Nucleotide Sequence of cDNA Containing the Complete Coding

THEJOURNALOF

Vol. 261. No. 1, Issue of January 5, pp. 5053,1986 Printed in U.S.A.

BIOLOGICAL CHEMISTRY

Nucleotide Sequence of cDNA Containing the Complete Coding Sequence forHuman Lysosomal Glucocerebrosidase* (Received for publication, June 17, 1985)

Shoji Tsuji, PrabhakaraV. Choudary, BrianM. Martin, Suzanne Winfield, JohnA. Barranger, and Edward I. Ginns From the Molecular and Medical GeneticsSection, Developmental and Metabolic NeurologyBranch, Intramural Research Program, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892

ComplementaryDNA clones for human glucocere- resolution in situ hybridization (10) and have subsequently brosidase were isolated from a human hepatoma li- isolated full length genomic clones for human glucocerebrosbrary in Xgtll. The complete nucleotide sequence of idase (11). In this communication, we describe the characterthe 1805-base pair cDNA insert has been determined. ization, including the complete nucleotide sequence, of cDNA In addition to 5’ and 3’ untranslated regions (51 and containing the complete coding sequence of the structural 206 base pairs, respectively), the cDNA insert contains protein for human glucocerebrosidase. 1548 base pairs that completely encode human glucocerebrosidase. All possible N-linked glycosylationsites EXPERIMENTAL PROCEDURES are identified. Examination of the 19 amino acids of Materials-Restriction endonucleases, reagents for dideoxy sethe leader polypeptide beginningwith the ATG at position 52 revealed a hydrophobic core and a carboxyl-quencing, T4DNA ligase, the Klenow fragment of DNA polymerase terminal glycine at the peptidase cleavage site, fea- I, as well as pBR322, M13mp18, and M13mp19 were obtained from either Bethesda Research Laboratories or New England Biolabs, tures consistent with the leader sequences described Beverly, MA. Polynucleotide kinase and reagents for Maxam-Gilbert Mr of sequencing were from DuPont-New England Nuclear, Boston, MA. forother human translocatedproteins.The 57,000 calculated from the 516 amino acids deduced The pUC13 was from P-L Biochemicals. Nitrocellulose membranes from cDNA sequence is in good agreement with that (BA 85) were from Schleicher and Schuell. [y3’P]ATP, [a-32P]dATP, identified by immunoprecipitation following in vitro and [a-?3]dATP were either from Amersham Corp. or DuPont-New England Nuclear. translation of humanplacental mRNA.

Glucocerebrosidase (EC 3.2.1.45) is a membrane-bound lysosomal enzyme whichcatalyzes the hydrolysis of glucocerebroside (1).Deficiency of this enzyme activity results in the sphingolipidosis known as Gaucher disease (2). On the basis of clinical signs and symptoms, this disease has been divided into three major phenotypes: Type 1 (chronic, non-neuronopathic), Type 2 (acute neuronopathic), and Type 3 (chronic neuronopathic) (2). Polymorphism of the cross-reactive material to glucocerebrosidase has allowed the identification of these phenotypes by Western analysis (3, 4). The polymorphism in the molecular weights of the protein in the 3 major phenotypes is the result of alteration of normal synthesis and/ or processing. Along withother data (5, 6) showing noncomplementation of phenotypes in cell hybrids, it is suggested that in Gaucher disease several different allelic mutations occur in thegene for glucocerebrosidase. However, the precise nature of the various mutations in the gene for glucocerebrosidase that result in the different types of Gaucher disease has not yet been determined. In order to betterunderstand the molecular genetics of this disease and to bring the possibility of gene transfer closer to a testable hypothesis, a cDNA clone for human glucocerebrosidase has been isolated in our laboratory (7) from a hepatoma library in Xgtll (8, 9). Using this cDNA as the probe, we determined the chromosomallocus of the gene codingforglucocerebrosidase (chromosome lq21) by high * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Preparation of cDNA Insert-The isolation of two cDNA clones for human glucocerebrosidasewas reported previously (7). The inserts of these X g t l l cDNA clones were further subcloned into pBR322 and pUC13, and thecDNA insert for glucocerebrosidasewas purified from EcoRI digests of the recombinants by preparative agarose gel electrophoresis (12). Sequence Analysis of the cDNA-The DNA sequence wa8 determined either by the dideoxynucleotide chain terminator method (13, 14) or by the method of Maxam and Gilbert (15). Plaque Hybridization-The cDNA insert was labeled with [a-32P] dCTP (3000 Ci/mmol) by nick translation. Phage were plated on Escherichia coli Y1088 and screened with nick-translated cDNA as described (12). Computer Analysis-DNA and protein sequences were analyzed using the programs, DNA/Protein Sequence Analysis Software and MicroGenie, from International Biotechnologies, Inc., New Haven, CT andBeckman Instruments., respectively. RESULTSANDDISCUSSION

The cDNA inserts of pBR322 subclones pGCl and pGC2 (derived from XGCl and XGC2 clones, respectively) are in opposite orientations but are both1.8 kilobase pairs long and hybridize with each other under stringent conditions. In addition, restriction endonuclease maps as well as 5’ and 3’ end terminal sequence of the cDNA frompGCl andpGC2 are the same. Since these findings demonstrated that these two clones were probably identical, further analysis was performed using the cDNA insert of pGC1. Fig. 1 shows the strategy for sequencing the cDNA insert of pGC1. Both strands were sequencedby the dideoxynucleotide chain terminator method (13, 14) using overlapping cDNA fragments. In theregion wherethere was ambiguity in the reading of the sequencing gel bythis method, the sequence was further confirmed by the method of Maxam and Gilbert (15).

50

Human Glucocerebrosidase cDNA Clone and Sequence

51

""FIG.1. Restriction map and sequencing strategy for theglucocerebrosidasecDNA clone pGC1. The positions of the restriction sites used in sequencing are indicated. The scale is in base pairs and begins with the first residue of the ATG coding for the initiation methionine. The solid box indicates the sequence coding for mature glucocerebrosidase.The cross-hatched box indicates the sequence coding for the leader polypeptide.Arrows indicate the direction and the extent of each seauence determination. The sequences determined by the method of Maxam-Gilbert are shownby dashed arrows. "

The sequence of the cDNA in pGC1 is shown in Fig.2 and contains only one open reading frame adequate to code for human glucocerebrosidase.This reading frame codes for 516 amino acids starting from the first ATG at position 52 and terminated byTGA at position 1600. In this open reading frame, an adenine occurs three residues before this ATG codon, and a guanine follows the ATG, one of the most common functional initiator codon patternsin eukaryotic mRNA (16). There is, however, an additional ATG out of frame from that of the glucocerebrosidase-coding region at position 21, followed 18 bases downstream by a termination codon. The M,calculated from these 516 amino acids is 57,000 and is in close agreement with the molecular weightof human glucocerebrosidase observedby immunoprecipitation following in vitro translation of human placental mRNA in a rabbit reticulocyte system (17,18). Although there are 206 nucleotides at the 3' untranslated end, this cDNA for glucocerebrosidaselacks both a consensus polyadenylation signal (AAUAAA) and a poly(A) tail. The poly(A) tail that would be expected 20-30 nucleotides downstream of a polyadenylation signal was probably lost during cDNA synthesis inthe construction of theXgtll library. Although another polyadenylation signal (AAUACA) for eukaryotic mRNA has been described(19),suggesting that there may besome heterogeneity in the polyadenylation signal, this glucocerebrosidase cDNA lacks any of the described polyadenylation signal sequences. The amino acidsequencededucedfrom the nucleotide sequence is presented in Fig. 2. Except for a single residue, Leu at 259, the amino acids from position 1 to 497 showed complete identity with chemically determined amino acid sequence from tryptic and cyanogen bromide-generatedpeptides of human placental glucocerebrosidase.' The chemically determined amino acid at this position is phenylalanine.' This discrepancy between cDNAand protein sequence may represent a silent variation in sequenceamong normal populations. Also possible but less likely is the occurrence of different species of mRNAs in human hepatoma and placental tissues or the aberrant synthesis of cDNA by reverse transcriptase during the X g t l l library construction. The codon usage freB. M. Martin, unpublished experiments.

quency (Table I) for each amino acid is similar to that reported for other human genes (20). Placental glucocerebrosidase contains approximately7% carbohydrate, and the structureof these N-asparagine-linked oligosaccharides has been determined (21) by carbohydrate analysis. The enzyme contains high mannose-type oligosaccharides as well as triantennary and biantennary complextype oligosaccharides (21). The potential glycosylation sites (Asn-X-Ser/Thr) are identified in the nucleic acid sequence of cDNA for glucocerebrosidase. Of these five possible sites for N-linked glycosylation,quantitative carbohydrate analysis indicates that carbohydrate addition does not occur at all of these residues (21). In addition to the amino acid sequence of mature human glucocerebrosidase, the reading frame of the cDNA insert codes for 19 additional amino acids that occur unstream of the sequence coding for the amino terminus of the mature enzyme. This proenzyme leader polypeptide contains a hydrophobic core consisting of Gly-Leu-Leu-Leu-Leu and in addition has Gly at its carboxyl terminus (Fig. 3).These features are consistent with the properties of leader sequencesof other human-translocated proteins (22-24). Although unlikely,it is still possible that an ATG upstream to the 5' terminus of our cDNA initiates translationof glucocerebrosidase.Further information regarding this will be available upon completionof genomic sequencing. However,Erickson et al. demonstrated by pulse-chase studies (17, 18) that cotranslationally glucocerebrosidase losesa peptide of approximately 2 kDa. This is consistent with the cleavage of the 19-amino acidleader polypeptide deduced fromthe sequence of the human lysosomal glucocerebrosidasecDNA. The molecular cloning of the low abundance mRNA for lysosomalhydrolases has beendifficult. Fukushima et al. reported the abundance of a-fucosidase cDNA in the human hepatoma library to be only 0.0018% (9). Usingthe 32P-labeled nick-translated cDNA insert from subclone pGC1 we found the abundance of glucocerebrosidase cDNA clones to be 0.0004%. The hybridization to thepBR322 subclones (pGC1) of a synthetic oligonucleotide probe derived fromthe amino acid sequenceof homogenoushuman placental glucocerebrosidase (7) as well as oligonucleotideprimed sequencing initially confirmed the identity of the clone (7). Southern analysis of

Human Glucocerebrosidase cDNA Clone and Sequence

52

GAATTCCCTTCCAGAGAGGAATGTCCCAAGCCTTTGAGTAGGGTAAGCATC ATG GCT GGC AGC CTC ACA GGA TTGCTTCTACTT CAG OCA GTG TCG TGG GCA N e tA l aG l yS e rL e uT h rG l yL e u leu L e uL e uG l nA l aV a lS e rT r pA l a 19 -10

102

-

4

TCA GGT GCC CGC CCC TGC ATC CCTAAA AGC T T C GGC TAC AGC TCGGTGGTGTGTGTCTGCAAT S a rG l yA l aA r gP r oC y s I l e P r oL y sS e rP h eG l yT y rS e rS e rV a lV a lC y sV a lC y s -1 1 10

GCC ACA TACTGT GAC TCCTTT GAC CCC A n A l aT h rT y rC y sA s pS e rP h aA s pP r o

1)

192

2o

CCG ACC T T T CCT GCC CTT GGT ACC TTC AGC CGC TAT GAG AGT ACA CGC AGT GGG CGA CGG ATG GAG CTG AGT ATG GGG CCC ATC CAG GCT P r oT h rP h eP r oA l aL e uG l yT h rP h aS e rA r gT y rG l uS e rT h rA r gS a rG i yA r gA r gN e tG l uL e uS e rN e tG i yP r oI l eG l nA l a 30 40 50

282

AAT CAC ACG GGC ACA GGC CTGCTACTGACCCTGCAG CCA GAA CAG AAG TTC CAG AAA GTG AAG GGA T T T GGA GGG GCC A n H i s T h rG l yT h rG l yL e u l e u L a uT h rL e uG l nP r oG l uG l nL y sP h eG l nL y sV a lL y sG l yP h eG l yG l yA l aM e tT h rA s pA l a 70

372

4

ATG ACA GAT GCT

80

6o

GCT GCT CTC AAC ATC CTT GCC CTGTCA CCC CCT GCC CAA AATTTG A l aA l aL e uA s nI l eL e uA l aL e uS e rP r oP r oA l aG l nA s nL e uL e uL e u 90 100

CTA CTT AAA TCG TACTTCTCT GAA GAA GGA ATC GGA TAT AAC ATC Lys S e r T y r P h e S e r G l u G l u G l y I l eG l y T y r A s n I e l 110

462

TTC CAG TTGCACAACTTC AGC H i s A a nP h eS e r 140

552

CTC CCA GAG GAA GAT ACC AAG CTC AAG ATA CCC CTG ATT CAC CGA GCA CTG CAG TTG GCC CAG CGT CCC GTT TCA CTCCTT GCC AGC CCC L e uP r oG l uG i uA s pT h rL y sL e u lys I l eP r o L e u I l e His A r qA l aL e uG l nL e uA l aG l nA r qP r oV a lS e rL e u leu A l aS e rP r o 150 160

642

TGG ACA TCA CCC ACT TGG CTC AAG ACC AAT GGA GCG GTG AAT GGG AAG GGG TCA CTC AAG GGA CAG CCC GGA GAC ATC TAC T r pT h rS e rP r oT h rT r pL e uL y sT h rA s nG l yA l aV a lA s nG l yL y sG l yS e rL e uL y sG l yG l nP r oG l yA s pI l eT y r 180 190 zoo

CAC CAG ACC H i s G l nT h r

732

TGG GCC AGA TACTTT GTG AAG TTC CTG GAT GCC TAT GCT GAG CAC AAG TTA CAG TTC TCG GCA GTG ACA GCT GAA AAT GAG CCT TCT GCT T r pA l aA r gT y rP h eV a lL y sP h eL e uA s pA l aT y rA l aG l u H i s L y sL e uG l nP h eT r pA l aV a lT h rA l aG l uA s nG l uP r oS e rA l a 210 220 230

822

GGG CTGTTG AGT GGA TAC CCC TTC CAG TGC CTG GGC TTC ACC CCT GAA CAT CAG CGA GAC T T AA T 1 GCC CGT GAC CTA GGT CCT ACC CTC G l yl e uL e uS e rG l yT y rP r oP h eG l nC y sL e uG l yP h eT h rP r oG l u His G l n A r g A s p m I l e A l a A r g A s p L e u G l y P r o T h r L e u 240 250 26 0

912

ATC CGG GTA CCC ATG GCC AGC TGT GAC TTC TCC ATC CGC ACC TAC ACC T A T GCA GAC ACCCCTGATGAT I l e A r qV a lP r oM e tA l aS e rC y sA s pP h eS e rI l eA r qT h rT y rT h rT y rA l aA s pT h rP r oA s pA s pP h eG l nL e u 120 130

GCC AAC AGT ACT CAC CAC AATGTC CGC CTACTCATGCTGGAT GAC CAA CGC TTG CTG CTG A l aA s nS e rT h r H i s H i s A s nV a lA r gL e uL e uM e tL e uA s pA s pG l nA r gL e uL e uL e uP r o 28 0

+

CCC CAC TGG GCA AAG GTG GTACTG ACA GAC H i s T r p A l a Lys V a l V a l L e u T h r A s p 290

%O CCA GAA GCA GCT AAA TATGTTCAT GGC ATT GCT GTA CAT TGG TAC CTG GAC TTT CTG GCT CCA GCC AAA GCC ACC CTA GGG GAG ACA CAC ProGluAlaAlaLyaTyrVal His G l y I l e A l a V a l H i s T r pT y rL e uA s pP h eL e uA l aP r oA l aL y sA l aT h rL e uG l yG l uT h r His 300 310 320

1002

1092

CGC CTGTTC CCCAACACCATGCTCTTT GCC TCA GAG GCC TGTGTG GGC TCCAAGTTC TOG GAGCAG AGT GTG CGG CTA GGC TCC TGG GAT A r gL e uP h eP r oA s nT h rM a t lau P h eA l aS a rG l uA l aC y sV a lG l yS e r lys P h eT r pG l uG l nS e rV a lA r qL e uG l yS e rT r pA s p 330 340 350

1182

CGA GGG ATG CAG TAC AGC CAC AGC ATC ATC ACG AAC CTC CTG TAC CAT GTG GTC GGC TGG ACC GAC TGG AAC CTT GCC CTG AAC CCC GAA l eT h r A s n L e u L e u T y r H i s V a l V a l G l y T r p T h r A s p T r p A s n L e u A l a L e u A s n P r o G l u A r g G l y N e t G l n T y r Ser His S e r I l e I 370 38 0 36 0

1272

GGA GGA CCC AAT TGG GTG CGT AAC T T T GTC GAC AGT CCC G l yG 1P r oA s nT r pV a lA r gA s nP h eV a lA s pS e rP r oI l e 3 d 400

1362

ATCATT

GTA GAC ATC ACC AAG GAC ACG TTTTAC

AAA CAG CCC ATG TTCTAC

110 V a l A s p I l e T h r L y s A s p T h r P h e T y r L y s G l n P r o N e t P h e T y r 410

TTCCTG GAG ACA ATCTCACCT GGC TAC TCC ATT CAC ACC TACCTG TGG CGT CGC CAG TGA TGGAGCAGATACTCAAGGAGGCACTGGGCTCAGCCTGGG PheLeuGluThr I l e S e r Pro G l y T y r S e r I l e His T h r T y r L e u T r p A r q A r g G l n T e r 480 490

(641

CATTAAAGGGACAGAGTCAGCTCACACGCTGTCTGTGACTAAAGAGGGCACAGCAGGGCCAGTGTGAGCTTACAGCGACGTAAGCCCAGGGGCAATGGTTTGGGTGACTCACTTTC~~~ (760 TCTAGGTGGTGCCAGGGGCTGGAGGCCCCTAGAAAAAGGGAATTC

FIG. 2. Nucleotide sequence of human glucocerebrosidase cDNA and the deduced amino acid sequence. Peptidase cleavage site is shown by an arrow. The region where there is a discrepancy with the chemically determined amino acid sequence is boxed. Potential carbohydrate binding Asn residues are shown by solid diamonds. EcoRI linker sequence (GGAATTCC)is included at both ends of the cDNA.

genomicDNAfrom somatic cell hybrids using 32P-nicked translated cDNA insert from pGC1 (10) showed appropriate hybridization patterns concordant with the presence of immunoprecipitable human glucocerebrosidase activity (25). The amino acids deduced from the cDNA insert reported in this work are identical to all but one chemically determined amino acid residue of human placental glucocerebrosidase,

thus conclusively confirming that these clones encode human glucocerebrosidase. The availability of a cDNA probe has enabled us to isolate and characterize genomic DNA (11)and determine by in situ hybridization the chromosomal locus for this enzyme (10). Restriction fragment length polymorphisms, as well as Northern analyses and S1 nuclease mapping of mRNA from normal

H u m a n Glucocerebrosidase cDNA Clone and Sequence

53

TABLE I Codon usage frequency forhuman glucocerebrosidase ~

l T T Phe

8 1.6% TTC Phe 17 3.3% .4% TTA Leu 2 TTG Leu 7 1.4%

TCT TCC TCA TCG

CTT Leu 9 1.7% CTC Leu 11 2.1% CTA Leu 8 1.6% CTG Leu 23 4.5% ATT ATC ATA ATG

5 1.0% 14 2.7% 0 --% 0 --%

TGT Cys .8% 4 TGC Cys .6% 3 TGA --1 .2% TGG Trp 13 2.5%

CCT Pro 11 2.1% CCC Pro 18 3.5% CCA Pro .8% 4 CCT Pro 1 .2%

CAT His 1.2% 6 CAC His 12 2.3% CAA Gln.4%2 CAG Gln 19 3.7%

CGT Arg .8% 4 CGC Arg 9 1.7% CGA Arg 4 .8% CGG Arg.6% 3

Ile 6 1.2% Ile 14 2.7% Ile 1 .2% Met 10 1.9%

ACT Thr .4% 2 ACC Thr 17 3.3% ACA Thr 10 1.9% ACG Thr 3 ,696

AAT Asn 8 1.6% AAC Asn 11 2.1% AAA Lys 1.2% 6 AAG Lys 16 3.1%

AGT AGC AGA AGG

4 .8% 5 1 .O% 4 3% 18 3.5%

GCT Ala 13 2.5% GCC Ala 19 3.7% GCA Ala 9 1.7% 1 .2% GCG Ala

GAT Asp 10 1.9% GAC Asp 16 3.1% GAA Glu 8 1.6% GAG Glu 10 1.9%

GGT Gly .6% 3 GGC Gly 14 2.7% GGA Gly 11 2.1% GGG Gly 1.7% 9

GTT Val GTC Val GTA Val GTG Val

Ser Ser Ser Ser

4 .8% 7 1.4% 1.4% 7 3 ,696

r

TAT Tyr TAC Tyr TTA --TTG ---

Ser 1.6% 8 Ser 10 1.9% Arg .4% 2 Arg 0 .O%

REFERENCES 1. Brady R. 0. Kanfer J. N., and Shapiro, D. (1965) Biochem. Biophys. Res.

Corhaun. is, 2211225 2. Brady, R. O., and Barran er,J A. (1983)in The Metabqlic Basis of Inherited Dlsease (Stanbury, J. W ngaarden J. B. Fredrlckson D. S., Goldstein, J. L., and Bo rwM !. eds) pp. h2-866, McGraw-hill, Inc., New York 3. Ginns E. I., Brad , R. O., Pirruccello, S., Moore, C., Sorrell, S., Furbish, Murray J., Ta er, J., and Barranger, J. A. (1982) Proc. Natl. F. Acad. Scr. U. A. 79 5gO7-5610 4. Ginns E. I Tegelaers fi. P. W Barneveld R. Galjaard H. Reuser A. J. J., brad;:R. 0..Ta’ger, J. M:, and BarrAngir, J. A. (i983) Clin. bhim. Acta 131,283-287 5. Gravel, R. A,, and b u n g A. (1983) Hum. Genet. 65 112-116 6. Wenger D. A. Roth S kudoh T., Grover, W. D., ‘fucker, S. H., Kaye, E. M., aAd Ullman, hh. 6. (1983) Pedmtr. Res. 17,344-348 7. Ginns, E. I., Chouda P. V Martin B. M Winfield, S. Stubblefield B. Mayor J. Merklezhmah, D., M h a ‘b J Bowed L. A. and bar: ranger’J.’A. (1984)Biochem. Bio hys k s . ‘Cohzmun. 123,534-580 8. DeWet, h., Fukushima H. Dewji N. Wilcox,E., O’Bnen, J. S., and Helinski D. R. (1984) D h A 3, i37-44i 9. Fukushima’ H. DeWet J. R and O’Brien, J. S. (1985) Proc. Natl. Acad. Sci. U. S ’ A . 82,1261-1262 10. Ginns E.I., Chouda P., Tsuji S., Martin, B., Stubblefield, B, Sa er, J., kozier, J., and %ranger, j.A. (1985) Proc. NatL Acad. Scr. U . Y A . 82 7101-7105 11. Chouhary P.V. Ginns E.I. and Barran er J A (1985) DNA 4, 74, 12. Maniatis,’T., Fdstch E!. F., a’nd Sambroof, j.(1982) Molecular Clonr Loboratory Manual Cold Spring Harbor Lsboratory, Cold Spnng Ha%: NY 13. San er, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. dS.A. 74,5463-5467 14. Wallace R. B Johnson M. J. Su gs, S. V., Miyoshi, K., Bhatt, R., and Itakda, K. (1981) Ge& 16, 31-25 15. Maxam A. M. and G~lbertW. (1980) Methods Enzymol. 65,499-560 (19h3) Microbial Reu. 47, 1-45 16. Kozak 17. Ericksbn, A. H.,Ginns, E. I., and Barranger, J. A. (1985) Fed. Proc. 44,

2,

s.,

- 34

t

peptidase ckavage

M A G S L T G L L L L Q A V S W A S G

,-I

leaderpolypeptide

1-

7

A R P C I F a m i n o terminus of human placental glucocerebrosidase

FIG. 3. Leader polypeptide of human glucocerebrosidase and the hydropathy plot. The hydropathy indices of 9 consecutive amino acids were calculated.

and mutant cells, should allow the description of gene mutations responsible for Gaucher disease. This direct observation of the gene alterations should further elucidate the mechanisms responsible for the clinical heterogeneity seen in this disorder. Furthermore, the availability of a full-length cDNA clone containing the complete coding sequence for human glucocerebrosidase should allow studies of the feasibility of correcting the enzyme deficiency in mammalian cells by gene transfer. Acknowledgments-We thank Drs. J. O’Brien, J. DeWet, and H. Fukushima for their suggestions and generosity in sharing the Xgtll hepatoma cDNA library. We acknowledgethe help given by Dr. Gary Murray in the preparation of homogenous glucocerebrosidase and of rabbit antiserum. We also thank B. Stubblefield, D. Merkle-Lehman, and G . Mook for their technical assistance, and L. Metz for typing the manuscript.

8.

A.

%.

k.

7n4

18. ErjiLson, A. H., Ginns, E. I., and Barranger, J. A. (1985) J. Biol. Chem. 260 14319-14324 19. Mason: P. J., Jones, M. B., Elkington, J. A., and Williams, J. G. (1985) EMBO J. 4,205-211 20. Grantham, R:, Gautier, C., Gouy, M.,Jacobzone, M., and Mercier, R. (1981) Nucleic Acrds Res. 9. r43-r74 21. Takasaki, S., Murray, G . J., Furbish, F. S., Brady, R. O., Barranger, J. A., and Kobata A. {1984]J. Biol. C k m . 259, 10112-10117 22. Watson M.E:E.1984 Nuclele Aclds Res. 12,5145-5164 23. Walter,’P., Gilmore R. and Blobel, G., (1984 Cell 38, 5-8 24. Rosenfeld, M. G.. kreibich. G.. Powv. D.. dato.. K... and Sabatini. D. D. ’ (1982) J Cell Bhl. 93, 135-143 25. Barneveld R. A. Keijzer W Tegelaers F. P. W. Ginns E. I. Geurts van Kessel ’A. Biady R. b. ‘Barran er ’J A. Ta’ger J. d a l . 4 H., Westehelh, A., a i d Reueb, A. J. (19M) Hum. cfenet. 64,217-2dl ’

~

5.

h