Cloning and Characterization of a Mammalian N-Acetylglucosamine-6-sulfotransferase That Is Highly...

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Biochemical and Biophysical Research Communications 263, 543–549 (1999)

Article ID bbrc.1999.1324, available online at http://www.idealibrary.com on

loning and Characterization of a Mammalian-Acetylglucosamine-6-sulfotransferase That

s Highly Restricted to Intestinal Tissue

in Kyu Lee,* Sunil Bhakta,† Steven D. Rosen,*,1 and Stefan Hemmerich†,1

Department of Anatomy and Program in Immunology, University of California, San Francisco, California 94143; andDepartment of Respiratory Diseases, Roche Bioscience, 3401 Hillview Avenue, Palo Alto, California 94304-1397

eceived August 13, 1999

rides is enormous (3). Since the sulfate modifications ofgqecnncghmrkthoiptwlGcig

ctl(its(aGk(1

Using the sequences of a galactose 6-O-sulfotrans-erase and an N-acetylglucosamine 6-O-sulfotransfer-se as probes in an EST approach, we have identified aighly related cDNA in human and an apparent ortho-

ogue in mouse. The cDNAs predict type II transmem-rane proteins that constitute new members of the Gal/alNAc/GlcNAc 6-O-sulfotransferase (GST) family. Mem-ers of this family have previously been implicated inhe sulfation of GAG chains within proteoglycans andhe sulfation of O-linked chains within sialomucin li-ands for L-selectin. Expression of the newly identifiedDNA in COS cells led to the addition of sulfate to C-6 oflcNAc in an acceptor glycoprotein. The tissue expres-

ion of transcripts corresponding to the cDNA wasighly restricted to the small intestine and colon in hu-ans. Based on these characteristics, the novel sulfo-

ransferase is designated I-GlcNAc6ST for intestinallcNAc 6-O-sulfotransferase. © 1999 Academic Press

Key Words: sulfotransferase; carbohydrate; N-acetyl-lucosamine; intestine.

The carbohydrates of glycoconjugates are highly di-erse structures with variation in monosaccharideomposition, glycosidic linkage positions, and branch-ng of chains (1). Further diversity is added by theovalent addition of sulfate (SO3

2) moieties to particu-ar hydroxyl groups and amino groups of saccharides2). The range of sulfate modifications to monosaccha-

Abbreviations used: BAC, bacterial artificial chromosome; C6ST,hondroitin-6-sulfotransferase; EST, expressed sequence tag; GlcNAc6ST,lcNAc-6-sulfotransferase; GlyCAM-1/IgG, IgG chimera of GlyCAM-1;ST, Gal/GalNAc/GlcNAc sulfotransferase; HEC-GlcNAc6ST, high-ndothelial-cell GlcNAc-6-sulfotransferase; HEV, high endothelialenules; I-GlcNAc6ST, intestinal GlcNAc-6-sulfotransferase; KSGal6ST,eratan sulfate Gal-6-sulfotransferase.

1 To whom correspondence may be addressed: (S.D.R.) Departmentf Anatomy, Box 0452, University of California, San Francisco, CA4143. Fax: (415) 476-4845. E-mail: [email protected]. (S.H.) E-mail:[email protected].

543

lycoproteins can be extensive in amount and fre-uently occur at high density, they can have a profoundffect on the physicochemical properties of the glyco-onjugates, at least in part through the addition ofegative charge (4). In addition, there is a growingumber of examples in which sulfation modificationsontribute to recognition determinants of glycoconju-ates (2–4). Critical roles for carbohydrate sulfationave been defined for binding of growth factors, hor-ones and chemokines, modulation of growth factor

eceptor activation, viral-host cell interactions and eu-aryotic cell-cell adhesion. In the last category, a sys-em receiving great attention in recent years is theoming of lymphocytes to lymph nodes, which dependsn the binding of blood-borne lymphocytes to special-zed high endothelial venules (HEV) in secondary lym-hoid organs (5). This process depends on the interac-ion of L-selectin, a lectin-like receptor on lymphocytes,ith specific mucin-like glycoproteins on the endothe-

ial lining of HEV (6). Carbohydrate sulfation (i.e.,lcNAc-6-SO4 and Gal-6-SO4), which is found in the

ontext of sialyl Lewis x, is required for optimal bind-ng between L-selectin and these HEV-associated li-ands (7).With the increasing realization of the importance of

arbohydrate sulfation in many biological processes,here has been considerable recent interest in the mo-ecular identification of carbohydrate sulfotransferases3, 4). The majority of the enzymes cloned to date arenvolved in the biosynthesis of the GAG chains of pro-eoglycans. Among the recently cloned carbohydrateulfotransferases are the members of the GST family3) consisting heretofore of 4 members in human: Thesere 6-O-sulfotransferases, which add sulfate to C-6 ofal, GalNAc or GlcNAc. Three of these enzymes,nown as KSGal6ST (CHST1) (8, 9, 10), GlcNAc6STCHST2) (9, 11, 12), and HEC-GlcNAc6ST (LSST) (10,3) are capable of sulfating HEV-associated ligands

0006-291X/99 $30.00Copyright © 1999 by Academic PressAll rights of reproduction in any form reserved.

Vol. 263, No. 2, 1999 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

544

and conferring L-selectin binding activity (10, 13, 14).Tsc

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ping to the 59 end of the murine open reading frame were found.CmisjOcfqA

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he fourth member of the family, C6ST, has beenhown to modify C-6 of GalNAc in chondroitin sulfatehains (15, 16).

In the present study, we report the sequence of aDNA that encodes an additional member of the GSTamily. We also provide the sequence of a highly ho-ologous cDNA from the mouse. We establish that theuman enzyme is a GlcNAc-6-sulfotransferase. North-rn analysis demonstrates highly restricted expressionf transcripts corresponding to this enzyme in themall intestine and colon.

ETHODS

Cloning of human and mouse I-GlcNAc 6-O sulfotransferases fromAC libraries. Two EST-databases, LifeSeq (InCyte Pharmaceuti-

als, Inc.), and dbEST (NIH) were probed with the protein sequencef human HEC-GlcNAc6ST using the tBLASTn algorithm (17). Ini-ially two human EST (Nos. 1846903 and 3219891) were identified inhe LifeSeq database Another murine EST was identified in dbESTGenBank Accession No. AA261202). Clones containing ESTs Nos.846903 and AA261202 were obtained and used as PCR templatesor the synthesis of probes. For amplification from No. 1846903, theollowing primer pair yielded a 237-bp amplicon: forward 59-CCA-GCGTCCGCACCCACGGGTCGGGGATCGGCAAGCCAATCGAG-39nd reverse 59-GTCTGGGCCTCGTGGCAGCACCAGATCCAGGGT-39.For amplification from No. AA261202, the following primer pair

ielded a 151-bp amplicon: forward 59-CACACGCTGCCCTTTGCC-AGATT-39 and reverse 59-TCTAAGATTCCGGTTGCTTCTCCGT-GAC-39.A 181-bp probe mapping to LifeSeq No. 3219891 was amplified

rom human small intestine cDNA (Quick Clone, Clontech) using therimer pair forward 59-ACATGGCCGTGCGCGACCTG-39 and re-erse 59-ATACGTCCTGCTTGCTGAT-39.The three probes described above were verified by direct sequenc-

ng and provided to Genome Systems Inc. for hybridization screeningn human (Nos. 1846903 and 3219891) and C57Bl/6 mouse (No.A261202) genomic BAC libraries (pBeloBac11 vector). While bothuman probes hybridized to a single BAC, the murine probe hybrid-

zed to three different BACs. DNA was isolated from the BAC clonessing the KB100 Magnum plasmid kit (Genome Systems) accordingo the manufacturer’s specifications. The BACs were sequenced di-ectly starting with primers derived from the appropriate ESTs andhen sequencing upstream and downstream with additional primers.wo of the murine BACs yielded identical sequences, while the thirdurine BAC did not yield sequence with the primers. The resulting

enomic sequences were confirmed by double stranded sequencingnd submitted to GenBank under the accession numbers AF176839human) and AF176841 (mouse).

Identification of cDNAs coding for human and mouse I-GlcNAc6ST.he open reading frames contained in the above genomic clones weresed to screen the EST databases using the BLASTn algorithm (17).he ESTs mapping to the human sequence are summarized in Table. Two mouse ESTs (Accession Nos. AI115260 and AI156825) map-

FIG. 1. Nucleotide and translated amino acid sequences of I-GlcNo. AF176838) and predicted protein sequences of human (h) and m

etters and the predicted amino acid sequence is indicated below theonger reading frame for human I-GlcNAc6ST beginning at the firsthe translations of the human and mouse cDNAs (c) and the human

ndicated in small capitals in order to demonstrate the divergenceomains are underlined and three potential N-linked glycosylation s

545

lones containing ESTs Nos. 3372492, 3126392, and AI282873 (hu-an) and the two mouse ESTs were retrieved, plasmid DNA was

solated and sequenced. The complete human I-GlcNAc6ST cDNAequence was assembled from ESTs Nos. 3372492 and AI282873oined tail to head at the internal NotI site at position 799 of the longRF and submitted to GenBank under Accession No. AF176838. The

omplete mouse I-GlcNAc6ST cDNA was determined from the twoull-length mouse ESTs Nos. AI115260 and AI156825 (identical se-uences) and submitted to GenBank under the Accession No.F176840.

Northern blot analysis. A 153-bp fragment corresponding to nt20-772 in Fig. 1 was labeled with [a-32P]dATP (Amersham Pharma-ia Biotech) and used to probe Northern blots of various humanissue RNAs (human multiple tissue northern blots, Clontech) asreviously described (10).

Expression of I-GlcNAc6ST. Human genomic BAC DNA contain-ng the entire I-GlcNAc6ST ORF was digested with EcoRI and XbaI,ractionated through 0.7% agarose and partially transferred to aitrocellulose filter. The 2.2 kb band containing the I-GlcNAc6STRF was identified by hybridization to the above 32P-labeled 153 bprobe. The corresponding DNA fragment in the agarose gel was thensed as template in amplification of the ORF with the followingrimer pair forward 59-GGGAATTCCAATGGGTAGGGTAGCCGA-TCTGAG-39 and reverse 59-GGGAAGCTTTCAGTCAGGCGATG-CCAGCTGAAGTGG-39.The 1315-bp amplicon containing EcoRI (59) and HindIII (39) com-

atible ends was subcloned into pCDNA3.1 (Invitrogen). The junc-ions and insert were confirmed by sequencing.

Transient transfection of COS-7 cells. For generation of recombi-ant GlyCAM-1/IgG fusion protein, COS-7 cells were grown to 80%onfluency in a T162 culture flask (Corning-Costar, Corning, NY)nd transfected with 8 mg of a plasmid encoding GlyCAM-1/IgG (10)nd 8 mg of pCDNA3.1 encoding either I-GlcNAc6ST, or lacZ (mockontrol) using Lipofectamine (Life Technologies) in Opti-MEM (Lifeechnologies) according to the manufacturer’s protocol. Recombi-ant GlyCAM-1/IgG was isolated as described previously (10). Oneercent of each protein sample was analyzed by SDS–PAGE and theemaining samples were lyophilized for subsequent acid hydrolysisnd positional analysis.

Analysis of sulfated GlyCAM-1/IgG carbohydrates. The lyophi-ized recombinant GlyCAM-1/IgG samples were subjected to partialcid hydrolysis, size and charge fractionation, and subsequent anal-sis by high pH anion-exchange chromatography (HPAEC) as de-cribed previously (7, 18).

ESULTS

Identification of a cDNA in human and mouseelated to the GSTs. We previously identified HEC-lcNAc6ST and KSGal6ST (10) through an expressed

equence tag (EST) approach in which we screeneduman EST databases for sequences that were homol-gous to a chicken chondroitin/keratan sulfate sulfo-ransferase (19). Several ESTs were found and used to

c6ST. cDNA sequence for human I-GlcNAc6ST (GenBank Accessionse (m) I-GlcNAc6ST. The open reading frame is denoted by capitalcleotide sequence. Capital italics denote the 59 portion of a possible

following the upstream stop codon (double underscored) at nt 218.nomic DNA (g) upstream of the presumed start codon at nt 344 arethese sequences 59 of this position. The putative transmembranes in each protein sequence are boxed.

Aounu

ATGgeofite

oegfGaEoNisqsB1stsNb

afsiBsMthlm1t

nucleotides 1 through 160 of the 1173-bp ORF as apimaEhiqtptwiAfcasAtBNdtcct23tgstardtiG5

iiiiAii

sbr

Rmp


btain full-length cDNAs. cDNAs for the same twonzymes have been independently described by otherroups (8, 13). To identify novel members of the GSTamily, we used the cDNA sequences of HEC-lcNAc6ST and KSGal6ST to probe the NCBI dbESTnd LifeSeq (InCyte Pharmaceuticals, Inc.) humanST databases for related sequences. Two non-verlapping ESTs (corresponding to LifeSeq clonesos. 1846903 and 3219891) were identified from two

ndependent human colon libraries derived from theame donor. Using the LifeSeq EST No. 1846903 se-uence as a probe, we then identified a highly relatedequence in the NCBI mouse dbEST database (Gen-ank Accession No. AA261202). The LifeSeq No.846903 and GenBank No. AA261202 clones were fullyequenced and found to be partial cDNAs which con-ained 39 poly(A) tails (882 and 869 bp in length, re-pectively). A 182-bp cDNA fragment of LifeSeq ESTo. 3219891 was generated from human colon cDNAy PCR as described under Methods.Previously, we cloned murine HEC-GlcNAc6ST frombacterial artificial chromosome (BAC) library and

ound the gene to be intron-less (10). We adopted aimilar strategy to obtain genomic clones correspond-ng to the human and mouse ESTs identified above.AC libraries from human and mouse (C57BL/6) werecreened with EST-derived probes as described underethods. Both human ESTs were found to hybridize to

he same single BAC clone, while the murine probeybridized to three different BACs from the mouse

ibrary. The genomic clone within the BAC from hu-an contained a long open reading frame (ORF) of

173 bp (GenBank Accession No. AF176839). No in-rons were detected. Using sequence corresponding to

FIG. 2. Northern blotting. Northern blots containing poly(A)1

NA from various human tissues were probed with a 153-bp frag-ent of the I-GlcNAc6ST cDNA. The blots were stripped and re-

robed with a 300-bp probe for b-actin (lower panels).

546

robe, we rescreened LifeSeq for overlapping ESTs anddentified ESTs Nos. 3372492 and 3373406 from a hu-

an skull ependymoma library and No. 3126392 fromhuman lung adenocarcinoma library. These three

ST mapped to the 59 end of the ORF identified in theuman BAC, with Nos. 3372492 and 3373406 contain-

ng the longest 59 untranslated region. Further se-uencing of the corresponding clones extended thesehree sequences to an internal NotI site located atosition 799 of the ORF. Using sequence correspondingo nucleotides 800 through 955 of the ORF as a probe,e rescreened the NCBI human dbEST database and

dentified a matching EST (GenBank Accession No.I282873) from a human colon adenocarcinoma. By

urther sequencing of the corresponding clone, thisDNA was shown to extend from the internal Not I sitet position 799 of the ORF to a 39 poly(A) tail. Theequence presented in Fig. 1 (GenBank Accession No.F176838) was compiled by joining the sequences from

he 59 EST (Lifeseq No. 3372492) and the 39 EST (Gen-ank Accession No. AI282873) clones at the internalot I site. The ORF agrees completely with the BAC-erived sequence. The complete human cDNA containswo possible start codons following an upstream stopodon located at position 218. Although neither of theseodons occurs in the consensus context for initiation,he presence of the GCCGCC motif in positions 29 to4 relative to the second codon (beginning at position44) favors it as the initiation site (20). Furthermore,he human genomic sequence, as well as the homolo-ous mouse cDNA sequence (see below), diverge con-iderably from the human cDNA sequence upstream ofhe second potential start codon (Fig. 1). Therefore, wessign the ATG at position 344 as the start of the openeading frame. On this basis, the human cDNA pre-icts a protein of 390 amino acid (Fig. 1). The nucleo-ide sequence of the predicted coding region is 63%dentical to the coding region of human HEC-lcNAc6ST while at the protein level, the similarity is9% and identity is 55% (Fig. 3). Compared to

TABLE 1

ESTs Mapping to Human I-GlcNAc-6ST

EST 59 end Library source

3372492 1 Skull soft tissue ependymoma3373406 1 Skull soft tissue ependymoma3216392 168 Lung adenocarcinoma3219891 555 Colonl282873 1139 Colon adenocarcinoma

1846903 1264 Colon0237696 1436 Small intestine

Note. i indicates LifeSeq ESTs (InCyte Pharmaceuticals) while theingle EST from dbEST is annotated by its Genbank accession num-er. 59 end denotes the beginning of the EST clone sequence witheference to the cDNA sequence in Fig. 1.

K3morgpn

lsstfoscpNwIc

ct

INrLla(w

b(smic

fit

a[togal(f7


SGal6ST, the similarity is 45.9% and identity is5.8%. The protein is predicted to be a type II trans-embrane protein with an N-terminal cytoplasmic tail

f nine residues and a transmembrane domain of 18esidues. There are three potential sites of N-linkedlycosylation. Based on the functional characterizationresented below, we term the enzyme encoded by thisovel gene as intestinal-GlcNAc6ST or I-GlcNAc6ST.Two of the three murine BACs contained the same

ong open reading frame of 1188 bp (GenBank Acces-ion No. AF176841). Using this sequence, we re-creened GenBank for matching ESTs and identifiedwo ESTs (Accession Nos. AI115260 and AI156825)rom whole mouse embryo. Both mapped to the 59 endf the predicted ORF derived from the BACs. Furtherequencing revealed that both ESTs were identical,ontaining the entire ORF plus a 39 extension to aoly(A) tail. This cDNA sequence (GenBank Accessiono. AF176840) predicts a protein of 395 amino acidshich is 80% similar and 76% identical to human

-GlcNAc6ST (Fig. 1). Though not proceeded by a stopodon, the first in-frame ATG in the mouse cDNA was

FIG. 3. Sulfation of GlyCAM-1/IgG conferred by I-GlcNAc6ST. Csecond plasmid encoding either human I-GlcNAc6ST or the lac

35S]sulfate and recombinant GlyCAM-1/IgG was isolated from the che captured material was subjected to SDS–PAGE and the remaindf recombinant GlyCAM-1/IgG isolated from COS cell transfectants.el (bottom panel) are shown. Densitometric quantification of thpproximately equal amounts of GlyCAM-1/IgG. (B) Analysis of sulfacZ. Sulfated mono- and disaccharides derived from [35S]sulfate labelF) or lacZ (E), as described above, were analyzed by high pH anion eollows: 1, GlcNAc-3SO3

2; 2, [35S]-SO422; 3, Galb1 3 4[SO3

2 3 6]G, GlcNAc-6SO3

2; 8, Gal-6SO32.

547

onsidered the true start codon for the reasons men-ioned above.

Expression of I-GlcNAc6ST. The expression of-GlcNAc6ST in human tissues was investigated byorthern analysis. As shown in Fig. 4, transcripts cor-

esponding to this gene were absent from most tissues.ow levels of a 2.8-kb transcript were apparent in fetal

iver. A prominent band of the same size was detectedt relatively high levels in colon and small intestineFig. 2). Transcripts of larger size (3.5, 4, 5, and 8 kb)ere also detected in these latter samples.The Northern analysis is consistent with the distri-

ution of ESTs in the NCBI and InCyte databasesTable 1). The only normal tissues that yielded corre-ponding ESTs were small intestine and colon. ESTsapping to the human I-GlcNAc6ST cDNA were also

dentified from several different tumors, including aolon adenocarcinoma.

Sulfation of GlyCAM-1 by I-GlcNAc6ST. To con-rm that I-GlcNAc6ST encoded a sulfotransferase, weransfected COS cells with a cDNA encoding GlyCAM-

cells were transfected with a plasmid encoding GlyCAM-1/IgG androtein (mock). Transfected cells were cultured in the presence ofitioned medium by passage over protein A–agarose. One percent ofas processed for analysis of sulfated carbohydrate. (A) SDS–PAGE

e autoradiograph (top panel) as well as the Coomassie blue-stainedoomassie blue-stained bands showed that both lanes contained

d carbohydrate in GlyCAM-1/IgG coexpressed with I-GlcNAc6ST orGlyCAM-1/IgG produced by COS cells transfected with I-GlcNAc6STange chromatography after acid hydrolysis. The standards were asAc; 4, [SO3

2 3 6]Galb1 3 4GlcNAc; 5, Gal-4SO32; 6, Gal-3SO3

2;

OSZ ponder wThe CateedxchlcN

1/IgG chimera and the human I-GlcNAc6ST cDNA.TpcpmiecfaoirArsGti

ahpFpGe

D

efmcsKtmbctftspiwtlpomguf

KhweItesghrswisaFtr

fbtgf

ctsssbao


he latter cDNA was obtained as a PCR product am-lified from a restriction fragment of one of the BAClones (see Methods). The cDNA extended 63 baseairs upstream from the predicted start codon and ter-inated immediately after the stop codon. GlyCAM-1

s one of the mucin-like ligands for L-selectin that arexpressed in HEV. We have previously used an IgGhimera of GlyCAM-1 to characterize the sulfotrans-erase activities of KSGal6ST, HEC-GlcNAc6ST (10)nd GlcNAc6ST (Hemmerich and Rosen, unpublishedbservations). The transfected COS cells were culturedn the presence of [35S]sulfate. GlyCAM-1/IgG was pu-ified from the conditioned medium on protein–agarose and analyzed by SDS–PAGE and auto-

adiography. As shown in Fig. 3A, there was sub-tantially elevated incorporation of [35S]sulfate intolyCAM-1/IgG when I-GlcNAc6ST cDNA was used in

he cotransfection compared to a control vector encod-ng the lacZ protein.

To establish the regiochemistry of the sulfation re-ction, radiolabeled GlyCAM-1/IgG was subjected toydrolysis and Dionex HPLC analysis according to ourreviously established procedures (18). As shown inig. 3B, transfection with I-GlcNAc6ST resulted inroducts that corresponded to [SO3 3 6]GlcNAc andalb1 3 4[SO3 3 6]GlcNAc. Thus, this enzyme wasstablished to be a novel GlcNAc-6-sulfotransferase.

ISCUSSION

We report here the cloning of a full-length cDNAncoding a novel human carbohydrate sulfotrans-erase. We also present the sequence of a highly relatedouse cDNA, which is likely to be the orthologue. The

loning was based on an EST approach in which wecreened for sequences related to HEC-GlcNAc6ST andSGal6ST. The predicted protein, I-GlcNAc6ST, is a

ype II transmembrane protein with a short cytoplas-ic tail, features that are typical of tyrosinyl and car-

ohydrate sulfotransferases (4). Consistent with theloning strategy, I-GlcNAc6ST is clearly a member ofhe Gal/GalNAc/GlcNAc 6-O-sulfotransferase (GST)amily (3). First, our activity analysis demonstratedhat the expressed protein catalyzes the addition ofulfate to C-6 of GlcNAc. Secondly, the sequence of theutative sulfotransferase domain (residues 41 to 390)s 35–56% identical to other members of the family,hereas its homology to other Golgi-associated sulfo-

ransferases of different carbohydrate specificities isess than 20% (3). Detailed inspection of the predictedrotein sequence of I-GlcNAc6ST reveals three regionsf high sequence conservation (50–95%) with the otherembers of the GST family (Fig. 4). Within these re-

ions, there are stretches of amino acids which arenique to the GST family and other regions which areound in all sulfotransferases.

548

Three other members of the GST family (C6ST,SGal6ST, and GlcNAc6ST) are widely expressed inuman tissues. However, I-GlcNAc6ST in commonith HEC-GlcNAc6ST, exhibits a very restricted tissuexpression. In human, the strongest expression of-GlcNAc6ST transcripts was observed in small intes-ine and colon. Further study is needed to determinexpression at the cellular level. In view of the expres-ion of HEC-GlcNAc6ST in peripheral lymphoid or-ans (lymph node HEV in mouse and tonsillar HEV inuman), one of the tissues of particular interest withespect to I-GlcNAc6ST is gut-associated lymphoid tis-ue. Selective expression of I-GlcNAc6ST in HEVould raise the possibility that this enzyme is involved

n the sulfation of L-selectin ligands. However, ithould be pointed out that glands of the gut elaboratenumber of mucins, many of which are sulfated (21).inally, members of the GST family have been showno be capable of sulfating GAG chains of proteoglycans,aising another potential function for I-GlcNAc6ST.I-GlcNAc6ST represents the 18th carbohydrate sul-

otransferase and the 5th member of the GST family toe molecularly cloned in mouse or human (3). Givenhe great diversity of sulfation modifications withinlycoconjugates, it is anticipated that other sulfotrans-erase genes remain to be identified. The identification

FIG. 4. Alignment of regions of high conservation among humanarbohydrate 6-sulfotransferases (Gal-6, GlcNAc-6 and GalNAc-6). Pro-ein sequences were aligned using the ClustalW algorithm (22). Blackhading indicates identity at that residue among at least four of theequences, gray shading indicates identity among three sequences orimilarity among three or more sequences. The regions indicated asinding sites for the 59 phospho-sulfate and 39 phosphate of PAPS weressigned based on their homologies to the equivalent regions defined inther sulfotransferases by Kakuta et al. (23).

of cDNAs encoding carbohydrate sulfotransferasessbt

A

HBtmtA

R

10. Bistrup, A., Bhakta, S., Lee, J. K., Belov, Y. C., Gunn, M. D., Zuo,

1

1

1

1

1

1

1

1

1

22

2

2


hould expedite rapid progress in the elucidation of theiological roles of specific sulfation modifications andhe contribution of particular enzymes to these functions.

CKNOWLEDGMENTS

This work was supported by grants from the National Institutes ofealth (Merit Awards R37GM23547 and RO1GM5741) and Rocheioscience to S.D.R. The LifeSeq EST database (InCyte Pharmaceu-

icals, Inc., Palo Alto, CA) was accessed through a licensing agree-ent with Roche Bioscience. Jin Kyu Lee is supported by a postdoc-

oral fellowship from the Arthritis Foundation. We thank Dr.nnette Bistrup for many helpful discussions.

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Cloning and Characterization of a Mammalian N-Acetylglucosamine-6-sulfotransferase That Is Highly...

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