THE JOURNAL OF BIOIDGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 14, Issue of April 8, PP. 10436-10443, 1994 Printed in U.S.A.

cDNA Cloning and Sequencing of Mouse Mastocytoma Glucosaminyl N-DeacetylaselN-Sulfotransferase, an Enzyme Involved in the Biosynthesis of Heparin*

(Received for publication, November 8, 1993, and in revised form, December 30, 1993)

Inger Eriksson, Dagmar SandbackS, Bo Ekl, Ul f LindahlS, and Lena yjellhn From the Department of Veterinary Medical Chemistry and the $Department of Cell Research, Swedish University of Agricultural Sciences, Box 7055, S-750 07 Uppsala, Sweden and the Wepartment of Medical and Physiological Chemistry, University of Uppsala, The Biomedical Centel; Box 575, S-75123 Uppsala, Sweden

A 110-kDa protein involved in heparin biosynthesis in mouse mastocytoma cells was previously shown to ex- press both glucosaminyl N-deacetylase and N-sulfo- transferase activity. In this study, the complete nucle- otide sequence corresponding to this protein is reported. The mF?NA, estimated to contain 3.9 kilobases encodes a protein with an Mr of 101,092. The predicted domain structure of the protein resembles those of pre- viously characterized Golgi proteins with an N-terminal cytoplasmic tail, a single membrane-spanning domain, and a large catalytic domain linked to the transmem- brane domain through a “stem region.” Comparison of the deduced amino acid sequence of the mouse masto- cytoma protein and a previously cloned similar enzyme from rat liver demonstrated that while large portions of the proteins, corresponding essentially to the putative catalytic domains, were closely related, other portions, in particular in the N-terminal parts, were markedly different. The divergence was not due to species differ- ences since two separate mouse transcripts could be identified that hybridized with probes specific for the two proteins. Also, functional differences were noted since the mastocytoma enzyme, contrary to the liver en- zyme, requires a polycation cofactor for expression of N-deacetylase activity. The results are discussed in re- lation to the structural properties of heparin and hepa- ran sulfate.

The biosynthesis of heparin and heparan sulfate is initiated by glycosylation reactions that generate saccharide sequences composed of alternating D-glucuronic and N-acetylglucosamine units (Lidholt and Lindahl, 1992). The resulting (GlcAp1,4- GlcNAcal,4-),’ disaccharide repeats are modified through a series of reactions that is initiated by N-deacetylation of N- acetylglucosamine residues. The generated free amino groups are then sulfated through the action of an N-sulfotransferase.

ish Medical Research Council, Grant BMH1-CT92-1766 from the Euro- * This work was supported by Grants 2309 and 6525 from the Swed-

pean Economic Community, and grants from Konung Gustaf Vs 80- Arsfond; the Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences; Italfarmaco S.p.A., Milan, Italy; and Polysacka- ridforskning AB, Uppsala, Sweden. The costs of publication of this article were defrayed in part by the payment of page charges. This

with 18 U.S.C. Section 1734 solely to indicate this fact. article must therefore be hereby marked ”advertisement” in accordance

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTMIEMBL Data Bank with accession number(s) X75885.

ll To whom correspondence should be addressed. Tel.: 46-18-174217;

The abbreviations used are: GlcA, D-glucuronic acid; MES, 2-(N- morpho1ino)ethanesulfonic acid; kb, kilobaseb); PCR, polymerase chain reaction; bp, base pairb); Mops, 4-morpholinepropanesulfonic acid.

Fax: 46-18-550762.

The subsequent modifications, involving 0-sulfation at various positions and C5-epimerization of D-glucuronic acid to L-idu- ronic acid, all occur in the vicinity of N-sulfate groups. Hence, N-deacetylationlN-sulfation has a key role in determining the extent of modification of the polysaccharide chain (Lindahl, 1989). Heparin, produced by connective tissue-type mast cells, is extensively N-sulfated (generally >80% of the GlcN units), whereas heparan sulfates, derived from virtually all other types of cells, usually show approximately equal amounts of N-acetylated and N-sulfated GlcN residues. Accordingly, hepa- rin contains more L-iduronic acid and 0-sulfate than heparan sulfate, as predicted from the substrate specificities of the cor- responding enzymes (Lindahl, 1989). However, why is N-deacetylationlN-sulfation more extensive in the mast cells than in other cells?

Proteins with glucosaminyl N-sulfotransferase activity have been purified from (heparan sulfate-producing) rat liver (Bran- dan and Hirschberg, 1988) as well as (heparin-producing) mouse mastocytoma (Pettersson et al., 1991). The cDNA- sequence and the deduced amino acid sequence of the rat liver protein have been published (Hashimoto et al., 1992). The mas- tocytoma protein was shown to catalyze also N-deacetylation, but only in the presence of an unidentified protein cofactor (Pettersson et al., 1991). While these results suggested that the two enzyme activities reside in the same protein, they did not exclude that the unidentified “cofactor” would actually harbor the active site for N-deacetylation (Pettersson et al., 1991). Recently, Wei et al. (1993) demonstrated that a purified soluble fusion protein containing the Golgi lumenal portion of the rat liver N-sulfotransferase expressed both N-deacetylase and N- sulfotransferase activity (see also Ishihara et ul. (1993)), thus suggesting that this protein contained both active sites.

This report describes the cDNA sequence and the deduced amino acid sequence for the mouse mastocytoma protein. Re- sults are presented that indicate that the liver and the masto- cytoma N-deacetylaselN-sulfotransferases are closely related but distinct proteins.

MATERIALS AND METHODS Peptide Purification and Sequencing-The 110-kDa protein (-20 pg),

purified from a detergent extract of mouse mastocytoma by affinity chromatography on wheat germ agglutinin-Sepharose, blue Sepharose, and 3’,5‘-ADP-agarose as previously described (Pettersson et al., 1991) was cleaved with a lysine-specific protease from Acromobacter lyticus (Waco) in the presence of 2 M guanidine HCI. The generated peptides were separated on a reverse phase C4 column (Brown-Lee) eluted at a flow rate of 100 pVmin with a 6-ml2-60% acetonitrile gradient in 0.1% trifluoroacetic acid and detected with a 990 Waters diode-array detector. Selected peptides were then analyzed with a model 470A protein se- quenator (Applied Biosystems) equipped with an on-line 120 phenyl- thiohydantoin Analyzer.

Probes for Screening-Single-stranded cDNA was synthesized using 1 pg of total mouse mastocytoma RNA in a 20-pl reaction mixture

10438

N-Deacetylase IN-Sulfotransferase cDNA Sequence 10439

containing 10 n" Tris-HC1, pH 8.3, 50 m~ KCl, 5 n" MgCh, 1 n" of each deoxynucleotide, 1 unit of RNase inhibitor (Perkin-Elmer Corp.), 2.5 p~ oligo(dT), and 1.25 units of murine leukemia virus reverse tran- scriptase (Perkin-Elmer Corp.). The reaction mixture was incubated for 42 "C for 15 min, a t 99 "C for 5 min, and finally a t 5 "C for 5 min. The sense and antisense degenerate oligonucleotides 1s and lA correspond- ing to peptide 1 (see Table I) were synthesized using a Pharmacia LKB Gene Assembler Plus. A 100-pl PCR mixture containing 10 pl of the single-stranded cDNA, 100 pmol of the oligonucleotides 1s and lA, 10 n" Tris-HC1, pH 8.3, 50 m~ KCl, 2.5 m~ MgCl,, 1 m~ of each de- oxynucleotide, and 1.25 units of Taq polymerase was incubated for 35 cycles in a thermal cycler (Gene ATAQ controller; Kabi-Pharmacia AB). Each cycle included denaturation at 95 "C for 1 min, annealing at 46 "C for 30 s, and extension at 70 "C for 1 min. The reaction products were separated on 12% polyacrylamide gels and a 48-bp band was cut out from the gel. After incubation of the gel slice in sterile H,O at 4 "C overnight, the eluted 48-bp product was reampliiied using the same conditions. After an additional polyacrylamide gel electrophoresis, the 48-bp product was isolated and 5'-end 32P-labeled using [Y-~~PIATP (DuPont NEN) and polynucleotide kinase (New England Biolabs).

Insert DNA of the 1.7-kb clone (see below) was 32P-labeled using [GI-~'P]~CTP (DuPont NEN) in the RPN.1601Y multiprime DNA label- ing system, obtained from Amersham International.

cDNA Libraries-Library 1 is a previously described A gtll mouse mastocytoma cDNA library (&ell& et al., 1989) comprising 0.7 x 10' independent recombinants. Library 2 was constructed using mouse mastocytoma polyadenylated RNA as template for cDNA synthesis in the cDNA synthesis kit from Pharmacia LKB Biotechnology Inc. The resulting cDNA was ligated to EcoRI-digested alkaline phosphatase- treated bacteriophage A g t l l DNA obtained from Pharmacia LKB Bio- technology Inc. Bacteriophage DNA was packaged using the Packagene system from Promega. Library 2 contains 4 x lo6 independent clones.

Screening of the cDNA Libraries-Nitrocellulose replicas of plaques from the bacteriophage A g t l l mouse mastocytoma cDNAlibraries were prehybridized in solution containing 6 x SSC (1 x SSC is 0.15 M NaCl, 0.015 M sodium citrate buffer, pH 7.0), 3 x Denhardt's solution, 2 m~ EDTA, 0.5% SDS, and 0.1 mg of denatured salmon D N N d for 3 h at 65 "C before hybridization for 12-15 h in the same buffer containing 32P-labeled probe. The filters were washed in 2 x SSC, 0.5% SDS at 42 "C and subsequently at 65 "C. When the 1.7-kb fragment was used as a probe, the filters were finally washed with 0.2 x SSC, 0.5% SDS at 65 "C.

Subcloning and Sequencing of cDNA Inserts-DNA inserts, isolated by preparative agarose gel electrophoresis (Sambrook et al., 1989) after EcoRI restriction cleavage of recombinant bacteriophage DNA, were subcloned into pUC 119 plasmid. The complete nucleotide sequence was determined independently on both strands using the dideoxy chain termination reaction with [GI-~SI~ATP and the modified T7 DNA po- lymerase (Sequenase, U. S. Biochemical Corp.) (Tabor and Richardson, 1987). The sequencing primers used were universal oligonucleotide primers, which bind close to the polylinker of pUC 119, and specific primers synthesized on the Gene Assembler Plus.

Generation of a Fusion Protein and Peptide Conjugates for Immunization-The insert from the 1.7-kb cDNA clone was ligated to the bacterial expression vector pGEX-3X (Pharmacia LKB Biotechnol- ogy), and transformed into JM 83 bacteria. The resulting fusion protein contains the C terminus of a 26-kDa glutathione S-transferase encoded by the parasitic helminth Schistosoma japonicum. The fusion protein is under control of the tac promoter, which is inducible by isopropyl-l- thio-0-D-galactopyranoside. After induction, the bacteria were lysed, and the solubilized proteins were purified by affinity chromatography on a glutathione-agarose column (sulphur linkage, Sigma). The fusion protein was eluted by competition with 5 n" free glutathione (Sigma) in 50 m~ Tris-HCI, pH 8.0 (Smith and Johnson, 1988). Synthetic peptides corresponding to peptides 1 and 2 (see Table I) were conjugated to bovine serum albumin with glutaraldehyde (Harlow and Lane, 1988). The fusion protein and the two peptide conjugates were homogenized in Freund's complete adjuvant before injection into rabbits.

SDS-Polyacrylamide Gel Electrophoresis and Immunoblotting- Purified 110-kDa protein and a crude mastocytoma microsomal fraction were subjected to polyacrylamide gel electrophoresis in SDS on 10-15% gradient gels according to the method of Blobel and Dobberstein (1975). Separated proteins were transferred to nitrocellulose filters using semi- dry electrophoretic transfer according to Kyhse-Andersen (1984). The filters were then blocked in 5% nonfat dry milk and incubated with 1/50 dilutions of the different sera. After washing, the specifically bound antibodies were allowed to react with 1261-labeled Protein A. The anti- gens recognized were detected by autoradiography.

TABLE I Peptide and primer sequences

A. Peptide

1s 1A 1. KFYHTGTEEEDAGDDM 2. KYFELFPQERSPL 3. KIIXVLINPADRAY 4. KGFWCQGLEGG

B. Rimer" Degeneracy

1s. 5"AARTTYTAYCAxACNGGNAC-3' x256 1A. St-CATRTCRTz?CCNGCRTc-3' " " x128

" R , A o r G x , T o r C ; N , A o r G o r T o r C .

RNA Purification--Total RNA was isolated from mouse mastocy- toma, rat liver, and mouse liver by the LiCVuredSDS method (Sam- brook et al., 1989). Polyadenylated RNA was purified using the poly- ATtract kit from Promega.

PCR of Rat Liver cDNA-A sense and an antisense primer corre- sponding to bp 3195-3216 and 3682-3661, respectively, in the rat liver cDNAsequence (Hashimoto et al., 1992) were synthesized. -To the 5'-end of the primers was added a dCCGAAlTC (where d is deoxy) extension to create an EcoRI site in the PCR product. For synthesis of single- stranded rat liver cDNA, 300 ng of total rat liver RNA in 10 n" Tris- HCl, pH 8.3,90 m~ KC1 was incubated at 70 "C for 5 min together with 0.5 p~ "downstream" antisense primer. After addition of MnC1, (final concentration 1 m ~ ) , the 4 deoxynucleotides (final concentration 0.2 m) and 5 units of thermostable rTth reverse transcriptaselDNA po- lymerase (Perkin-Elmer Corp.), the 2 0 4 reaction mixture was incu- bated at 60 "C for 15 min. The cDNA was amplified using 35 cycles of incubation aRer addition of 80 pl of 5% glycerol, 10 n" Tris-HC1, pH 8.3, 0.1 M KCl, 1.5 m~ MgCl,, 0.75 m~ EGTA, 0.05% Tween 20, and 0.1 p~ "upstream" sense primer. Each cycle included denaturation at 95 "C for 1 min, annealing at 64 "C for 1 min, and extension at 72 "C for 1 min. After a final 10-min incubation at 72 "C, the 504-bp product was recov- ered by 2% agarose electrophoresis, cleaved with EcoRI, and subcloned into pUC 119 plasmid for nucleotide sequence analysis.

Northern Blot Hybridization-Polyadenylated RNA from mouse mas- tocytoma, mouse liver, and rat liver was denatured in 20 n" Mops buffer, pH 7.0, containing 50% (v/v) formamide and 2.2 M formaldehyde at 70 "C for 3 min before being loaded on 1.2% (vh) formaldehyde- containing agarose gels. ARer electrophoresis, the gel was soaked in 50 m~ NaOH for 30 min and rinsed with water and 20 x SSPE ( 1 x SSPE is 0.15 M NaCl, 10 n" NaH,PO,~H,O, 1 m~ EDTA, pH 7.4) before transfer to the nylon membrane, Hybond N' (Amersham Corp.), in 20 x SSPE. The membranes were hybridized at 50 "C in a solution contain- ing 5 x SSPE, 5 x Denhardt's solution, 0.5% SDS, 100 pg of denatured salmon DNA/ml and washed in 2 x SSC, 0.5% SDS at 50 "C for 2 x 20 min then at 65 "C for 2 x 20 min.

The probes used were: (a) the 1.7-kb clone, (b) a 356-bp cDNAfrag- ment from the 3'-end of the mouse mastocytoma cDNA (mouse masto- cytoma probe), generated from pUC 19 containing the 5B clone a h r cleavage with EcoRI (cleavage site in the polylinker) and BamHI (cleav- age site a t bp 2941, see Fig. 3), and ( c ) a 284-bp cDNAfragment from the 3'-noncoding part of the rat liver cDNA (rat liver probe) corresponding to bp 3396-3679 (see Hashimoto et al. (1992)). This fragment was gen- erated by cleavage with Hinff of the 504-bp rat liver cDNAPCR product obtained as described above. The three cDNA fragments were 32P- labeled with the multiprime DNA labeling system described above.

N-Deacetylase Assay-Purified mastocytoma N-deacetylase (110-kDa protein) was incubated at 37 "C with 10,000 cpm of substrate ([3Hlacetyl-labeled Escherichia coli K5 capsular polysaccharide) in a total volume of 200 pl of 50 m~ MES, pH 6.3, 10 m~ MnCI,, 1% Triton X-100. Polybrene (Janssen Chimica, Belgium; synthetic polycation) was added as indicated. After 1 h, reactions were terminated by addition of 200 pl of 1 M monochloroacetic acid, 0.5 M NaOH, 2 M NaCl. The released [3Hlacetate was determined by scintillation counting in a biphasic sys- tem (for a more detailed description see Navia et al. (1983) and Petters- son et al. (1991)).

-

RESULTS

Generation of a Probe and Screening of Library 1"TO obtain amino acid sequence data for the 110-kDa protein, highly pu- rified enzyme was digested with lysine-specific protease, and the generated peptides were separated on a reverse phase col-

N-DeacetylaselN-Sulfotransferase cDNA Sequence

FIG. 1. Nucleotide sequence and predicted amino acid me-

on the right and the left indicate the nucleotide residue and the amino quence of the 110-kDa mouse mastocytoma protein. The numbers

umn. Sequence was obtained from four peptides (Table I). A cDNA probe was produced by PCR using mouse mastocytoma cDNA and degenerated sense and antisense primers based on the sequence of peptide 1 (see Table I). The 48-bp product was purified by polyacrylamide gel electrophoresis, reamplified us- ing the same primers, and finally isolated after polyacrylamide gel electrophoresis. The 48-bp PCR product was labeled with 32P and used for screening of a mouse mastocytoma A g t l l library (library 1). One hybridizing clone, containing a 1.7-kb insert was identified. Nucleotide sequence analysis indicated that the insert corresponded to a region entirely within the coding part of the transcript. Peptides 1,2, and 3 (see Table I) were all identified in the deduced amino acid sequence.

The 1.7-kb fragment was subcloned into the procaryotic ex- pression vector PGEX-~X, and the resulting glutathione S-transferase fusion protein was used to produce a rabbit poly- clonal antiserum. Synthetic peptides with the structures of peptides 1 and 2 were also used for ~muniza t ion of rabbits after conjugation to bovine serum albumin. All three antisera recognized the 110-kDa protein in immunoblotting (data not shown), demonstrating that the 1.7-kb cDNA is derived from the transcript encoding the 110-kDa protein.

Characterization of cDNA Corresponding to the Entire Cod- ing Region-Screening of a larger mouse mastocytoma A g t l l library (library 2) using the 1.7-kb fragment as a probe identi- fied 13 additional clones. Based on restriction patterns using ApaI and PssI alone or in combination, two clones, 6B (1.3 kb) and 5B (1.75 kb), extending from the known sequence into the 5' and 3' directions, respectively, were selected for subcloning and sequence determination.

The cDNA sequence obtained comprises 3306 bp (Fig. 1). The first ATG, found at position 158, is preceded by a guanosine at position -3 and thus conforms to the consensus initiation se- quence (Kozak, 1989). The coding region contains 2646 bp en- coding a protein of 882 amino acids. Northern blot analysis of mastocytoma mRNA revealed a strongly predominant -3.9-kb transcript (Fig. 2).

Predicted Protein Structure-The mouse mastocytoma cDNA encodes an 882 amino acid protein with a predicted molecular mass of 101,092 daltons. The deduced primary structure con- tains regions that correspond to the four sequenced peptides (see Table I, Fig. 1). The predicted protein has typical features of a resident Golgi protein (see Machamer (1991)): a short N- terminal cytoplasmic tail (18 amino acids) followed by a mem- brane-spanning region (24 amino acids) and a large luminal domain. Seven potential N-glycosylation sites are present. At least some of these are likely to be glycosylated, since the mas- tocytoma enzyme binds to wheat germ agglutinin (Pettersson et al., 1991). In addition, the purified enzyme showed an apparent M, of 110,000 on SDS-polyacrylamide gel electrophoresis (Pet- tersson et al., 1991), -9 kDa larger than the estimated mass (see above), hence allowing for the presence of oligosaccharides.

Comparison with the Rat Liver N-Sulfotransferase- Recently, the cDNA sequence of a rat liver N-sulfotransferase was published (Hashimoto et at., 1992). Comparison of the pre- dicted amino acid sequences of this protein and the mouse mastocytoma enzyme reveals that the two proteins are closely related with 71% amino acid sequence identity (72% at the nucleotide level) (Fig. 3). However, the N-terminal regions (amino acids 1-81) are quite different (only 30% identity at the amino acid level and 46% at the nucleotide level). In addition,

acid residue in the respective sequence. The four sequenced peptides are underlined. Amino acids in the potential membrane-spanning do- main are shown in italics. The asparagine residues that may be glyco- sylated are marked by asterisks. The two potential polyadenylation signals in the 3'-noncoding region are underlined.

N-Deacetylase I N-Sulfotransferase cDNA Sequence 10441

kb

9.4- 6.2- 3.9- 2.8- 1.8-

0.9-

FIG. 2. Size of the mouse mastocytoma transcript. Polyadeny- lated RNA from the mouse mastocytoma was separated by agarose electrophoresis and hybridized with 32P-labeled 1.7-kb fragment as de- scribed under “Materials and Methods.” Sizes of RNA markers (Life Technologies, Inc.) are indicated in kilobases.

the 5’- and 3’-untranslated regions seem unrelated (both 37% identity). To investigate whether these discrepancies could be due to species difference, probes were produced that would hybridize with the 3’-ends of the rat liver and the mouse mas- tocytoma transcripts, respectively. Northern blotting of rat liver, mouse liver, and mouse mastocytoma mRNA, demon- strated that the mouse mastocytoma probe hybridized with the -3.9-kb band in mouse mastocytoma mRNA as expected (Fig. 4, compare with Fig. 3). Small amounts of this transcript, de- tected with the mouse mastocytoma probe after prolonged ex- posures, also occurred in rat liver and mouse liver mRNA (not evident in Fig. 4A). In contrast, the rat liver cDNA probe rec- ognized an 8-kb transcript in both rat and mouse liver mRNA, while no such band was apparent in the mouse mastocytoma mRNA preparation (Fig. 4). Thus, different transcripts appear to be utilized for the synthesis of the two proteins.

Effect of Polybrene on N-Deacetylase Activity-Purification of the mastocytoma N-deacetylaselN-sulfotransferase yielded a 110-kDa protein that expressed N-sulfotransferase activity, but lacked N-deacetylase activity unless supplemented with a crude protein fraction that was separated from the 110-kDa protein by chromatography on immobilized wheat germ agglu- tinin (Pettersson et al., 1991). Thus it could not be established whether the active site for N-deacetylation was actually located in the 110-kDa component or in one of the added proteins. I t was accidentally found that basic proteins, such as histones (data not shown) or, indeed, a synthetic polycation, Polybrene, could substitute for the crude mastocytoma protein fraction as a N-deacetylase “cofactor” (Fig. 5; see also Kjellen et al. (1992)). While no N-deacetylation occurred in the absence of Polybrene, enzyme activity was readily detected in the presence of this compound, with maximal effect at -25 pg/ml. At higher Poly- brene concentrations the stimulatory effect decreased. The mechanism behind the stimulation remains unclear. However, the findings clearly demonstrate that the catalytic sites for N-deacetylation and N-sulfation both reside in the 110-kDa

mouse 1 MLQLWKVVRPARQLELHRLILLLIGFSLVSMGFLAYYVSTSPKAKEPLPL rat 1 PA ACLR LC H SPQAVLF FV C F VFVS LYGWNRGL ....

51 PLGDCSSSGAAGPGPARPP..VPPRPQRPPETTRTEPWLVFVESAYSQL 47 SA A E DCGD P VA SRLL IK VQAVAPS D L L

99 G Q E I V A I L ESSRFRYSTELVFGRGDMPTLTDHTHGRWLVI 97 v K R IA K KGR FA I I

149 LDAWSRELLDRYCVEYGVGIIGFFRAREHSLLSAQLKGFPLFLHSNLGLR 147 N K A K N N K

199 DYQVNPSAPLLHLTRPSRLEFGPLFGDDWTIFQSNHSTYEPVLIASHRPA 197 CSI KS W EV K V E V L KT S S

249 ELSMP .... GPVLRRARLPTWQDLGLHDGIQRVLFGHGLSFWLHKLVFV 247 . I HLGADAG HA LHA NNLN

295 DAVAYLTKRLCLDLDRYILVDIDDIFVGKEGTRMKVADVEALLTTQNKL 296 F S P E K F D E

345 RTLVPNFTFNLGFSGKFYHTTEEEDAGDDMLLKHRREFWWFPHMWSHMQ 346 HI Y F DA L S W K

395 P.LFHNRSVLADQMRLNKQFALEHGIPTDLGYAVAPHHSGWP1HSQLYE 3 9 6 H Q E A K V M v v 444 AWKSVWGIQVTSTEEYPHLRPARYRRGFIHNGIMVLPRQTCGLFTHTIFY 446 Q N R K

494 NEYPGGSRELDRSIRGGELFLTVLLNPISVFMTHLSNYGNDRLGLYTFES 496 KI N KH

544 LVRFLQCWTRLRLQTLPPVPLAQKYFELFPQERSPLWQNPCDDKRHKDIW 546 HS N Q QI SE KD D E

594 SKEKTCDRLPKFLIVGPQKTTTAIHFFLSLHPAVTSSFPSPSTFEEIQF 596 F L I LYL GM DLS NY SE

644 FNGPNYHKGIDWYMDFFPVPSNASTDFLFEKSATYFDSEWPRRGAALLP 646 H E I W S Y N A A

694 RAKIITVLINPADRAYSWYQHQRAHGDPIALNYTFYQVISASSQAPLLLR 696 K VL I D V K HE T GPD SSK

744 SLQNRCLVFGYYSTHLQRWLTYYPSGQLLIMDGQELRVNPAASMEIIQKF 746 A WYA IE SAFHAN I VL KL TE KV DTV

794 LGITPFLNYTRTLRFDEDKGFWCQGLEGGKTRCLGRSKGRRYPDMDMESR 796 V STVD HK A PK L K K K E L D

844 LFLTDFFRNHNLELSKLLSRLGQPAPLWLREELQHSSVG 846 A K YY D I YKM TL T D NTR..

50 46

98 96

148 146

198 196

248 246

294 295

344 345

394 395

443 445

493 495

543 545

593 595

643 645

693 695

743 745

793 795

843 845

882 882

FIG. 3. Comparison of the deduced amino acid sequences of the mouse mastocytoma and rat liver enzymes. Only the amino acid residues in the rat liver N-sulfotransferase (Hashimoto et al., 1991) that differ from those in the mouse enzyme are shown. The numbers indicate the amino acid residues of the proteins. Gaps (.) inserted to optimize the alignment of the sequences were identified by the com- mand “BESTFIT” of the GCG program (Devrew et al., 1984).

protein. Similar conclusions have been put forward with regard to the rat liver enzyme (Wei et al., 1993). By contrast, the N-deacetylase activity of the latter enzyme did not depend on the presence of additional polycation (in support of the notion that the mastocytoma and liver enzymes are not only structur- ally but also functionally different).

DISCUSSION

Current notions on the structural organization of Golgi pro- teins derive largely from studies on glycosyltransferases (Jozi- asse, 1992; Shaper and Shaper, 1992). These enzymes all show the orientation of type I1 membrane-bound proteins (see Wick- ner and Lodish (1985)), with the N-terminal regions projecting into the cytoplasm. The short cytoplasmic tail is positively charged and is followed by a single membrane-spanning do- main. The large catalytic domain, which includes the C termi- nus, is linked to the transmembrane domain through a peptide segment (“stem region”) composed of 40-60 amino acid resi- dues. The stem region is flexible and largely devoid of second- ary structure. It contains a high proportion of proline and glycine residues and often consensus sites for N-linked carbo- hydrate side chains. Recent studies on the targeting of Golgi proteins (reviewed in Machamer (1991) and in Shaper and Shaper (1992)) suggested that, while sequences located within the transmembrane domains appear to be essential for Golgi retention, the cytoplasmic tail and the stem region may contain

10442 N-Deacetylase IN-Sulfotransferase cDNA Sequence

a b c d e f

9.4- 6.2- 3.9-

2.8

1.8

0.9- 0.6-

FIG. 4. Northern blot analysis with probes specific for the mouse mastocytoma and rat liver transcripts. Polyadenylated RNA from rat liver (a and d) , mouse liver ( b and e ) , and mouse masto- cytoma tissue (c and f ) was separated by agarose electrophoresis and hvbridized with 32P-labeled mouse mastocvtoma urobe (a-c:) and rat lke r probe (d-f), recognizing regions in thk 3’-noncoding parts c transcripts (see ‘Materials and Methods”). Sizes of RNA markers Technologies, Inc.) are indicated in kilobases.

50 100 154

Polybrene concentration lg/ml

If t (L

he i fe

FIG. 5. N-Deacetylase activity as a function of Polybrene con- centration. Purified mouse mastocytoma 110-kDa protein (-40 ng) was assayed for N-deacetylase activity as described under “Materials and Methods” in the presence of increasing concentrations of Polybrene. The given values represent the mean of two determinations.

information of importance in this regard. The deduced amino acid sequence of the mouse mastocytoma

N-deacetylaselN-sulfotransferase implies a protein domain structure similar to that ascribed to the glycosyltransferases. The putative transmembrane domain of 24 amino acid residues (Fig. 1) is preceded by an N-terminal region of 18 residues that contain 4 positively charged (1 lysine and 3 arginine) amino acids and 1 negatively charged (glutamic acid). The postulated stem region contains a large proportion of proline (30% of the

first 40 amino acids C-terminal to the transmembrane domain), whereas the glycine content is moderate (10%). No potential N-glycosylation site is seen within this region.

A comparison of the deduced amino acid sequence of the mouse mastocytoma N-deacetylaselN-sulfotransferase with that of the corresponding rat liver enzyme reveals close homol- ogy between large portions of the two proteins (Fig. 3). How- ever, the N-terminal regions comprised by the 81 first amino acid residues (corresponding to the cytoplasmic tail), the trans- membrane domain, and 39 amino acid residues of the stem region appear unrelated. Analogous variants, with closely simi- lar catalytic domains but discrepant N-terminal regions, have been noted also- for certain glycosyltransferases (see Joziasse (1992)). In the case of a murine al,3-galactosyl transferase gene, it has been determined that the transition from homolo- gous to unrelated regions coincides with the boundary between two exons (Joziasse et al., 1992). I t was suggested that a portion of an ancestral gene, encoding much of the catalytic domain of the enzyme, had been reutilized in a number of genes (Joziasse, 1992). The relationship at the gene level between the mouse mastocytoma and the rat liver N-deacetylaselN-sulfotrans- ferases remains to be elucidated. However, Northern blots us- ing probes based on selected divergent nucleotide sequences indicated that the two enzymes are encoded by transcripts of markedly different size (Fig. 4). Moreover, both of these tran- scripts could be demonstrated in mouse tissues, thus excluding simple species difference. The two transcripts may represent the products of two different genes or differently spliced vari- ants originating from the same gene. However, since differ- ences were found at two distantly located parts of the tran- scripts, the latter possibility is less likely.

The structural divergence between the two N-deacetylaselN- sulfotransferases primarily involves regions of the proteins that have been implicated in intracellular targeting (see above), and it is, therefore, conceivable that the enzymes are differently located within the Golgi network. However, the pre- sent work also revealed a more clearcut, functional difference. The mastocytoma enzyme expresses N-deacetylase activity, but only in the presence of a macromolecular cofactor (presumably a protein in the intact cell (Pettersson et al., 1991) that can be replaced by a synthetic polycation (Polybrene) in a purified system (Fig. 5; see also Kjell6n et al. (1992)). Experiments along the same line demonstrated, on the other hand, that the re- combinant rat liver protein does not require any such cofactor in order to catalyze N-deacetylation and that the activity is insensitive to the addition of Polybrene (Wei et al., 1993). It is recalled that the mast cell N-deacetylaselN-sulfotransferase initiates the series of polymer modification reactions that ulti- mately yields heparin, a heavily N- and 0-sulfated polysaccha- ride with few residual, usually isolated, N-acetyl groups. The liver enzyme (and, presumably, analogous enzymes in most other cell types) is involved in the biosynthesis of heparan sulfate, which generally contains extended regions of consecu- tive, N-acetylated disaccharide units. I t is tempting to specu- late that the demonstrated distinctive features of the isolated enzymes entail different functional properties in the intact cell that are ultimately reflected in the structural characteristics of heparin versus heparan sulfate.

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