuchi N. Nakayama K. Narimatsu H. Jigami Y. Aoki T. Nishihara S. J. Biol. Chem. 2004; 279: 26469-26474Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The cells were harvested and suspended in 10 mm HEPES-Tris (pH 7.4) containing 0.25 m sucrose, 1 mm phenylmethylsulfonyl fluoride, and 1 μg/ml each of leupeptin, aprotinin, and pepstatin A. The cells were then homogenized using a Dounce homogenizer. The lysate was centrifuged at 1,000 × g for 10 min to remove the unlysed cells and cell wall debris. The supernatant was then centrifuged at 7,700 × g for 10 min at 4 °C, and the supernatant was further centrifuged at 100,000 × g to yield a pellet of P100 membrane fraction. Subcellular Fractionation of Yeast and Transport Assay—Yeast (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 (22Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar) using a yeast expression vector, YEp352GAP-II (23Nakayama K. Maeda Y. Jigami Y. Glycobiology. 2003; 13: 673-680Crossref PubMed Scopus (52) Google Scholar). These transformed yeast cells were grown at 30 °C in a synthetic defined medium, which did not contain uracil, for selecting transformants. Subcellular fractionation and nucleotide sugar transport assays were performed as described previously (3Kamiyama S. Suda T. Ueda R. Suzuki M. Okubo R. Kikuchi N. Chiba Y. Goto S. Toyoda H. Saigo K. Watanabe M. Narimatsu H. Jigami Y. Nishihara S. J. Biol. Chem. 2003; 278: 25958-25963Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 19Suda T. Kamiyama S. Suzuki M. Kikuchi N. Nakayama K. Narimatsu H. Jigami Y. Aoki T. Nishihara S. J. Biol. Chem. 2004; 279: 26469-26474Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The cells were harvested, washed with ice-cold 10 mm NaN3, and 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/g of cells). The spheroplasts were pelleted using a refrigerated centrifuge and washed twice with 1.0 m ice-cold sorbitol to remove traces of zymolyase. The cells were suspended in ice-cold lysis buffer (0.8 m sorbitol in 10 mm triethanolamine (pH 7.2), 5 μg/ml of pepstatin A, and 1 mm phenylmethylsulfonyl fluoride) and subsequently homogenized using a Dounce homogenizer. The lysate was centrifuged at 1,000 × g for 10 min to remove the unlysed cells and cell wall debris. The supernatant was then centrifuged at 10,000 × g for 15 min at 4 °C, which yielded a pellet of P10 membrane fraction. The supernatant was further centrifuged at 100,000 × g to yield a pellet of P100 membrane fraction. Each fraction (200 μg of protein) was then 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) that contained 1 μm radiolabeled substrate at 30 °C for 5 min. After incubation, the radioactivity incorporated in the microsomes was trapped using a 0.45-μm nitrocellulose filter and measured using liquid scintillation. The amount of incorporated radioactivity was calculated as the difference from the background value obtained from the same assay at 30 °C for 0 min for each sample. Western Blot Analysis—Fifty micrograms of protein from each sample was added to 3× sodium dodecyl sulfate (SDS) sample buffer (New England Biolabs Inc., Beverly, MA) and subsequently incubated at room temperature for 2 h. The samples were fractionated on a 2-15% SDS-polyacrylamide gel gradient (Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan). The separated proteins were electrotransferred onto a polyvinylidene difluoride membrane. The 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) in accordance with the manufacturer's instructions. Quantitative Analysis of the PAPST2 Transcript in Human Tissues by Real-time PCR—The amount of PAPST1 and PAPST2 transcripts in human tissues was determined by real-time PCR. Total RNA was extracted from human tissues by the method of Chomczynski and Sacchi (24Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). First strand cDNA was synthesized using a Superscript II First Strand Synthesis kit (Invitrogen) in accordance with the manufacturer's instructions. Real-time PCR was performed using a qPCR Mastermix (QuickGoldStar; Eurogentec, Seraing, Belgium) and ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster, CA). The PCR primer pair sequences and TaqMan probes used for each gene were as follows. For the detection of PAPST1, the forward primer 5′-GGCAGGCCCTGAAGCT-3′, reverse primer 5′-TGCGGGTCATCACTCTTTC-3′, and probe 5′-CCACAGGGCTCCAGGTGTCTTATCTG-3′ were used. For the detection of PAPST2, the forward primer 5′-GATTAGGCCCTGCAGTAACATT-3′, reverse primer 5′-ATCCAGTGAGGGAAAAAAGGA-3′, and probe 5′-TGTGCAAAGAATCCAGTTCGGACCTA-3′ were used. The probes were labeled at the 5′-end with the reporter dye FAM and at the 3′-end with the quencher dye TAMRA. The relative amounts of PAPST1 and PAPST2 transcripts were normalized with respect to the human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) transcripts present in the same cDNA. Northern Blot Analysis—PAPST2 cDNA probe (full-length open reading frame) was prepared by random priming using [α-32P]dCTP and BcaBest™ labeling kit (Takara Bio Inc., Shiga, Japan) in accordance with the manufacturer's instructions. Poly(A)+ RNA was isolated using Oligotex-dT30 (super) mRNA purification kit (Takara Bio Inc.) in accordance with the manufacturer's instructions. Poly(A)+ RNA derived from each sample was separated by 1.2% agarose gel containing 2.2 m formaldehyde and then transferred onto Hybond-XL nylon membrane (Amersham Biosciences). The membranes were prehybridized in the hybridization solution (5× SSPE (standard saline phosphate with EDTA; 150 mm NaCl, 10 mm NaH2PO4, 1 mm EDTA), 5× Denhardt's solution, and 0.2 mg/ml of sermon sperm DNA) for 2 h at 42°C. The membranes were hybridized overnight in hybridization solution containing 2 × 106 cpm/ml of each radiolabeled probe at 42 °C. Following hybridization, the membrane was washed in 2× SSPE containing 0.1% SDS at room temperature and in 0.2× SSPE containing 0.1% SDS at 50 °C. The radiolabeled materials were detected by using a BAS-2000 imaging analyzer (Fuji Photo Film). The membrane was reprobed with a human GAPDH cDNA probe. RNAi of PAPST2 Gene—A sequence of small interfering RNA (siRNA) for each gene was designed as described previously (25Ui-Tei K. Naito Y. Takahashi F. Haraguchi T. Ohki-Hamazaki H. Juni A. Ueda R. Saigo K. Nucleic Acids Res. 2004; 32: 936-948Crossref PubMed Scopus (614) Google Scholar). Twenty-five base pairs of stealth RNAs were designed and purchased from Invitrogen. The PAPST1-813 siRNA (initiated at the 813 nucleotide position from the start codon) has the sequence 5′-CCUCAUCUUACUGGCAGGUUAUAUU-3′. The sequences of PAPST2-342 siRNA (initiated at the 342 nucleotide position from the start codon) and PAPST2-513 siRNA (initiated at the 513 nucleotide position from the start codon) are 5′-CCUUACCUUAGUGCAGUUUGCCUUU-3′ and 5′-CCAAGUCAUCUUCAAGUGCUGCAAA-3′, respectively. As control siRNA, stealth RNAi negative control duplex (Invitrogen) was used. The HCT116 cells were subcultured onto 6-cm dishes at a concentration of 1 × 106 cells/dish in Dulbecco's modified Eagle's medium/Ham's nutrient mixture F-12 (1:1) containing 10% fetal bovine serum 24 h prior to the transfection. The cells were transfected with 10 or 100 nm siRNA by using Lipofectamine 2000 reagent. RNA was extracted using TRIzol reagent (Invitrogen), and the first strand cDNA was synthesized using a Superscript II First Strand Synthesis kit (Invitrogen). Metabolic Labeling of Colon Cancer Cell Line—Radiolabeling of sulfated residues in cell macromolecules was performed using procedures similar to those described previously (26Tsuiji H. Hayashi M. Wynn D.M. Irimura T. Jpn. J. Cancer Res. 1998; 89: 1267-1275Crossref PubMed Scopus (17) Google Scholar). HCT116 cells were subcultured onto 10-cm dishes at a concentration of 1.5 or 2 × 106 cells/dish in the inorganic sulfate-free Dulbecco's modified Eagle's medium/Ham's nutrient mixture F-12 (1:1) containing 10% fetal bovine serum and 100 μCi/ml of carrier-free [35S]H2SO4 48 h after transfection and 48 h prior to the analysis. The cells were rinsed with PBS and detached with PBS containing 0.02% EDTA for 5 min. The cells were rinsed twice with PBS and suspended in 4 volumes of lysis buffer (10 mm Tris-HCl (pH 7.4), 0.5% Nonidet P-40, 1 mm EDTA, and 0.5 mm phenylmethylsulfonyl fluoride) and incubated on ice for 1 h. The solution was centrifuged at 15,000 × g for 30 min, and the supernatants were used as cell lysates. Fifty micrograms of protein from each sample were added to 3× SDS sample buffer (New England Biolabs Inc.) and boiled at 100 °C for 5 min. The samples were fractionated on a 2-15% SDS-polyacrylamide gel gradient (Daiichi Pure Chemicals Co., Ltd.). Gels were stained with Coomassie brilliant blue and dried on Whatman 3MM paper (Whatman International Ltd.). The radiolabeled materials were detected by using a BAS-2000 imaging analyzer (Fuji Photo Film). Flow Cytometric Analysis—The HCT116 cells were subcultured onto 10-cm dishes at a concentration of 1.5 or 2 × 106 cells/dish 72 h after transfection and 24 h prior to the analysis. These cells were then harvested with PBS containing 1 mm EDTA and washed with a wash buffer (PBS containing 0.1% bovine serum albumin and 0.1% sodium azide). Cell suspensions of 100 μl (0.5 × 106 cells) were incubated with G72 mAb (27Mitsuoka C. Sawada-Kasugai M. Ando-Furui K. Izawa M. Nakanishi H. Nakamura S. Ishida H. Kiso M. Kannagi R. J. Biol. Chem. 1998; 273: 11225-11233Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar) for 1 h on ice and washed twice with 0.5 ml of the wash buffer. The cells were then resuspended in 100 μl of FITC-conjugated goat anti-mouse IgM and incubated on ice for 30 min. The cells were washed twice with wash buffer and resuspended in 500 μl of PBS containing 0.1% sodium azide. Flow cytometric analysis was performed using FAC-SAria flow cytometer (BD Biosciences) and WinMDI 2.8 software (The Scripps Research Institute Cytometry software page). Molecular Cloning of Human PAPST2 cDNA—We identified a cDNA sequence (GenBank™ Accession number NM_015948) homologous to the putative nucleotide sugar transporter gene UGTrel1 by using the same procedure that was employed for the PAPST1 gene (3Kamiyama S. Suda T. Ueda R. Suzuki M. Okubo R. Kikuchi N. Chiba Y. Goto S. Toyoda H. Saigo K. Watanabe M. Narimatsu H. Jigami Y. Nishihara S. J. Biol. Chem. 2003; 278: 25958-25963Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Initially, it was described as CGI-19 protein in the GenBank™ data base. We named it PAPST2 and cloned the open reading frame as described under "Experimental Procedures." The phylogenetic tree of PAPS and the nucleotide sugar transporter genes in humans and Drosophila indicated that PAPST1, PAPST2, and UGTrel1 are classified in the same group (Fig. 1A). An alignment of the amino acid sequences of these genes is shown in Fig. 1B. PAPST2 comprised 401 amino acids with a calculated mass of 44.6 kDa. Hydrophobicity analyses of the amino acid sequences indicate that the PAPST2 protein is a type III transmembrane protein with nine transmembrane domains similar to that of PAPST1, although the transmembrane topology has yet to be verified experimentally. PAPST2 showed 22.4 and 21.7% identity to UGTrel1 and PAPST1, respectively. The structural similarity suggested that PAPST2 is a PAPS transporter gene similar to PAPST1. There are eight potential N-glycosylation sites in the PAPST2. The PAPST2 gene is mapped on human chromosome 6p24.3, and the mRNA comprises 11 exons. PAPST2 Is a Golgi-localized Protein—PAPST1 is a membrane protein that is localized on the Golgi apparatus (3Kamiyama S. Suda T. Ueda R. Suzuki M. Okubo R. Kikuchi N. Chiba Y. Goto S. Toyoda H. Saigo K. Watanabe M. Narimatsu H. Jigami Y. Nishihara S. J. Biol. Chem. 2003; 278: 25958-25963Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Because PAPST2 shares similarity to PAPST1, we expected that PAPST2 would also be a Golgi-localized PAPS transporter. First, we investigated the subcellular localization of the PAPST2 protein. Human colon cancer HCT116 cells were transiently transfected with a mammalian expression vector, pCXN2, that contained HA-tagged PAPST2 or HA-tagged PAPST1 gene and were double immunostained with anti-HA mAb and anti-β1,4 galactosyltransferase 1 mAb. The results of immunofluorescence microscopy of the cells are shown in Fig. 2A. The HA-tagged PAPST1 protein was observed to be colocalized with β1,4 galactosyltransferase 1, which is a protein that is typically localized in the trans-Golgi (21Uemura M. Sakaguchi T. Uejima T. Nozawa S. Narimatsu H. Cancer Res. 1992; 52: 6153-6157PubMed Google Scholar), and this observation is consistent with a previous report (3Kamiyama S. Suda T. Ueda R. Suzuki
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