Molecular cloning and characterization of a novel human ... · mg/ml gel) was purchased from E-Y Laboratories, Inc. (San Mateo, CA). Psathyrella velutina lectin (PVL) was prepared

1

Molecular cloning and characterization of a novel human galactose 3-O-sulfotransferase that

transfers sulfate to Galβ1→3GalNAc residue in O-glycans*

Akira Seko, Sayuri Hara-Kuge, and Katsuko Yamashita‡

Department of Biochemistry, Sasaki Institute, 2-2, Kanda-Surugadai, Chiyoda-ku, Tokyo 101-

0062, and CREST (Core Research for Evolutional Science and Technology) of the Japan Science

and Technology Corporation, 2-3, Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan.

Key words: Gal-3-O-sulfotransferase, mucin-type-O- glycan, cDNA cloning, sulfated glycan,

core1 oligosaccharide, core 2 oligosaccharide, periodate oxidation

Sub title: cloning of Galβ1→3GalNAc:→3′ sulfotransferase.

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on May 1, 2001 as Manuscript M101558200 by guest on January 23, 2020

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Footnotes

*This work was supported in part by the Grants-in-aid for Scientific Research on Priority Areas

(#10178104 and #12217156) from the Ministry of Education, Science, Sports and Culture of

Japan, and the Grants-in aid from the Public Trust Nishi Cancer Research Fund.

The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBank™/EBI

Data Bank with accession number AF316113.

‡To whom correspondence should be addressed: at the Department of Biochemistry, Sasaki

Institute, 2-2 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan. Fax: 81-3-3294-2656; E-

mail: [email protected]

1The abbreviations used are: Bn, benzyl; core 2, Galβ1→3(GlcNAcβ1→6)GalNAcα1→; EST,

expressed sequence tags; Gal, galactose; GalCer, galactosylceramide; GalDG,

galactosyldiacylglycerol; GalNAc, N-acetylgalactosamine; GalNAcOH, N-acetylgalactosaminitol;

GlcNAc, N-acetylglucosamine; HEPES, N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid;

LacCer, lactosylceramide; PAPS, 3′-phosphoadenosine-5′-phosphosulfate; PNA, peanuts

agglutinin; pNP, p-nitrophenyl; RCA-I, Ricinus communis agglutinin-I; PVL, Psathyrella

velutina lectin; TLC, thin layer chromatography; type 1, Galβ1→3GlcNAc; type 2,

Galβ1→4GlcNAc.

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ABSTRACT

We have identified a novel galactose 3-O-sulfotransferase, termed Gal3ST-4, by analysis of

expression sequence tag using the amino acid sequence of human cerebroside 3′-

sulfotransferase(Gal3ST-1). The isolated cDNA contains a single open reading frame coding

for a protein of 486 amino acids with a type II transmembrane topology. The amino acid

sequence of Gal3ST-4 revealed 33 %, 39 % , and 30 % identity to human Gal3ST-1,

Galβ1→3/4GlcNAc:→3′ sulfotransferase(Gal3ST-2), and Galβ1→4GlcNAc:→3′

sulfotransferase(Gal3ST-3), respectively. Gal3ST-4 gene comprised at least four exons and was

located on human chromosome 7q22. Expression of Gal3ST-4 in COS-7 cells produced a

sulfotransferase activity which catalyzes the transfer of [35S]sulfate to the C-3′ position of

Galβ1→3GalNAcα1-O-Bn. Gal3ST-4 recognizes Galβ1→3GalNAc and

Galβ1→3(GlcNAcβ1→6)GalNAc as good substrates, but not Galβ1→3GalNAcOH or

Galβ1→3/4GlcNAc. Asialofetuin is also a good substrate and the sulfation was exclusively

found in O-linked glycans which consists of Galβ1→3GalNAc moiety, suggesting that the

enzyme is specific for O-linked glycans. The Northern blot analysis revealed that 2.5 kb mRNA

for the enzyme is extensively expressed in various tissues. These results suggest that Gal3ST-4

is the fourth member of a Gal:→3 sulfotransferase family, and that the four members, Gal3ST-1,

Gal3ST-2, Gal3ST-3 and Gal3ST-4, are responsible for sulfation of different acceptor substrates.


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INTRODUCTION

Sulfation is one of the most extensive modifications for glycan chains in various

glycoconjugates. Sulfated glycans are associated with the physiological functions of

glycoproteins in the mucosal barrier system, regulation of their half-life, and cell-to-cell

interaction and the content of sulfated glycans in mucus glycoproteins is modified in cystic

fibrosis and colon cancer (reviewed in 1-3). However, the precise molecular mechanisms in

these phenomena and other significances of sulfation in various glycoproteins remain unclear.

SO3–→3Gal structure in O-linked and N-linked glycans has been found in various

glycoproteins including thyroglobulin (4-6), meconium glycoproteins (7), respiratory mucous

glycoproteins from patients with cystic fibrosis (8-10) and chronic bronchitis (11), an ovarian

cystadenoma glycoprotein (12), LS174T-HM7 colon carcinoma mucin (13), Tamm-Horsfall

glycoprotein (14), sulfomucins (15), and oviducal mucins (16). Sulfated residues in these

glycoproteins are attached to C-3′ of Galβ1→3/4GlcNAc, Galβ1→3GalNAc, or Galβ1→3Gal

structure. The occurrence of SO3–→3Gal structure has not been fully elucidated to date, because

of the difficulty of structural studies for sulfated glycans. However, the sulfated structure seems

to be extensively distributed, since the glycoprotein-specific β-Gal-3′-sulfotransferase (Gal3ST-

2) gene is ubiquitously expressed in various human tissues (17).

In relation to the biosynthesis of sulfated glycans, cDNA cloning of more than twenty

sulfotransferases has been achieved up to date (reviewed in 18). Among these sulfotransferases,

the C-3 sulfation of galactose is catalyzed by Gal: →3 sulfotransferase. Three Gal: →3

sulfotransferases have so far been cloned; one is glycolipid-specific cerebroside sulfotransferase


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(CST or Gal3ST-1)(19), which utilizes galactosylceramide (GalCer1), lactosylceramide (LacCer)

and galactosyldiacylglycerol (GalDG)(20), the second is GP3ST or Gal3ST-2 which utilizes not

only Galβ1→3/4GlcNAc but also Galβ1→3GalNAc (17), and the third is Gal3ST-3 which

utilizes Galβ1→4GlcNAc (21). Three enzymes share about 40 % identity of the amino acid

sequences, indicating the existence of a family of Gal:→3sulfotransferases (17, 21). To search

for a novel member of the family, we investigated the expressed sequence tags (EST) data bases

using Gal3ST-1 amino acid sequence and found the fourth member of Gal:→3sulfotransferase.


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EXPERIMENTAL PROCEDURES

Materials ————3′-Phosphoadenosine-5′-phospho[35S]sulfate (PAPS, 96.9

GBq/mmol) and UDP-[3H]Gal[4,5-3H(N), 1110 GBq/mmol] were purchased from NEN Life

Science Products, Inc. (Boston, MA). Galβ1→3GalNAcα1-O-p-nitrophenyl (pNP), N-

acetyllactosamine (type 2), Galβ1→3GalNAc, and Galβ1→3(GlcNAcβ1→6)GalNAcα1-O-pNP

(core 2-O-pNP) were purchased from Funakoshi Co., Ltd. (Tokyo, Japan). Lacto-N-biose I

(type 1), Galβ1→3GalNAcα1-O-benzyl (Bn), GlcNAcβ1-O-Bn, GalNAcα1-O-Bn, UDP-Gal,

galactosylceramide (GalCer) from bovine brain, lactosylceramide (LacCer) from bovine, and

galactosyl diacylglycerol (GalDG) from whole wheat flour, were purchased from Sigma

Chemical Co. (St.Louis, MO). Streptococcus 6646K β-galactosidase was purchased from

Seikagaku Co. (Tokyo, Japan). Ricinus communis agglutinin-I(RCA-I)-agarose (4 mg/ml gel)

was purchased from Hohnen Oil Co. (Tokyo, Japan). Peanuts agglutinin(PNA)-agarose (4.5

mg/ml gel) was purchased from E-Y Laboratories, Inc. (San Mateo, CA). Psathyrella velutina

lectin (PVL) was prepared according to the method (22) and the lectin was conjugated to CNBr-

activated Sepharose 4B (Pharmacia) according to the manufacturer′s instructions.

Galβ1→3GlcNAcβ1→3Galβ1→4Glc was isolated from human milk (23).

Galβ1→4GlcNAcβ1→2Manα1→3(6)Manβ1→4GlcNAc was obtained from the urine of GM1-

gangliosidosis patients (24). Galβ1→4GlcNAcβ1→2Manα1→3(Galβ1→4GlcNAcβ1→2

Manα1→6)Manβ1→4GlcNAcβ1→4GlcNAc was prepared from egg yolk SGP (25) by

hydrazinolysis-reacetylation and mild acid hydrolysis.

SO3–→3Galβ1→3(Galβ1→4GlcNAcβ1→6)GalNAcα1- -Bn was a kind gift from Dr. S.


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Yazawa (Japan Immunoresearch Laboratories, Takasaki)(26). The sulfated oligosaccharide was

digested with Streptococcus 6646K β-galactosidase (10 mU) and jack bean β-N-

acetylhexosaminidase (3 U) in 0.1 M sodium acetate buffer (pH 5.3)-10 mM MnCl2. The digest

was applied on a RCA-I-agarose column (1.5 × 2.8 cm) and the pass-through fraction was further

applied on a PVL-Sepharose column (1.5 × 2.8 cm, 4.5 mg/ml gel). The pass-through,

SO3–→3Galβ1→3GalNAcα1- -Bn, was desalted with Sep-Pak C18 Cartridge (Waters, Milford,

MA) and used as an authentic compound.

cDNA Cloning of Gal3ST-4 —————— Based on the amino acid sequence of human

Gal3ST-1 (19), we found one sequence (GenbankTM accession number AW961058) with high

similarity in the EST data bases at the National Center for Biotechnology Information (National

Institutes of Health, Bethesda, MD). We used GeneTrapper cDNA Positive Selection System

(Life Technologies, Rockville, MD) to obtain the cDNA clone according to the manufucturer′s

instructions. Briefly, an oligonucleotide, 5′-GCCCTAGCGAAACATTGTCTGGTA-3′ (the

nucleotide sequence corresponding to 1440-1463; see Fig.1A) was biotinylated and then used as

a probe. SuperScriptTM human testis cDNA library (Life Technologies) in which testis cDNA

was cloned into the eukaryotic expression vector PCMV-SPORT, was degraded to single-

stranded cDNA by Gene II and Exonuclease III digestion. The single-stranded cDNA was

hybridized with the biotinylated probe and target cDNA was captured to streptavidin-conjugated

paramagnetic beads. The target cDNA was released, re-double-stranded, and then transformed

into DH5α cells. Among 60 colonies, one colony contained cDNA for Gal3ST-4. The cDNA

for Gal3ST-4 contained in pCMV-SPORT, named pCMV-SPORT-Gal3ST-4, was sequenced


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using ABI PRISM 310 Genetic Analyzer (PE Biosystems).

Expression of Gal3ST-4 in COS-7 cells —————The plasmid (1 µg) was transfected

into COS-7 cells on 35-mm dishes using LIPOFECTIN Reagent (Life Technologies) according to

the manufacturer′s instructions. After 48 h, the cells were washed once with PBS, scraped off

from the dishes in 10 mM HEPES-NaOH (pH 7.2)-0.25 M sucrose, and homogenized. The

homogenate was ultracentrifuged at 100,000 × g for 1 h. The precipitated crude membranes

were suspended in 20 mM HEPES-NaOH (pH 7.2) and kept at –80 °C until use.

Assay of sulfotransferase activity ————Twenty µl of reaction mixture consisting of

0.1 M sodium cacodylate (pH 6.3), 10 mM MnCl2, 0.1 % (v/v) Triton X-100, 0.1 M NaF, 2 mM

ATP-Na2, 6.5 µM [35S]PAPS (2.8 × 105 dpm), 1 mM Galβ1→3GalNAcα1-O-pNP, and the crude

membrane fraction appropriately diluted, was incubated at 37 °C for 1 h. The [35S]-labeled

products were purified by paper electrophoresis (pyridine/acetic acid/water=3:1:387, pH 5.4).

The Rf values of [35S]sulfated Galβ1→3GalNAcα1-O-pNP and PAPS are 0.69 and 1.89,

respectively, when the Rf value of bromophenol blue is taken as 1.0. After extraction with

water, the radioactivities were counted. In the case of glycolipids used as acceptor substrates,

the detection of the [35S]-labeled products was performed according to the methods reported by

Kawano et al. (27).

Characterization of the [35S]-labeled product—————The [35S]-labeled product was

subjected to periodate oxidation (28). The labeled oligosaccharides were dissolved in 20 µl of

75 mM sodium metaperiodate-75 mM sodium acetate (pH 5.3) and incubated at 4 °C for 24 h in

the dark. Excess periodate was destroyed by adding 2 µl of 20 % ethylene glycol. After 1 h


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at room temperature, 300 µl of 0.1 M sodium borate (pH 9.0) containing 0.1 M sodium

borohydride was added and the solutions were stood for 1 h at room temperature. The solutions

were acidified by adding acetic acid and passed through a column (0.5 × 3 cm) of Bio-Rad

AG50W-X8(H+ form). The eluates were evaporated and residual boric acid was removed by

repeated evaporation with methanol. The residues were hydrolyzed in 100 µl of 0.05 N H2SO4

at 80 °C for 1 h. After being neutralized with NaOH, the mixtures underwent paper

electrophoresis. The [35S]-labeled compounds were extracted with water, applied on a thin

layer plate (Kieselgel 60F254, Merck, Darmstadt, Germany), and developed with solvents,

pyridine/ethyl acetate/acetic acid/water=5:5:1:3, or 1-butanol/ethanol/water=4:1:1. The

radioactivities were monitored by a radiochromatogram scanner.

[3H]Galβ1→3GalNAcα1-O-Bn as a positive control for periodate oxidation was

prepared as follows; twenty µl of solution containing 50 mM HEPES-NaOH (pH 7.2), 10 mM

MnCl2, 0.5 % (v/v) Triton X-100, 5 mM GalNAcα1-O-Bn, 2.5 µM UDP-[3H]Gal (3.3 × 106 dpm),

250 µM UDP-Gal, and crude membrane fractions from porcine colonic mucosa, was incubated at

37 °C for 1 h. The reaction mixture underwent paper electrophoresis, then the neutral fraction

further underwent paper chromatography which was developed with the solvent, pyridine/ethyl

acetate/acetic acid/water=5:5:1:3. A radioactive fraction ([3H]Galβ1→3GalNAcα1-O-Bn) with

the Rf value 0.72 was extracted with water. Linkage position of [3H]Gal residue was confirmed

by its binding to a PNA-agarose column, because PNA specifically recognizes Galβ1→3GalNAc

structure (29). Synthesis of 6-[35S]sulfo-GlcNAcβ1-O-Bn was performed using human

GlcNAc:→6sulfotransferase as described previously (30).


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[35S]sulfation of asialofetuin by Gal3ST-4—————— Fourty µg of asialofetuin (Sigma) was

dissolved in the enzyme reaction solution described above without Galβ1→3GalNAcα1-O-pNP

and incubated at 37 °C for 16 h. Half of the reactant was subjected to mild alkaline treatment in

50 µl of 1 M NaBH4-0.05N NaOH at 37 °C for 24 h. After acidification by acetic acid, the

solution was applied on an AG50W-X8 column and the eluate was evaporated as described above.

Another half of the reactant was subjected to N-glycanase digestion by the Glycopeptidase F De-

N-glycosylation Set (Takara Shuzo Co., Kyoto, Japan). Liberated [35S]-labeled oligosaccharides

were analyzed by paper electrophoresis.

Northern blot analysis ——————Human Multiple Tissue Northern Blot membranes

(CLONTECH, Palo Alto, CA) were used according to the manufacturer′s instructions. The

mRNA content in each lane of the Northern blot membrane is normalized to the mRNA

expression level of β-actin. [32P]-labeled probe was prepared from the cDNA fragment (1-1423;

see Fig.1A) by Random Primed DNA Labeling Kit (Roche Dignostics GmbH) using [α-32P]dCTP

(NEN Life Science Products Inc.) according to the manufacturer′s instructions. The

membranes were prehybridized in ExpressHyb Solution (CLONTECH) at 68 °C for 2 h, and then

hybridized with the [32P]-labeled probe in the same solution at 68 °C for 16 h. The Northern

blot membranes were washed in 2 × SSC-0.05% SDS at room temperature and then in 0.1 × SSC-

0.1% SDS at 50 °C. The radioactivity was detected with FLA-2000 (Fuji Photo Film Co. Ltd.,

Tokyo).


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RESULTS

Molecular cloning of a cDNA homologous to human Gal3ST-1–––––––– We found

small sequences (GenBankTM accession number AW961058) similar to the sequence of human

Gal3ST-1 (19) in the EST data bases. We prepared a sense oligonucleotide, 24 nucleotides in

length, the sequence of which is present in the EST, and used it as a probe for GeneTrapper

cDNA positive selection system to screen a human testis cDNA library. One clone was obtained

and the nucleotide sequence was determined (Fig. 1A). The 2460 bp cDNA had a 5′-

untranslated region of 236 bp, a single open reading frame of 1458 bp, and a 3′-untranslated

region of 766 bp including a poly (A)+ tail. The translation initiation site conformed to Kozak′s

rule (31), and the upstream region contained an in-frame stop codon. The open reading frame

predicts a protein of 486 amino acid residues with a molecular mass of 54,173Da with one

potential N-linked glycosylation site. Hydropathy plot analysis of the deduced amino acid

sequence revealed one prominent hydrophobic segment of 22 amino acid residues in length in the

N-terminal region, predicting that the protein has a type II transmembrane topology (Fig.1B).

The cDNA sequence was compared to the Human Genome Project Data Base, and the genomic

organization and the chromosomal localization were revealed (Fig. 1C). The gene comprises at

least four exons and spans about 10 kb in human chromosome 7q22. The intron/exon junctions

followed the GT/AG rule (33). The coding region is located in three exons (exons 2, 3, and 4),

and two introns were inserted between nucleotide 361 and 362 (at Arg 42), and nucleotide 665

and 666 (between Glu143 and Val144) of the cDNA (Fig. 1A).

The amino acid sequence of the protein (named Gal3ST-4) showed 33 %, 39 % and

Fig.1


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30 % identity with human Gal3ST-1 (19), human Gal3ST-2 (17), and human Gal3ST-3 (21),

respectively (Fig.2). The two highly conserved regions (Fig.2, underline and double line)

contain putative PAPS binding motifs (34), which are commonly conserved in all

sulfotransferases cloned so far (18). There exist other relatively conserved regions in the C-

terminal domain (299-314 and 358-374 of Gal3ST-4, in Fig. 2). In searching other sequences

homologous to the cDNA for Gal3ST-4, we found one cDNA clone (GenBankTM accession

number AK022178), with 2176 bp in length, which includes the full length of the open reading

frame of cDNA for Gal3ST-4.

Characterization of the putative sulfotransferase as Gal 1 3GalNAc: 3

sulfotransferase––––––––– The putative sulfotransferase was expressed in COS-7 cells, and the

crude membrane fraction was prepared as an enzyme source. The membrane fraction from the

cells transfected with pCMV-SPORT-Gal3ST-4, pCMV-SPORT vector containing the cDNA for

Gal3ST-4, had a sulfotransferase activity (5.6 pmol/min/mg protein) using Galβ1→3GalNAcα1-

-Bn as acceptor. The membrane fractions derived from the transfectant with pCMV-SPORT

and wild type had no sulfotransferase activity. The [35S]sulfated product,

[35S]SO3–→(Galβ1→3GalNAcα1- -Bn), purified by paper electrophoresis was resistant to

6646K β-galactosidase digestion and passed through a RCA-I-agarose column (data not shown).

Since authentic Galβ1→3GalNAcα1- -Bn was retarded to RCA-I agarose column, these results

suggested that the [35S]sulfate was transferred to the galactose residue. The migrating position

of the [35S]sulfated product in paper electrophoresis was close to that of authentic

[35S]SO3–→6GlcNAcβ1→2Man, suggesting that the product is monosulfated disaccharide. To

Fig.2


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determine the linkage position of [35S]sulfate residue, the [35S]sulfated product was subjected to

periodate oxidation. [3H]Galβ1→3GalNAcα1- -Bn was also subjected to the oxidation as a

positive control of the reaction; when subjected to TLC developed in the solvent system, 1-

butanol/ethanol/water=4:1:1, [3H] Galβ1→3GalNAcα1- -Bn and the oxidative product migrated

to positions with the Rf values of 0.45 and 0.56, respectively. The latter compound is

theoretically [3H]glycerol, because galactose is [3H]-labeled on the ring proton at the C-4 and 5.

The result indicates that the oxidative reaction went well. The reaction product from

[35S]SO3–→(Galβ1→3GalNAcα1- -Bn) underwent paper electrophoresis (Fig. 3A). It migrated

to the same position as the untreated [35S]labeled product. If [35S]sulfate is transfered to the C-2,

C-4, or C-6 position of galactose residue, or N-acetylgalactosamine residue, CH2OH-CH(OSO3–)-

CH2OH, CH2OH-CH(OSO3–)-CHOH-CH2OH, CH2OH-CHOH-CH2(OSO3

–), or

SO3–→GalNAcα1-O-Bn should be produced by periodate oxidation, respectively.

[35S]SO3–→6GlcNAcβ1-O-Bn and its periodate-oxidized and reduced product, [35S]SO3

–-OCH2-

CHOH-CH2OH, migrated faster in paper electrophoresis than [35S]SO3–→(Galβ1→3GalNAcα1-

-Bn)(Fig. 3A). The result suggests that [35S]sulfate does not bind to GalNAc residue or the C-

2, 4, or 6 position of Gal, but the C-3 position of Gal. Moreover, the reaction product was

subjected to TLC using different solvent systems (Fig.3B and C). The reaction product was

developed to the same position as the untreated product, in both solvent systems. The structure

of the reaction product was further confirmed by TLC with authentic

SO3–→3Galβ1→3GalNAcα1- -Bn (Fig. 3D). The reaction product was developed to the same

position as the authentic oligosaccharide and the oligosaccharide treated with periodate oxidation.

Fig.3


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These results suggest that [35S]sulfate binds to the C-3 position of Gal residue and that the

enzyme cloned here is a Gal:→3sulfotransferase.

The optimal pH of the enzyme was 6-7, using Galβ1→3GalNAcα1-O-pNP as acceptor

substrate (Fig.4). In the presence of MnCl2, the activity increased about 2.2-fold (Table I).

EDTA had no effect on the activity, suggesting that Gal3ST-4 does not essentially require

divalent cations. NEM and DTT had weak inhibitory effect on the activity, when these assays

were performed in the presence of MnCl2.

The acceptor substrate specificity of Gal3ST-4 is shown in Table II. Galβ1→3GalNAc,

Galβ1→3GalNAcα1-O-pNP, Galβ1→3GalNAcα1-O-Bn, and core2-O-pNP oligosaccharides, all

of which contain Galβ1→3GalNAc structure, are good substrates for Gal3ST-4. In contrast,

Galβ1→3GlcNAc (type 1), Galβ1→4GlcNAc(type 2), Galβ1→3GalNAcΟΗ and GalCer are poor

substrates. The results suggest that Gal3ST-4 is highly specific for Galβ1→3GalNAc

pyranoside structure. The specificity of Gal3ST-4 is sharply different from those of Gal3ST-1,

Gal3ST-2, and Gal3ST-3 which recognize GalCer, Galβ1→3/4GlcNAc, and Galβ1→4GlcNAc

structure as good substrates, respectively (17, 20, 21).

Gal3ST-4 activity was inhibited by high concentrations of acceptor substrates (Fig.5).

The activities for core2-O-pNP and Galβ1→3GalNAcα1-O-pNP were maximum at 1.5 and 1

mM and decreased significantly above 3 and 1.5 mM, respectively. The Km values for

Galβ1→3GalNAcα1-O-pNP and core2-O-pNP, which were estimated using only the data

obtained with lower concentrations of the two substrates, were 0.24 and 0.23 mM, respectively

(Table III).

Table II

Fig.4

Fig.5

Table I

Table III


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Incorporation of [35S]sulfate into asialofetuin by Gal3ST-4 –––––––––––– It was

investigated whether Gal3ST-4 can specifically add sulfate to Galβ1→3GalNAc in asialofetuin,

which contains both bi-, tri-antennary N-glycans and O-glycans consisting of Galβ1→3GalNAc

(35, 36). After incubating asialofetuin with Gal3ST-4, half of [35S]sulfated products were

subjected to mild alkaline treatment which specifically releases O-glycans, and another half of

the products were digested with N-glycanase. As shown in Table IV, [35S]sulfated

oligosaccharides were released by alkaline treatment, but not by N-glycanase digestion. These

results also support that Gal3ST-4 specifically acts on Galβ1→3GalNAc residue in O-glycans.

Northern blot analysis––––––– Among various human tissues, mRNA for Gal3ST-4 with 2.5 kb

was expressed mainly in placenta, thymus, testis, ovary, spinal cord, trachea, and adrenal gland,

and moderately in brain, lung, spleen, prostate, small intestine, colon, stomach, thyroid, and

lymph node (Fig. 6).

Table IV

Fig.6


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DISCUSSION

The present study demonstrates the isolation of a novel cDNA encoding

Galβ1→3GalNAc-specific Gal: →3 sulfotransferase by searching an EST homologous to human

Gal3ST-1 (19). The amino acid sequence of Gal3ST-4 reveals 33 %, 39 % and 30 %, identity

with Gal3ST-1, Gal3ST-2, and Gal3ST-3, respectively. This indicates that Gal3ST-4 is the

fourth member of a Gal:→3 sulfotransferase family. These enzymes are in common with

transferring sulfate to non-reducing terminal Gal, but selectively recognize aglycon moieties of β-

Gal. Gal3ST-1 acts on GalCer, LacCer, and GalDG, but not on lactose and

Galβ1→4GlcNAcβ1→3Galβ1→4Glcβ1-Cer (20). Gal3ST-2 acts on

Galβ1→3/4GlcNAcβ1→R and Galβ1→3GalNAcα1-O-Bn, but not on GalCer and LacCer (17).

Gal3ST-3 acts on Galβ1→4GlcNAcβ1→R (21). In contrast, Gal3ST-4 specifically recognizes

Galβ1→3GalNAcα1→ structure and Galβ1→3/4GlcNAc, GalCer, LacCer, and GalDG are not

substrates (Table II). The results indicate that the four sulfotransferases are utilized in

accordance with distinct acceptor glycoconjugates.

Gal3ST-4 gene comprises at least four exons (Fig.1C). The coding region is inserted

with two introns at Arg 42 and between Glu143 and Val 144. The putative transmembrane

domain and two putative PAPS binding domains (5′PSB and 3′PB) (34) are localized in exons 2,

3, and 4, respectively. We investigated the intron/exon alignment of Gal3ST-2 gene using the

Human Genome Project Data Base, and found that the coding region for Gal3ST-2 is also

inserted with two introns, at Pro40 and between Gln125 and Val 126. The insertion positions of

Gal3ST-2 gene are very close to those of Gal3ST-4 (Fig. 1C). In contrast, the coding region for


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Gal3ST-1 is inserted with one intron at Thr 44 (37); the position is close to Arg 42 of Gal3ST-4

and Pro40 of Gal3ST-2. As for 5′-untranslated region, it has been shown that there exist at least

seven exons for the 5′-untranslated region of human Gal3ST-1 gene and that these exons are

alternatively utilized in a cancer-associated manner (37). Whether or not there exist alternative

forms of mRNA for Gal3ST-4 remains unclear, but this is an important issue for elucidating

transcriptional regulation of the Gal3ST-4 gene.

Gal3ST-4 is highly specific for Galβ1→3GalNAcα1→ structure.

Galβ1→3(GlcNAcβ1→6)GalNAcα1-O-pNP is also a good substrate and the Km and Vmax

values for the core 2 oligosaccharide are similar to those for Galβ1→3GalNAcα1-O-pNP (Table

III), indicating that the substitution of β-GlcNAc at the C-6 of GalNAc does not affect the

substrate recognition of Gal3ST-4. Kuhns et al., (38) showed that Galβ1→3GalNAc:β1→6 N-

acetylglucosaminyltransferases in acute myeloid leukemia cells and rat colon can act on

Galβ1→3GalNAcα1-O-Bn, but not on SO3–→3Galβ1→3GalNAcα1-O-Bn, and suggested that

the substitution of β-GlcNAc at the C-6 of Galβ1→3GalNAc should precede sulfation at the C-3′.

Our result that Gal3ST-4 can utilize core 2 oligosaccharide as a good substrate, is consistent with

their results, with regard to biosynthesis of sulfated core 2 glycans.

The enzyme activity of Gal3ST-4 is inhibited by higher concentrations of acceptor

substrates (Fig.5). Similar inhibitory effects have been reported for β1→4galactosyltransferase

I, II, and III (39), β1→4galactosyltransferase from human colonic mucosa (40), and

GlcNAc: →6sulfotransferase (30). These transferase activities are inhibited at concentrations in

excess of 2-5 mM, except for β1→4galactosyltransferase II, the activity of which is inhibited


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even at ~0.6 mM of GlcNAcβ1-O-Bn (39). The inhibitory effects of the two substrates for

Gal3ST-4 (Fig. 5) appear at similar concentrations with those for the transferases described above,

although the molecular mechanism and biological significance remain unclear.

The result in Fig. 6 showed a relatively extensive expression of the mRNA for Gal3ST-4.

It is important whether or not SO3–→3Galβ1→3GalNAcα1→ structure is present in the tissues

examined in Fig. 6. Although Chance and Mawhinney (10) showed the occurrence of

SO3–→3Galβ1→3(R→GlcNAcβ1→6)GalNAcα1→ structure in tracheobronchial mucous

glycoproteins from a patient with cystic fibrosis, information about the existence of the sulfated

glycan has so far been scarce. On the other hand, Gal:→3 sulfotransferase activities for

Galβ1→3GalNAcα1→ structure have been reported in rat colonic mucosa (38), human breast,

colon, and several tumor tissues (41). To assess whether or not SO3–→3Galβ1→3GalNAcα1→

structure is widely distributed, a lectin or antibody which specifically recognizes the sulfated

glycan needs to be explored.

Chandrasekaran et al., (41) reported the occurrence of two distinct Gal:3-O-

sulfotransferases (groups A and B) in human various tumors and normal tissues with a tissue-

dependent distribution. Group A sulfotransferase recognizes Galβ1→3GalNAcα-O-allyl and 3-

O-MeGalβ1→4GlcNAcβ1→6(Galβ1→3)GalNAcα-O-Bn as good acceptors, but not

Galβ1→4GlcNAcβ-O-allyl and Galβ1→3GlcNAcβ-O-allyl, while group B sulfotransferase has

rather broad substrate specificity (41). The substrate specificity of group A sulfotransferase is

similar to that of Gal3ST-4. They also showed that group A Gal:→3sulfotransferase is

dominant in breast tumor, some ovarian tumor, and some metastatic ovary, and that the specific


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activities in breast tumor are higher than those in breast normal tissues (41). In contrast,

Brockhausen et al.,(42) showed that a sulfotransferase activity for Galβ1→3GalNAcα1-O-Bn in

a human mammary epithelial cell line, MTSV1-7, is higher than those in three human breast

cancer cell lines. We are in the process of investigating changes in expression level of mRNA

for Gal3ST-4 in these cancerous tissues.


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39. Almeida, R., Amado, M., David, L., Levery, S.B., Holmes, E.H., Merkx, G., van Kessel, A.G.,

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Legends for figures

Fig. 1. Nucleotide and deduced amino acid sequence of human Gal3ST-4 cDNA (GenBank

accession number AF316113), hydropathy plot of the protein, and genomic organization of

human Gal3ST-4 gene.

A, the predicted amino acid sequence for Gal3ST-4 is shown below the nucleotide sequence.

The putative membrane spanning domain is underlined, and one potential N-glycosylation site is

marked by dot. The presumptive polyadenylation signal is boxed. B, the hydropathy plot was

calculated by the method of Kyte and Doolittle (32) with a window of 11 amino acids. C, the

5′- and 3′- untranslated regions are shown as open boxes. Shaded boxes represent the coding

sequences. The black horizontal lines denote the introns.

Fig. 2. Comparison of the predicted amino acid sequences of human Galβ1→3GalNAc: →3′

sulfotransferase (Gal3ST-4) with human Gal3ST-1(19), Gal3ST-2 (17), and Gal3ST-3 (21).

The predicted amino acid sequences were aligned using GENETYX-MAC (Ver.11) computer

program. Black boxes and shaded boxes indicate that the predicted amino acid is identical

among three and two sequences, respectively. Two putative PAPS binding domains (5′PSB and

3′PB) (34) are indicated by underline and double line, respectively.

Fig. 3. Analysis of the sulfate linkages of [35S]SO3–→(Galβ1→3GalNAcα1-O-Bn) synthesized

by human Gal3ST-4. A, paper electrophoresis at pH 5.4 of periodate oxidation product of

[35S]SO3–→(Galβ1→3GalNAcα1-O-Bn). B and C, silica-gel TLC of periodate oxidation


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product of [35S]SO3–→(Galβ1→3GalNAcα1-O-Bn) using ethylacetate/pyridine/acetic

acid/water=5:5:1:3 (B), or 1-butanol /ethanol/water=4:1:1 (C). D, silica-gel TLC of

Galβ1→3GalNAc (lane 1), GalNAcα1-O-Bn (lane 2), SO3–→3Galβ1→3GalNAcα1-O-Bn (lane

3), and periodate oxidation product of SO3–→3Galβ1→3GalNAcα1-O-Bn (lane 4) using 1-

butanol/ethanol/water=4:1:1. The chromatogram was visualized by 5% H2SO4/MeOH.

Arrows indicate the front of the developing solvent and the positions of standard compounds and

enzymatic reaction products; a, [35S]sulfate: b, [35S]PAPS; c, the periodate oxidation product of

[35S]SO3–→6GlcNAcβ1-O-Bn; d, bromophenol blue; e, [35S]SO3

–→6GlcNAcβ1-O-Bn; f,

[35S]SO3–→6GlcNAcβ1→2Man; g, [35S]SO3

–→(Galβ1→3GalNAc); h,

[35S]SO3–→(Galβ1→3GalNAcα1-O-Bn); i, [35S]SO3

–→[Galβ1→3(GlcNAcβ1→6)GalNAcα1-O-

pNP]; j, the front of the developing solvent; k, [3H]Galβ1→3GalNAcα1-O-Bn; l, the periodate

oxidation product of [3H]Galβ1→3GalNAcα1-O-Bn.

Fig. 4. Effect of pH on Gal3ST-4 activity. Buffers used were 0.1 M of sodium acetate (•),

sodium cacodylate (○)、and HEPES-NaOH（▲）. Ion strength was adjusted to 0.1 with NaCl.

Fig. 5. Effect of acceptor concentrations on Gal3ST-4 activity. Galβ1→3GalNAcα1-O-pNP (•)

and Galβ1→3(GlcNAcβ1→6)GalNAcα1-O-pNP (○) were used as acceptor substrates.

Fig. 6. Northern blot analysis of Gal3ST-4 transcript. The amount of poly (A)+ RNA was

normalized to the mRNA expression levels of β-actin. The blots were hybridized with a 32P-


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labeled cDNA probe specific for human Gal3ST-4. On the left, the migration positions of

standards are indicated.


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Table I. Effect of various compounds on Gal3ST-4 activity

Compound Relative activity (%)a

None 100

MnCl2b 222

CaCl2b 126

MgCl2b 92

EDTAb 124

NEMc 132

DTTc 97

a Galβ1→3GalNAcα1-O-pNP was used as a substrate. The values represent the percentage of the activity compared with that in the absence of compounds.

b The concentrations of cations and EDTA were 10 mM.c The concentrations were 5 mM. The activities were assayed in the presence

of 10 mM MnCl2.

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Table II. Substrate specificity of human Gal3ST-4

Acceptor Relative activitiy (%)

Galβ1→3GalNAcα1-O-pNP 100a

Galβ1→3GalNAcα1-O-Bn 52

Galβ1→3GlcNAc < 1

Galβ1→4GlcNAc 1

Galβ1→3GalNAc 105

Galβ1→3GalNAcOH < 1

Galβ1→3(GlcNAcβ1→6)GalNAcα1-O-pNP 127

Galβ1→3GlcNAcβ1→3Galβ1→4Glc < 1

Galβ1→4GlcNAcβ1→2Manα1→3(6)Manβ1→4GlcNAc < 1Gal2·GlcNAc2·Man3·GlcNAc2 < 1

GalCer < 1

LacCer < 1

GalDG < 1

a Relative ratios are taken with the value of Galβ1→3GalNAcα1-O-pNP as 100.


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Table III. Kinetic properties of human Gal3ST-4

Substrate Km Vmax

mM pmol/min/mg protein

Galβ1→3GalNAcα1-O-pNPa 0.24 7.2

Galβ1→3(GlcNAcβ1→6)GalNAcα1-O-pNPa 0.23 6.3

PAPSb 0.00029 4.2

Enzyme source was the crude membrane fraction derived from COS-7 cells

transfected with pCMV-SPORT-Gal3ST-4.

a The concentration of PAPS was 4 µM.

b Acceptor substrate was 3 mM of Galβ1→3GalNAcα1-O-pNP.


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Table IV. Action of Gal3ST-4 on asialofetuin

Total radio- Released [35S]oligosaccharides by activity (dpm)a Alkaline treatment (dpm)b N-glycanase digestion (dpm)c

14115 12280 0

a The enzyme reaction was performed using 40 µg of asialofetuin as described in

"EXPERIMENTAL PROCEDURES".b Half amount of [35S]-labeled protein was treated with 1 M NaBH4-0.05N NaOH at 37°C

for 24 h and released [35S]-labeled oligosaccharides were detected by paper

electrophoresis.c Half amount of [35S]-labeled protein was digested with 1 mU of N-glycanase following

denaturation in 0.5 M Tris-HCl (pH 8.6)-0.5 % SDS-0.1 M mercaptoethanol at 100 °C for

3 min and then addition of 1 % Nonidet P-40 as stabilizer.


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Fig. 1A


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Fig. 1B


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238166666536236149481

3'5'6.2 K1.3 K

0.16 K

Exon 1 Exon 2 Exon 3 Exon 4

Fig. 1C


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[GENETYX-MAC: Multiple Alignment]Date : 2001.04.27

Gal3ST-4 1 ---MGPLSPARTLRLWGPRSLGVALGVFM TI-GFALQLLGGPFQRRLPGLQLRQPSAPS- 55Gal3ST-1 1 -----MLPPQKKPWESMAK--GLVLGALF TSFL LLVYSYAVPPLHAGLASTTPEAAASCS 53Gal3ST-2 1 ------MMSLLGGLQRYFRVILLLLLAL- TL-L LL----AG-FLHSDLE LDTP------- 40Gal3ST-3 1 MPPILQRLQQATKMMSRRKILLLVLG--CSTVSLLI--------HQGAQ LSWYPK----- 45

Gal3ST-4 56 -----------LRPALPSCPPRQRLV FLKTHKSGSSSVLSL LHRYGDQ HGLRFALPA--- 101Gal3ST-1 54 PPALEPEAVIRANGSAGECQPRRNIV FLKTHKTASSTLLNILFRFGQK HRLKFAFPNG-- 111Gal3ST-2 41 -----------LFGGQAEGPPVTNIM FLKTHKTASSTVLNILYRFAET HNLSVALPAGS- 88Gal3ST-3 46 ---LFP-LSCPPLRNSPPRPKHMTVAFLKTHKTAGT TVQNILFRFAER HNLTVALPHPSC 101

Gal3ST-4 102 RYQ FGYPKLFQASR-- VKGYR PQGGGTQLPFHILCHHMRFNLK EVLQV MPSDSFFFSIVR 159Gal3ST-1 112 RND FDYPTFF-A-RSL VQDYR P-G-AC---FNIICNHMRFHYD EVRGLV PTNAIFITVLR 164Gal3ST-2 89 RVHLG YPWLFLA-R-Y V-----E GVGSQQRFNIMCNHLRFNLPQ VQKV MPNDTFYFSILR 141Gal3ST-3 102 EHQFCYPRNFSA-H-F V---H P---ATRPP-HVLASHLRFDRA ELERL MPPSTVYVTILR 152

Gal3ST-4 160 DPAALAR SAFSYYK-STSSAFRKS--P SLAAFLANP-RGF YRPGAR-GDHYARNLLWFDF 214Gal3ST-1 165 DPARLF ESSFHYFGPVV PLTWKLSAGDKLTEFLQDPDR-Y Y-DPNGFNAHYLR NLLFFDL 222Gal3ST-2 142 NPVFQL ESSFIYYKTYA P-AFR-GA-P SLDAFLASP-RTF YNDSRHLRNVYAKNNMW FDF 197Gal3ST-3 153 EPAAMF ESLFSYYNQYC P-AFRRVPNA SLEAFLRAPE-AY YRAGEHF-AMFAHNTLAY DL 209

Gal3ST-4 215 GLPFPPEKRAKRGNIHPPRDPNPPQLQVLPSGAGPRAQTLNPNALIHPVSTVTDHRSQIS 274Gal3ST-1 223 GYDN-------------------------------------------------------- 226Gal3ST-2 198 GFD--------------------------------------------------------- 200Gal3ST-3 210 GGDN-------------------------------------------------------- 213

Gal3ST-4 275 SPASFDLGSSSFIQWGLAWLDSVFDLVMVAEYFDESLVLLADA LCWGLDDVVGFMH NAQA 334Gal3ST-1 227 ---SLD-PSSPQVQEHILE VERR FHLVLLQ EYFDESLVLLKDL LCWELEDVLYFKLNA-- 280Gal3ST-2 201 ---PNAQCEEGYVRARIAE VERR FRLVLIAEHLDESLVLLRRR LRWALDDVVAFRLNS-- 255Gal3ST-3 214 --ERSPRDDAAYLAGLIRQ VEEV FSLVMIAEYFDESLVLLRRL LAWDLDDVLYAK LNA-- 269

Gal3ST-4 335 GHKQGLSTVSNSG LTAEDRQ LTA RA-RAWNNLDWALYVHFNRSL WA-- R-IEKY GQGRLQ 390Gal3ST-1 281 -RRD-- SPV--P RLSGE--- LYG RA-TA WNMLDSHLYRHFNASF W--- RKVEAF GRERMA 328Gal3ST-2 256 --RSAR S-VA-- RLSPETR---ERA-RSWCALDWRLYEHFNRTL WAQL R-AEL- GPRRL- 303Gal3ST-3 270 --RAA- SS---- RLAAIPAA L-A RAARTWNALDAGLYDHFNATF W--- RHVARA GRACVE 318

Gal3ST-4 391 T-AVAE LR-A--R-REALAKH CLV- GGEASDPKYITDRRFR PFQF-GSAKV LGYILRSGL 443Gal3ST-1 329 R-EVAA LRHANERMR----TI CID- GGHAVDAAAIQDEAMQ PWQPLGTKS ILGYNLKKSI 382Gal3ST-2 304 RGEVER LR-A--R-RRELASL CLQD GGALKNHTQIRDPRLR PYQ-SGKAD ILGYNLRPGL 358Gal3ST-3 319 R-EARE LREA--RQR--LLRR CFGDEPLLRPAAQIRTKQLQ PWQPSRKVD IMGYDLPGGG 373

Gal3ST-4 444 PQD--QECERLAT PELQYKDK LDAKQFPPTVSLPLKTS RP--- LSP 484Gal3ST-1 383 GQRHAQLCRRMLT PEIQYLMD LGANLWVTK--LW-KFI RD--F LRW 423Gal3ST-2 359 DNQTLGVCQRLVM PELQYMAR LYALQFPEK---PLKNI- P--F LGA 398Gal3ST-3 374 AGPATEACLKLAM PEVQYSNY LLRK QKRRGG---AR-ARPEPV LDNPPPRPIRVLPRGPQ 429

Gal3ST-4 484 484Gal3ST-1 423 423Gal3ST-2 398 398Gal3ST-3 430 GP 431

Fig. 2


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A

B

C

Distance from origin (cm)

abc

jkh

defghi

g c

jkhg c l

l

( ) (+)

5 10 150

1

2

3

4

D jh

Fig. 3


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100

pH6

78

50

50

Relative activity (%)F

ig. 4


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0.5 2 1050 10.20.1

2

4

6

Concentration of acceptor substrate (mM)

Fig.5


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Akira Seko, Sayuri Hara-Kuge and Katsuko Yamashita1-3GalNAc residue in O-glycansβ3-O-sulfotransferase that transfers sulfate to Gal

Molecular cloning and characterization of a novel human galactose

published online May 1, 2001J. Biol. Chem.

10.1074/jbc.M101558200Access the most updated version of this article at doi:

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Molecular cloning and characterization of a novel human ... · mg/ml gel) was purchased from E-Y Laboratories, Inc. (San Mateo, CA). Psathyrella velutina lectin (PVL) was prepared