THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 16, pp. 10945–10953, April 21, 2006
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Molecular Cloning and Characterization of a Novel 3ⴕPhosphoadenosine 5ⴕ-Phosphosulfate Transporter, PAPST2*
Received for publication, August 15, 2005, and in revised form, February 8, 2006 Published, JBC Papers in Press, February 21, 2006, DOI 10.1074/jbc.M508991200

Shin Kamiyama‡§, Norihiko Sasaki‡¶, Emi Goda‡¶, Kumiko Ui-Tei储, Kaoru Saigo储, Hisashi Narimatsu**,
Yoshifumi Jigami**, Reiji Kannagi‡‡, Tatsuro Irimura§§, and Shoko Nishihara‡¶1
From the ‡Laboratory of Cell Biology, Department of Bioinformatics, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577,§
Research Association for Biotechnology, 3-9, Nishi-Shinbashi 2-chome, Minato-ku, Tokyo 105-0003, ¶Core Research for
Evolutional Science and Technology of Japan Science and Technology Agency, Kawaguchi Center Building, 4-1-8, Hon-cho,
Kawaguchi, Saitama 332-0012, 储Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, **Research Center for Glycoscience, National Institute of Advanced Industrial Scienceand Technology, 1-1-1 Umezono, Tsukuba, Ibaraki, 305-8586, ‡‡Program of Molecular Pathology, Aichi Cancer Center, 1-1
Kanokoden, Chikusa-ku, Nagoya 464-8681, and §§Laboratory of Cancer Biology and Molecular Immunology, Graduate School of
Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

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 theuniversal sulfate donor, namely, 3⬘-phosphoadenosine 5⬘-phosphosulfate (PAPS).2 In higher organisms, PAPS is synthesized in the cytosol or
nucleus by PAPS synthetases (1, 2) and is subsequently translocated into

* This work was supported by Core Research for Evolutional Science and Technology of
the Japan Science and Technology Agency and the New Energy and Industrial Technology Development Organization. The costs of publication of this article weredefrayed 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 toindicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB231931.
1
To whom correspondence should be addressed. Tel./Fax: 81-426-91-8140; E-mail:shoko@t.soka.ac.jp.
2
The 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 ␤1,4 (fucose ␣1,3) N-acetyl D-glucosamine;
Lea, Lewis a antigen, galactose ␤1,3 (fucose ␣1,4) N-acetyl D-glucosamine; mAb,

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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 bothhumans and Drosophila (3). 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 isessential for viability in vivo (3). Furthermore, Lüders et al. (4) 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 avariety of developmental signaling pathways, and the functions of
HSPGs are dependent on their sulfation state (5–10). A mutation in thesll gene caused defects in multiple signaling pathways, including Wingless and Hedgehog signaling, probably because of the lack of HSPG
sulfation (4).
Despite the low expression of the PAPST1 gene in the colon (3),human colonic tissues highly express many sulfated glycoconjugatessuch as proteoglycans and sulfomucins. For example, the 3⬘-sulfo Lewis
a epitope (3⬘-sulfo Lea: Gal ␤1,3 (fucose ␣1,4) GlcNAc that is sulfated at
the C-3 position of Gal) is strongly expressed in the normal colonicepithelium but diminishes considerably in primary colon carcinomas(11–13). The sialyl 6-sulfo Lewis x epitope (sialyl 6-sulfo Lex: Sia ␣2,3
Gal ␤1,4 (fucose ␣1,3) GlcNAc that is sulfated at the C-6 position of
GlcNAc) is also expressed in normal human colonic tissues but not incancerous tissues (14). These sulfated glycoconjugate epitopes are
believed to regulate many biological processes in the colon (11–17).
In the present study, we attempted to identify the PAPS transporterthat is responsible for the sulfation of glycoconjugates in the colon tissue. We found a gene that is closely related to the human PAPST1 geneby 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.

monoclonal antibody; PBS, phosphate-buffered saline; RNAi, RNA interference;siRNA, small interfering RNA; sll, slalom.

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Sulfation is an important posttranslational modification associated with a variety of molecules. It requires the involvement of thehigh energy form of the universal sulfate donor, 3ⴕ-phosphoadenosine 5ⴕ-phosphosulfate (PAPS). Recently, we identified a PAPS
transporter gene in both humans and Drosophila. Although humancolonic 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 wasshown to have PAPS transport activity with an apparent Km value of
2.2 ␮M, which is comparable with that of PAPST1 (0.8 ␮M). Overexpression of either the PAPST1 or PAPST2 gene increased PAPS
transport activity in human colon cancer HCT116 cells. The RNAinterference 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.

PAPST2, a Novel Human PAPS Transporter
EXPERIMENTAL PROCEDURES

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were expressed simultaneously in the HCT116 cells. After 24 h of subculturing, the cells were transfected with 0.25 ␮g/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-conjugatedanti-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 ␮g of pCXN2 vector,
pCXN2 inserted with HA-tagged PAPST1, or pCXN2 inserted with
HA-tagged PAPST2 by using Lipofectamine 2000 reagent in accordancewith the manufacturer’s protocol. The transfectants were selected by
the addition of 600 ␮g/ml of geneticin (Invitrogen) to the medium andcultured for 1 month after 48 h.
Subcellular fractionation was performed as described previously (3,19). The cells were harvested and suspended in 10 mM HEPES-Tris (pH7.4) containing 0.25 M sucrose, 1 mM phenylmethylsulfonyl fluoride,and 1 ␮g/ml each of leupeptin, aprotinin, and pepstatin A. The cells
were then homogenized using a Dounce homogenizer. The lysate wascentrifuged at 1,000 ⫻ g for 10 min to remove the unlysed cells and cellwall 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 (22) using a yeast expression vector,
YEp352GAP-II (23). 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 (3, 19). 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 washedtwice 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 ␮g/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 apellet of P10 membrane fraction. The supernatant was further centrifuged at 100,000 ⫻ g to yield a pellet of P100 membrane fraction. Eachfraction (200 ␮g of protein) was then incubated in 100 ␮l 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 ␮M radiolabeled substrate at 30 °C for 5 min. After incubation, the radioactivity
incorporated in the microsomes was trapped using a 0.45-␮m nitrocellulose filter and measured using liquid scintillation. The amount ofincorporated radioactivity was calculated as the difference from thebackground value obtained from the same assay at 30 °C for 0 min foreach sample.
Western Blot Analysis—Fifty micrograms of protein from each sample was added to 3⫻ sodium dodecyl sulfate (SDS) sample buffer (NewEngland Biolabs Inc., Beverly, MA) and subsequently incubated at room

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Downloaded from www.jbc.org at University of Tokyo Library, on October 17, 2010Materials—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)-conjugatedanti-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 ofExpression Plasmids—The human PAPST2 gene was identified andcloned using the same procedures as described previously (3). The
amino acid sequence of the open reading frame of UGTrel1 (18) wasused as a query sequence for the TBLASTN search that was performed
to detect novel genes. To obtain the cDNA of NM_015948, a humangene that was identified in this study, and to create recombination sites
for the GATEWAYTM cloning system (Invitrogen), we used two steps of
attB adaptor PCR and prepared attB-flanked PCR products. For the firstgene-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威 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 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 GATEWAYTM cloning system in accordance with the
manufacturer’s protocol. A 3 ⫻ HA epitope tag or a c-Myc tag wasinserted 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 (3, 19). 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, theHCT116 cells were transfected with 0.25 ␮g/well of pCXN2 (20),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 apermeabilizing 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-␤1,4-galactosyltransferase 1
mAb (21) as described previously (3, 19). Finally, the cells were washedfour 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PAPST2, a Novel Human PAPS Transporter

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centration 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 extractedusing 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 (26). 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
␮Ci/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 byusing 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 ␮l (0.5 ⫻ 106 cells) were incubated with G72
mAb (27) for 1 h on ice and washed twice with 0.5 ml of the wash buffer.
The cells were then resuspended in 100 ␮l of FITC-conjugated goatanti-mouse IgM and incubated on ice for 30 min. The cells were washedtwice with wash buffer and resuspended in 500 ␮l of PBS containing
0.1% sodium azide. Flow cytometric analysis was performed using FACSAria flow cytometer (BD Biosciences) and WinMDI 2.8 software (The
Scripps Research Institute Cytometry software page).

RESULTS
Molecular Cloning of Human PAPST2 cDNA—We identified acDNA sequence (GenBankTM Accession number NM_015948) homologous to the putative nucleotide sugar transporter gene UGTrel1 byusing the same procedure that was employed for the PAPST1 gene (3).
Initially, it was described as CGI-19 protein in the GenBankTM database. 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 ofthese genes is shown in Fig. 1B. PAPST2 comprised 401 amino acids
with a calculated mass of 44.6 kDa. Hydrophobicity analyses of theamino acid sequences indicate that the PAPST2 protein is a type III
transmembrane protein with nine transmembrane domains similar tothat of PAPST1, although the transmembrane topology has yet to beverified 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-glyco-

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temperature for 2 h. The samples were fractionated on a 2–15% SDSpolyacrylamide 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 withthe manufacturer’s instructions.
Quantitative Analysis of the PAPST2 Transcript in Human Tissues by
Real-time PCR—The amount of PAPST1 and PAPST2 transcripts inhuman tissues was determined by real-time PCR. Total RNA wasextracted from human tissues by the method of Chomczynski and Sacchi (24). 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 PCRprimer 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 withthe 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 [␣-32P]dCTP
and BcaBestTM labeling kit (Takara Bio Inc., Shiga, Japan) in accordancewith 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 ahuman GAPDH cDNA probe.
RNAi of PAPST2 Gene—A sequence of small interfering RNA(siRNA) for each gene was designed as described previously (25). 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 fromthe start codon) are 5⬘-CCUUACCUUAGUGCAGUUUGCCUUU-3⬘
and 5⬘-CCAAGUCAUCUUCAAGUGCUGCAAA-3⬘, respectively. As
control siRNA, stealth RNAi negative control duplex (Invitrogen) wasused. The HCT116 cells were subcultured onto 6-cm dishes at a con-

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FIGURE 1. Amino acid sequence analysis of
PAPST2. A, by using the ClustalX program, a dendrogram showing the relationships among thetransporters was constructed based on amino acidsequences. The human (Hs) and Drosophila (Dm)transporter genes are identified by their GenBankTM accession numbers. The branch length
indicates the evolutionary distance between theindicated members. The scale at the bottom represents evolutionary distance. Tr., transporter. B,
Clustal multiple sequence alignment of human
PAPST2, PAPST1, UGTrel1, and SLC35B4. A multiple sequence analysis was performed using theClustalX program. Introduced gaps are indicatedby hyphens. Asterisks indicate positions of aminoacids that are identical among all proteins. Colons
indicate fully conserved amino acids of “strong”
groups (defined by a score ⬎0.5). Dots indicate
fully conserved amino acids of “weaker” groups(defined by a score ⱕ0.5). Bold letters indicate theprobable N-glycosylation sites in the sequences.
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.

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PAPST2, a Novel Human PAPS Transporter
sylation sites in the PAPST2. The PAPST2 gene is mapped on humanchromosome 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 (3). Because PAPST2 shares
similarity to PAPST1, we expected that PAPST2 would also be a Golgilocalized PAPS transporter.
First, we investigated the subcellular localization of the PAPST2 protein. Human colon cancer HCT116 cells were transiently transfectedwith a mammalian expression vector, pCXN2, that contained
HA-tagged PAPST2 or HA-tagged PAPST1 gene and were double

FIGURE 3. Tissue distribution of the PAPST1 and PAPST2 transcripts. A, quantitative analysis of the PAPST1 and PAPST2 transcripts in human tissues by real-time PCR. The
expression levels of the PAPST1 and PAPST2 transcripts were normalized with respect to those of the GAPDH transcript, which was measured in the same cDNAs. The indicated values
are the mean ⫾ S.E. obtained from four measurements. Open bars, PAPST1; Solid bars, PAPST2. B, Northern blot analysis of PAPST2 in the human colon. The poly(A)⫹ RNAs preparedfrom two human colon cancer cell lines, namely, HCT116 and DLD-1, and normal human colon tissue were blotted on a nylon membrane and hybridized with the probe for PAPST2(left panel) as described under “Experimental Procedures.” The right panel shows the signal detected by the probe for GAPDH on the same membrane. The positions of 18 and 28 S
ribosomal RNA are indicated. Lanes 1, HCT116; lanes 2, DLD-1; lanes 3, normal colon. Arrow indicates the approximate location of the PAPST2.

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FIGURE 2. Both PAPST1 and PAPST2 are Golgi-localized proteins. A, HCT116 cells
were transfected with HA-tagged PAPST2 (a–d) or HA-tagged PAPST1 (e–h) and analyzed
using indirect immunofluorescence. Double staining was performed for the HA tag (band f) and ␤1,4 galactosyltransferase 1 (c and g). The images of HA and ␤1,4 galactosyltransferase 1 were merged (d and h). B, HCT116 cells were simultaneously transfectedwith c-Myc-tagged PAPST1 and HA-tagged PAPST2. Double staining was performed forthe c-Myc tag (b) and HA tag (c). The images of HA and ␤1,4 galactosyltransferase 1 weremerged (d). The scale at the bottom represents 20 ␮m.

immunostained with anti-HA mAb and anti-␤1,4 galactosyltransferase
1 mAb. The results of immunofluorescence microscopy of the cells areshown in Fig. 2A. The HA-tagged PAPST1 protein was observed to becolocalized with ␤1,4 galactosyltransferase 1, which is a protein that is
typically localized in the trans-Golgi (21), and this observation is consistent with a previous report (3). In this study, HA-tagged PAPST2 wasalso observed to be colocalized with ␤1,4 galactosyltransferase 1,
thereby indicating trans-Golgi localization.
To confirm whether PAPST1 and PAPST2 proteins possess identicalsubcellular localization, an experiment was performed wherein HAtagged PAPST2 and c-Myc-tagged PAPST1 were expressed simultaneously in HCT116 cells. The cells were double immunostained withrhodamine-conjugated anti-HA mAb and FITC-conjugated anti-c-Myc
mAb. As shown in Fig. 2B, PAPST2-HA was completely colocalizedwith PAPST1-c-Myc. These results indicate that the PAPST2 protein
localizes on the Golgi apparatus like the PAPST1 protein.
Tissue Distribution of PAPST1 and PAPST2 Transcripts—Next, weanalyzed the tissue distribution of PAPST2 transcripts using real-time
PCR. All transcript levels are shown relative to that of GAPDH. As
shown in Fig. 3A, PAPST2 is highly expressed in the colon, whereas theexpression level of PAPST1 is low. PAPST2 transcripts are also widely
expressed in other tissues when compared with that of PAPST1 (Fig.
3A). To confirm the expression of PAPST2 transcripts in the colon, we
performed Northern blot analysis of RNAs that were derived from coloncancer cell lines HCT116 and DLD-1 and normal human colon tissue.
The PAPST2 mRNA was detected as a single band corresponding to 2
kbp (Fig. 3B), which is consistent with the length of the identified
sequence, namely, NM_015948 (2068 bp). We expected that PAPST2
would be a PAPS transporter gene that functions by compensating forthe insufficient expression of PAPST1 during the synthesis of sulfatedglycoconjugates in the colon.
PAPST2 Is a PAPS Transporter Gene—The substrate specificity of the
PAPST2 protein was examined by yeast expression in a manner similar
to that used for PAPST1 (3). The yeast expression vector YEp352GAP-II
was inserted with HA-tagged PAPST2 and introduced into W303–1ayeast for the preparation of the Golgi-enriched P100 membrane fractionthat expressed the PAPST2 protein. The HA-tagged PAPST2 protein
was detected in the yeast P100 membrane fraction by Western blotting
using an antibody against the HA epitope tag (Fig. 4A).

PAPST2, a Novel Human PAPS Transporter

The substrate specificity of the PAPST2 protein was examined using
the P100 membrane fraction and radiolabeled substrates. The transport
activity of PAPS into the P100 membrane fraction is shown in Fig. 4B.
The P100 membrane fraction prepared from yeast cells that expressed
PAPST2 showed PAPS transport activity that was significantly higher
than that observed in the mock cells (1.7 ⫾ 0.2 versus 2.6 ⫾ 0.3 pmol/mg
of protein, respectively, mean ⫾ S.E. from six independent experiments;p ⬍ 0.05, Student’s t-test), although the yeast Golgi-enriched fraction
had relatively high endogenous PAPS transport activity. The substrateconcentration dependence of PAPS transport by PAPST2 is shown in
Fig. 4C. PAPST2 showed a saturated PAPS transport activity with anapparent Km value that was estimated to be 2.2 ␮M.
Both PAPST1 and PAPST2 Transport PAPS in the Human Colon
Cancer Cell Line—We also tested the PAPS transport activity of
PAPST2 protein by using a mammalian cell line. For measuring the
PAPS transport activity, we analyzed the Golgi-enriched fractions ofcolon cancer HCT116 cells that stably expressed HA-tagged PAPST1,
HA-tagged PAPST2, or the mock vector (pCXN2 vector alone).
As shown in Fig. 5A, the levels of PAPST1 and PAPST2 transcripts in
the PAPST1 and PAPST2 transfectant cells, respectively, showed amarked increase over those obtained from the mock transfectants. No
difference was observed between the mock and PAPST1 transfectants
with respect to PAPST2 transcript levels or between the mock and
PAPST2 transfectants with respect to PAPST1 transcript levels (Fig.
5A). In each transfectant, HA-tagged PAPST1 or HA-tagged PAPST2
protein was detected in the Golgi-enriched fraction by Western blotting
using an antibody against the HA epitope tag (Fig. 5B). The PAPS transport activity that was obtained from each Golgi-enriched fraction is10950 JOURNAL OF BIOLOGICAL CHEMISTRY

FIGURE 5. Both PAPST1 and PAPST2 transport PAPS in human colon cancer cell line.
A, expression levels of PAPST1 and PAPST2 transcripts in each transfectant. The relative
amounts of each transcript were normalized with respect to those of the GAPDH transcript, which was measured in the same cDNAs. The indicated values are the mean ⫾ S.E.
obtained from three independent measurements. B, expression states of the PAPST1
and PAPST2 proteins in each transfectant. Each Golgi-enriched fraction (10 ␮g of protein) prepared from the HCT116 cells that stably expressed mock vector (lane 1), HAtagged PAPST2 (lane 2), or HA-tagged PAPST1 (lane 3) was subjected to SDS-polyacrylamide electrophoresis. Western blot analysis was performed using a monoclonal
antibody against a HA epitope. Arrow and arrowhead indicate HA-tagged PAPST2 andHA-tagged PAPST1, respectively. C, PAPS transport activity. Each Golgi-enriched fraction(100 ␮g of protein) was incubated in 100 ␮l of the reaction buffer containing 1 ␮M
[35S]PAPS at 30 °C for 5 min, and the incorporated radioactivity was measured. Values
shown are the mean ⫾ S.E. obtained from three independent experiments.

shown in Fig. 5C. These Golgi-enriched fractions also showed relatively
high endogenous PAPS transport activity; however, the value observedin the PAPST2 transfectants was significantly higher than that observedin the mock transfectants (1.9 ⫾ 0.2 versus 4.2 ⫾ 0.3 pmol/mg of protein, respectively, mean ⫾ S.E. from three independent experiments;p ⬍ 0.05, Student’s t-test). These results indicate that both PAPST1 and
PAPST2 act as PAPS transporters in the human colon cancer cell line.
PAPST2 Is Involved in the Synthesis of Sulfated Glycoconjugates in the
Human Colon—We revealed that PAPST2 is a PAPS transporter that isexpressed in the colon. To elucidate the role of PAPST2 in the synthesisof sulfated glycoconjugates in the colon, we performed a flow cytometric analysis of the colon cancer cell line by using an antibody, G72,
recognizing sialyl 6-sulfo N-acetyllactosamine (sialyl 6-sulfo galactose
␤1,4 N-acetyl D-glucosamine: Sia ␣2,3 Gal ␤1,4 GlcNAc that is sulfatedat the C-6 position of GlcNAc) (27). We attempted to reduce PAPST2
expression in the colon cancer cell line HCT116, which is moderately
reactive to G72 antibody, by using RNAi. Based on their ability to suppress gene expression, two siRNA sequences that would target the
PAPST1 and PAPST2 genes, i.e. PAPST1– 813 and PAPST2–513,
respectively, were selected. These siRNAs were synthesized as doublestranded stealth RNAs (Invitrogen) possessing 25 nucleotides, and thestealth RNAi negative control duplex (Invitrogen) was used as a control
siRNA. Lipofection was used to transfect the HCT116 cells with 10 nM
PAPST1– 813 and PAPST2–513 siRNAs.

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FIGURE 4. PAPST2 is a PAPS transporter. A, expression state of PAPST2 protein in the
Golgi-enriched fraction. Each P100 fraction (50 ␮g of protein), prepared from yeast cells
expressing either the mock vector (lanes 1 and 2) or HA-tagged PAPST2 (lanes 3 and 4),
was subjected to SDS-polyacrylamide electrophoresis. Western blot analysis was performed using a monoclonal antibody against the HA epitope. Arrow indicates HA-tagged
PAPST2. B, substrate specificity of PAPST2. Each P100 fraction (200 ␮g of protein) was
incubated in 100 ␮l of reaction buffer containing 1 ␮M substrate at 30 °C for 5 min, andthe incorporated radioactivity was measured. The indicated values are the mean ⫾ S.E.
obtained from six independent experiments. Each experiment was performed with twotransformant clones. Open bars, Mock; solid bars, PAPST2. C, substrate concentration
dependence. Each P100 fraction (200 ␮g of protein) was incubated in 100 ␮l of reaction
buffer containing different concentrations of [35S]PAPS at 30 °C for 5 min, and the incorporated radioactivity was measured. Specific incorporation was calculated by subtracting the value of the mock transfection from each of the corresponding values. Right panel
shows the double-reciprocal plot used to determine the Km value.

PAPST2, a Novel Human PAPS Transporter

The cells were harvested and immunostained with G72 antibody 96 h
after transfection. The efficiency of gene silencing was determined by realtime PCR. As shown in Fig. 6A, transfection using the PAPST2–513 and
PAPST1–813 siRNAs resulted in a 45 and 76% knockdown of the corresponding mRNAs, respectively. Results of the flow cytometric analysis of
the cells treated with siRNAs are shown in Fig. 6B. The cells treated withPAPST2–513 siRNA showed significantly reduced G72 reactivity whencompared with that of control siRNA (averaged relative mean fluorescenceintensity for mock, 10.4 ⫾ 1.0 and for PAPST2, 6.8 ⫾ 0.5; mean ⫾ S.E. fromthree independent experiments, p ⬍ 0.05, Student’s t-test). On the other
hand, transfection of PAPST1–813 siRNA showed similar moderatedecrease in G72 reactivity despite the greater reduction of the PAPST1
gene. This indicates that PAPST2 plays a role in the synthesis of the sialyl
6-sulfo galactose ␤1,4 N-acetyl D-glucosamine epitope in this cell line. We
obtained similar results when another siRNA sequence targeted the
PAPST2 gene (PAPST2–342) at a concentration of 100 nM (averaged
mean fluorescence intensity for mock, 4.0 ⫾ 1.0; for PAPST2–513,
2.4 ⫾ 0.2; for PAPST2–342, 2.1 ⫾ 0.4; and for PAPST1– 813, 2.8 ⫾ 0.3,
mean ⫾ S.E. from four independent experiments).
We also determined total sulfate incorporation into cellular proteins in
the HCT116 cells by metabolic labeling with [35S]sulfate. As shown in Fig.
7A, transfection of both PAPST2–513 and PAPST1–813 siRNAs reduced
the [35S]sulfate-labeled proteins. The density of radioactivity in each lane of
the SDS-PAGE is shown in Fig. 7C. The PAPST1–813- and PAPST2–513transfected cells resulted in a radioactivity density of 83.0% ⫾ 2.3 and 90.2%⫾ 3.7 as compared with that of mock siRNA, respectively (mean ⫾ S.E.
from three independent experiments; p ⬍ 0.05 for PAPST2–513 versus
mock, Student’s t-test). Transfection using the PAPST2–513 and
PAPST1–813 siRNAs resulted in a 30 and 69% knockdown of the corre-

APRIL 21, 2006 • VOLUME 281 • NUMBER 16

FIGURE 7. Metabolic labeling of HCT116 cells transfected with siRNAs. A, HCT116
cells were transfected with mock siRNA, PAPST2 siRNA (PAPST2–513), or PAPST1 siRNA(PAPST1– 813) at a concentration of 10 nM by using Lipofectamine 2000 reagent. Fortyeight hours after transfection, cells were metabolically labeled with [35S]sulfate and cultured for 48 h. The cell lysate of each transfectant (50 ␮g of protein) was subjected to
SDS-polyacrylamide electrophoresis. The radioactive compounds were detected as
described under “Experimental Procedures.” B, the Coomassie brilliant blue-stained
image of the same gel. C, the density of radioactive bands in each lane was calculatedusing the public domain NIH image program (developed by the U. S. National Institutesof Health) and normalized with respect to those of the Coomassie brilliant blue-stained
image. Values shown are the mean ⫾ S.E. obtained from three independent experiments. Values obtained for the mock siRNA are presented as 100%.

sponding mRNAs, respectively. These results demonstrated that PAPST2 isa PAPS transporter gene that is involved in the synthesis of sulfated glycoconjugates in the colon.

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FIGURE 6. Flow cytometric analysis of HCT116 cells transfected with siRNAs. HCT116 cells were transfected with mock siRNA, PAPST2 siRNA (PAPST2–513), or PAPST1 siRNA(PAPST1– 813) at a concentration of 10 nM by using Lipofectamine 2000 reagent. The cells were harvested and analyzed 96 h after transfection. A, knockdown efficiency of each siRNAfor the PAPST1 and PAPST2 transcripts. Relative amounts of each transcript were quantified by real-time PCR. The indicated values are the mean ⫾ S.E. obtained from three
independent experiments. B, relative mean fluorescent intensity. Cells treated with each siRNA were immunostained with G72 mAb and analyzed by flow cytometry. The values ofmean fluorescent intensity were adjusted with respect to those of the negative control cells. The indicated values are the mean ⫾ S.E. obtained from three independent experiments.
C, a representative result of flow cytometric analysis. Faint, dotted lines indicate the negative controls stained with FITC-conjugated anti-mouse IgM without the primary antibody.

PAPST2, a Novel Human PAPS Transporter
DISCUSSION

3

E. Goda and S. Nishihara, unpublished observation.

10952 JOURNAL OF BIOLOGICAL CHEMISTRY

incorporation into cellular proteins. The sulfotransferases have long
been believed to be the rate-limiting components of the sulfation process; however, sulfation is also controlled by the components involved in
the earlier steps, such as the sulfate transporters and PAPS synthetases(42). For instance, PAPS synthetases modulate PAPS levels in highendothelial venules and thus play a key role in the control of the sulfation state of L-selectin ligands and its functional activities (43– 45). Furthermore, mutations in some genes involved in PAPS synthesis, such as
diastrophic dysplasia sulfate transporter (DTDST) (46 – 49) and PAPS
synthetase 2 (PAPSS2) (2, 50), are responsible for a form of humaninherited osteochondrodysplasias that results because of down-regulated sulfation of chondroitin sulfate in the cartilage. This evidence
implies that PAPS production is a rate-limiting step for sulfation incertain tissues. Our investigation also demonstrated that the PAPS
transporter is a rate-limiting factor in the sulfation of glycoconjugates in
the colon cells. The PAPST2 protein showed a relatively low apparent
Km value for PAPS (2.2 ␮M), although the precise kinetics of PAPST2
protein, excluding other factors, remains uncertain. The effect of the
PAPS transporter would be significant if the Km value for PAPS would
be lower than the Km value of the sulfotransferases in the Golgi lumen.
In the previous study, we reported that sll, the PAPST1 ortholog in
Drosophila, is essential for viability of the fly (3). Lüders et al. (4) demonstrated that sll gene is involved in the signaling pathways and that the
mutation of sll gene exhibits disrupted Wingless gradient and reduced
Hedgehog signaling activity. Here, both PAPST1 and PAPST2 areshown to be Golgi-localized PAPS transporters. Most sulfation reactions of glycoconjugates, including proteoglycans (51), glycoproteins(52), and glycolipids (53), occur in the Golgi apparatus. Furthermore,tyrosine O-sulfation of the proteins occurs in the trans-Golgi network(54, 55). In most human tissues, the expression of PAPST2 is comparable with that of PAPST1 (Fig. 3). The PAPST1 and PAPST2 proteins
have comparable Km values for PAPS (0.8 versus 2.2 ␮M, respectively).
PAPST1 and PAPST2 may function in a manner that is complementary
to each other. Further investigations are required to clarify the involvement and significance of PAPST1 and PAPST2 in these pathways.
REFERENCES
1. Li, H., Deyrup, A., Mensch, J. R., Jr., Domowicz, M., Konstantinidis, A. K., andSchwartz, N. B. (1995) J. Biol. Chem. 270, 29453–29459
2. Kurima, K., Warman, M. L., Krishnan, S., Domowicz, M., Krueger, R. C., Jr., Deyrup,A., and Schwartz, N. B. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8681– 8685
3. Kamiyama, 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., and Nishihara, S.
(2003) J. Biol. Chem. 278, 25958 –25963
4. Lüders, F., Segawa, H., Stein, D., Selva, E. M., Perrimon, N., Turco, S. J., and Häcker,
U. (2003) EMBO J. 22, 3635–3644
5. Reichsman, F., Smith, L., and Cumberledge, S. (1996) J. Cell Biol. 135, 819 – 827
6. Zioncheck, T. F., Richardson, L., Liu, J., Chang, L., King, K. L., Bennett, G. L., Fugedi,
P., Chamow, S. M., Schwall, R. H., and Stack, R. J. (1995) J. Biol. Chem. 270,
16871–16878
7. Olwin, B. B., and Rapraeger, A. (1992) J. Cell Biol. 118, 631– 639
8. Yayon, A., Klagsbrun, M., Esko, J. D., Leder, P., and Ornitz, D. M. (1991) Cell 64,
841– 848
9. Rapraeger, A. C., Krufka, A., and Olwin, B. B. (1991) Science 252, 1705–1708
10. Aviezer, D., and Yayon, A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12173–12177
11. Irimura, T., Wynn, D. M., Hager, L. G., Cleary, K. R., and Ota, D. M. (1991) Cancer Res.
51, 5728 –5735
12. Yamori, T., Ota, D. M., Cleary, K. R., Hoff, S., Hager, L. G., and Irimura, T. (1989)
Cancer Res. 49, 887– 894
13. Matsushita, Y., Yamamoto, N., Shirahama, H., Tanaka, S., Yonezawa, S., Yamori, T.,
Irimura, T., and Sato, E. (1995) Jpn. J. Cancer Res. 86, 1060 –1067
14. Izawa, M., Kumamoto, K., Mitsuoka, C., Kanamori, C., Kanamori, A., Ohmori, K.,
Ishida, H., Nakamura, S., Kurata-Miura, K., Sasaki, K., Nishi, T., and Kannagi, R.
(2000) Cancer Res. 60, 1410 –1416
15. Nakayama, T., Watanabe, M., Katsumata, T., Teramoto, T., and Kitajima, M. (1995)
Cancer Res. 75, 2051–2056
16. Nakamori, S., Kameyama, M., Imaoka, S., Furukawa, H., Ishikawa, O., Sasaki, Y.,

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We identified a novel human PAPS transporter that is predominantly
expressed in the colon. Human colonic epithelial tissues express many
sulfated glycoconjugates. Silencing of the PAPST2 gene by RNAi caused
a reduction in the amount of sulfated structures in the colon cancer cellline. Expression of small amounts of PAPST1 mRNA in the colon suggests that PAPST2 plays a major role in the sulfation of glycoconjugatesin this organ.
The PAPST2 gene is closely related to the PAPST1 gene, and the
genomic locus of the PAPST2 gene is located close to that of PAPST1(6p24.3 and 6p11.2–12.1, respectively). Despite their functional conservation, the genomic structures of PAPST2 and PAPST1 are not similar(eleven exons versus four exons, respectively). Furthermore, both
PAPST1 and PAPST2 have a single ortholog in Drosophila, namely, sll
and NM_140697, respectively (Fig. 1A).3 These findings indicate thatduring the evolutionary process, the PAPST1 and PAPST2 genes
diverged relatively early and thereafter evolved independently.
Recently, the nucleotide sugar transporter genes have been classified
into the solute carrier family 35 (SLC35) (28). The PAPST1 and PAPST2
genes have also been classified into the SLC35 subfamily B. In GenBankTM, UGTrel1, PAPST1, and PAPST2 have been defined as members of B1 (SLC35B1), B2 (SLC35B2), and B3 (SLC35B3), respectively.
More recently, Ashikov et al. (29) reported that another member ofsubfamily B, namely, SLC35B4, is a UDP-xylose/UDP-GlcNAc transporter gene. Data bases mention that SLC35B4 is a human ortholog ofyeast UDP-GlcNAc transporter genes, namely, Kluyveromyces lactis
mnn2–2 (30) and S. cerevisiae yea4 (31). This implies that the subfamily
B comprises members with entirely different substrate specificities
despite their structural conservation.
Silencing of PAPST2 gene expression in the colon cancer cell line
revealed the involvement of PAPST2 in the synthesis of a sulfatedepitope, sialyl 6-sulfo galactose ␤1,4 N-acetyl D-glucosamine (Fig. 6).
The fucosylated derivative, namely, sialyl 6-sulfo Lex, is a major L-selectin ligand present on the high endothelial venules of the human peripheral lymph nodes for the initial adhesion of leukocytes to endothelial
cells (27, 32–34). It has been proposed that sialyl 6-sulfo Lex serves as aligand for selectins in routine trafficking of leukocytes, whereas nonsulfated sialyl Lex plays a major role in the inflammatory response (35,36). PAPST2 may be involved in the function of these physiologicalroutine processes, including the recruitment of gut-homing helper
memory T lymphocytes, by regulating the sulfation of carbohydrate
epitopes.
It is common knowledge that the risk of malignancy and recurrenceof colorectal cancer is correlated with an increase in sialylation (13, 15,16) and a decrease in the sulfation of carbohydrate epitopes (11–14).
The sialyl 6-sulfo Lex (14) and 3⬘-sulfo-Lea (13, 17) are predominantly
expressed in nonmalignant tissues but are not detected in the malignant
tissues in colorectal cancers. Kumamoto et al. (37) reported that incolorectal cancers the expression of the UDP-Gal transporter increases
significantly and results in the supply of the donor substrate for thesynthesis of sialyl Lea and sialyl Lex. On the other hand, the colon cancer
cell line HCT116 showed relatively low PAPST2 expression when compared with that of normal colon tissue. We could not find any coloncancer cell line that highly expressed PAPST2. The possibility that
PAPST2 decreases the expression associated with the progression ofcolorectal cancer, as reported in the case of certain sulfotransferases(38 – 40) and a sulfate transporter (41), may be considered.
The RNAi of PAPST2 gene in the HCT116 cells reduced total sulfate

PAPST2, a Novel Human PAPS Transporter

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36. Kannagi, R. (2002) Curr. Opin. Struct. Biol. 12, 599 – 608
37. Kumamoto, K., Goto, Y., Sekikawa, K., Takenoshita, S., Ishida, N., Kawakita, M., andKannagi, R. (2001) Cancer Res. 61, 4620 – 4627
38. Seko, A., Sumiya, J., Yonezawa, S., Nagata, K., and Yamashita, K. (2000) Glycobiology
10, 919 –929
39. Uchimura, K., El-Fasakhany, F. M., Hori, M., Hemmerich, S., Blink, S. E., Kansas, G. S.,
Kanamori, A., Kumamoto, K., Kannagi, R., and Muramatsu, T. (2002) J. Biol. Chem.
277, 3979 –3984
40. Seko, A., Nagata, K., Yonezawa, S., and Yamashita, K. (2002) Jpn. J. Cancer Res. 93,
507–515
41. Antalis, T. M., Reeder, J. A., Gotley, D. C., Byeon, M. K., Walsh, M. D., Henderson,
K. W., Papas, T. S., and Schweinfest, C. W. (1998) Clin. Cancer Res. 4, 1857–1863
42. Kamiyama, S., and Nishihara, S. (2004) Trends Glycosci. Glycotech. 16, 109 –123
43. Hemmerich, S., Butcher, E. C., and Rosen, S. D. (1994) J. Exp. Med. 180, 2219 –2226
44. Girard, J. P., Baekkevold, E. S., and Amalric, F. (1998) FASEB J. 12, 603– 612
45. Imai, Y., Lasky, L. A., and Rosen, S. D. (1993) Nature 361, 555–557
46. Superti-Furga, A., Neumann, L., Riebel, T., Eich, G., Steinmann, B., Spranger, J., andKunze, J. (1999) J. Med. Genet. 36, 621– 624
47. Superti-Furga, A., Hästbacka, J., Wilcox, W. R., Cohn, D. H., van der Harten, H. J.,
Rossi, A., Blau, N., Rimoin, D. L., Steinmann, B., Lander, E. S., and Gitzelmann, R.
(1996) Nat. Genet. 12, 100 –102
48. Hästbacka, J., Superti-Furga, A., Wilcox, W. R., Rimoin, D. L., Cohn, D. H., andLander, E. S. (1996) Am. J. Hum. Genet. 58, 255–262
49. Hästbacka, J., de la Chapelle, A., Mahtani, M. M., Clines, G., Reeve-Daly, M. P., Daly,
M., Hamilton, B. A., Kusumi, K., Trivedi, B., Weaver, A., Coloma, A., Lovet, M.,
Buckler, A., Kaitila, I., and Lander, E. S. (1994) Cell 78, 1073–1987
50. ul Haque, M. F., King, L. M., Krakow, D., Cantor, R. M., Rusiniak, M. E., Swank, R. T.,
Superti-Furga, A., Haque, S., Abbas, H., Ahmad, W., Ahmad, M., and Cohn, D. H.
(1998) Nat. Genet. 20, 157–162
51. Salmivirta, M., Lidholt, K., and Lindahl, U. (1996) FASEB J. 10, 1270 –1279
52. Lane, N., Caro, L., Otero Vilardebo, L. R., and Godman, G. C. (1964) J. Cell Biol. 21,
339 –351
53. Fleischer, B., and Zambrano, F. (1974) J. Biol. Chem. 249, 5995– 6003
54. Moore, K. L. (2003) J. Biol. Chem. 278, 24243–24246
55. Bauerle, P. A., and Huttner, W. B. (1987) J. Cell Biol. 105, 2655–2664

JOURNAL OF BIOLOGICAL CHEMISTRY

10953

Downloaded from www.jbc.org at University of Tokyo Library, on October 17, 2010

Kabuto, T., Iwanaga, T., Matsushita, Y., and Irimura, T. (1993) Cancer Res. 53,
3632–3637
17. Yamachika, T., Nakanishi, H., Inada, K., Kitoh, K., Kato, T., Irimura, T., and Tatematsu, M. (1997) Virchows Arch. 431, 25–30
18. Ishida, N., Miura, N., Yoshioka, S., and Kawakita, M. (1996) J. Biochem. 120,
1074 –1078
19. Suda, T., Kamiyama, S., Suzuki, M., Kikuchi, N., Nakayama, K., Narimatsu, H., Jigami,Y., Aoki, T., and Nishihara, S. (2004) J. Biol. Chem. 279, 26469 –26474
20. Niwa, H., Yamamura, K., and Miyazaki, J. (1991) Gene 108, 193–199
21. Uemura, M., Sakaguchi, T., Uejima, T., Nozawa, S., and Narimatsu, H. (1992) Cancer
Res. 52, 6153– 6157
22. Ito, H., Fukuda, Y., Murata, K., and Kimura, A. (1983) J. Bacteriol. 153, 163–168
23. Nakayama, K., Maeda, Y., and Jigami, Y. (2003) Glycobiology 13, 673– 680
24. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156 –159
25. Ui-Tei, K., Naito, Y., Takahashi, F., Haraguchi, T., Ohki-Hamazaki, H., Juni, A., Ueda,
R., and Saigo, K. (2004) Nucleic Acids Res. 32, 936 –948
26. Tsuiji, H., Hayashi, M., Wynn D. M., and Irimura, T. (1998) Jpn. J. Cancer Res. 89,
1267–1275
27. Mitsuoka, C., Sawada-Kasugai, M., Ando-Furui, K., Izawa, M., Nakanishi, H., Nakamura, S., Ishida, H., Kiso, M., and Kannagi, R. (1998) J. Biol. Chem. 273, 11225–11233
28. Ishida, N., and Kawakita, M. (2004) Pflugers Arch. 447, 768 –775
29. Ashikov, A., Routier, F., Fuhlrott, J., Helmus, Y., Wild, M. K., Gerardy-Schahn, R., andBakker, H. (2005) J. Biol. Chem. 280, 27230 –27235
30. Abeijon, C., Robbins, P. W., and Hirschberg, C. B. (1996) Proc. Natl. Acad. Sci. U. S. A.
93, 5963–5968
31. Roy, S. K., Chiba, Y., Takeuchi, M., and Jigami, Y. (2000) J. Biol. Chem. 275,
13580 –13587
32. Mitsuoka, C., Kawakami-Kimura, N., Kasugai-Sawada, M., Hiraiwa, N., Toda, K.,
Ishida, H., Kiso, M., Hasegawa, A., and Kannagi, R. (1997) Biochem. Biophys. Res.
Commun. 230, 546 –551
33. Hemmerich, S., Bertozzi, C. R., Leffler, H., and Rosen, S. D. (1994) Biochemistry 33,
4820 – 4829
34. Kimura, N., Mitsuoka, C., Kanamori, A., Hiraiwa, N., Uchimura, K., Muramatsu, T.,
Tamatani, T., Kansas, G. S., and Kannagi, R. (1999) Proc. Natl. Acad. Sci. U. S. A. 96,
4530 – 4535
35. Kannagi, R., and Kanamori, A. (1999) Trends Glycosci. Glycotech. 11, 329 –344