THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 267, No. 10, Issue of April 5, pp. 6570-6575, 1992 Printed in U. S. A.

Cloning and Characterization of cDNA Encoding Canine a-L-Iduronidase mRNA DEFICIENCY IN MUCOPOLYSACCHARIDOSIS I DOG*

(Received for publication, June 7,1991)

Knoxville, Tennessee 37916 -“ I

a-L-Iduronidase is a lysosomal enzyme, the defi- ciency of which causes mucopolysaccharidosis I (MPS I); a canine MPS I colony has been bred to test thera- peutic intervention. The enzyme was purified to ap- parent homogeneity from canine testis and found to consist of two electrophoretically separable proteins that had common internal peptides but differed at their amino termini. A 57-base oligonucleotide, correspond- ing to the most probable codons of the longest peptide, was used to screen a canine testis cDNA library. Three cDNAs were isolated, two of which lacked the 5‘-end whereas the third was full-length except for a small internal deletion. The composite sequence encodes an open reading frame of 655 amino acids that includes all sequenced peptides. The amino terminus of the larger protein, glutamic acid 26, is at the predicted signal peptide cleavage site, whereas the amino ter- minus of the smaller protein is leucine 106. There are six potential N-glycosylation sites and a non-canonical polyadenylation signal, CTTAAA. A search of Gen- Bank showed that the amino acid sequence of a - ~ - iduronidase has similarity to that of a bacterial B- xylosidase. A full-length cDNA corresponding to the composite sequence was constructed (pcIdu) and in- serted into the pSVL expression vector (pSVcIdu). Two days after Cos-1 cells were transfected with pSVcIdu, their intracellular and secreted level of a-L-iduroni- dase activity had increased 8- and 22-fold, respec- tively, over the endogenous activity. Fibroblasts of MPS I dogs, which have no a-L-iduronidase activity, lacked the normal a-L-iduronidase mRNA of 2.2 kilo- bases and contained instead a trace amount of a 2.8- kilobase species. Isolation and characterization of an

* This work was supported in part by Grant DK38857 from the National Institutes of Health (to E. F. N.), Grant DIR86-18937 from the National Science Foundation (to D. B. T.), a fellowship from the Bank of America-Giannini Foundation (to L. J. S.), a fellowship from the Lucille P. Markey Program in Cellular Biochemistry (to K. P. M.), and a medical student research training fellowship from the Howard Hughes Medical Institute (to S. M. M.). The costs of publi- cation of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertise- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in thispaper has been submitted

M81893. to the GenBankTM/EMBL Data Bank with accession numbercs)

ll Present address: Center for Neurologic Diseases, Brigham and Women’s Hospital, Boston, MA 02115.

I( To whom correspondence should be addressed Dept. of Biological Chemistry, UCLA School of Medicine, Los Angeles, CA 90024-1737.

Lori J. Stoltzfus, Beatriz Sosa-Pineda, Samuel M. Moskowitz, Kaushiki P. Menon, Bonnie Dlott, Lucilla HooperS, David B. TeplowQlf, Robert M. ShullS, and Elizabeth F. NeufeldII From the Department of Biological Chemistry and Brain Research Institute, School of Medicine and Molecular Biology Institute, University of California, Los Angeles, California 90024, the §Division of Biology, California Institute of Technology, Pasadena, California 91 125. and the iDeoartment of Pathobioloev. College of Veterinary Medicine, University of Tennessee,

expressible a-L-iduronidase cDNA represents the first step toward mutation analysis and replacement ther- apy.

a-L-Iduronidase (EC 3.2.1.76) is a lysosomal enzyme re- quired for the degradation of dermatan sulfate and heparan sulfate. Genetically determined deficiency of this enzyme is the cause of a group of lysosomal storage diseases known as mucopolysaccharidosis I or MPS I’ (reviewed in Ref. 1). These include the Hurler syndrome, with severe retardation of men- tal and physical growth, characteristic skeletal deformities, joint stiffness, cloudy corneas, cardiovascular disease, and death usually in late childhood. At the other end of the clinical spectrum is the Scheie syndrome, with corneal opacities and joint stiffness but normal stature and intelligence and a potentially normal life span. Forms of intermediate severity, with major physical handicaps but retention of intellect and survival to adulthood, are generally known as Hurler/Scheie. There exist canine and feline models of a-L-iduronidase de- ficiency disease, which clinically resemble most closely the intermediate form of the human MPS I.

a-L-Iduronidase has been purified to near homogeneity from human kidney (2), lung (3,4), and liver (5-7), as well as from porcine liver (8); its natural history has been examined by metabolic labeling of cultured human (9, 10) and canine (11) fibroblasts. The enzyme has figured prominently in the discovery of receptor-mediated endocytosis of lysosomal en- zymes; the activity originally described as “Hurler corrective factor” (12) proved to be a-L-iduronidase with a mannose 6- phosphate marker for binding to a receptor on the surface of fibroblasts and subsequent delivery to lysosomes (13-15).

The ease with which glycosaminoglycan accumulation in fibroblasts cultured from Hurler patients can be corrected by administration of exogenous enzyme has served as the basis for therapeutic attempts (reviewed in Ref. 1). Bone marrow transplantation is currently performed, with some indication of success, as an experimental therapy for Hurler syndrome (16); a positive outcome has been observed in three MPS I dogs transplanted with bone marrow from their unaffected littermates (17, 18). The treatment of a systemic disease by transplanted hematopoietic tissue may be attributed to trans- fer of enzyme from marrow-derived cells as they circulate in the blood or take up residence in tissues. As a result of the

’ The abbreviations used are: MPS I, mucopolysaccharidosis I; kb, kilobase(s); SDS, sodium dodecyl sulfate; PMSF, phenylmethylsul- fonyl fluoride.

6570

cDNA Encoding a-L-Iduronidase 6571

partial success of bone marrow transplantation, MPS I has become a candidate for gene replacement therapy via hema- topoietic cells.

We undertook to clone and characterize canine cDNA encoding a-L-iduronidase in order to have a reagent that is indispensable for studying the structure and function of the normal enzyme and the mutations underlying MPS I, as well as for exploring therapeutic strategies such as enzyme or gene replacement in the canine model. While this work was in progress, the isolation of human a-L-iduronidase cDNA was reported and the gene was mapped to human chromosome 4p16.3 (19).

A preliminary account of this work has been presented in abstract form (20).

EXPERIMENTAL PROCEDURES~

Amino Acid Sequence Analysis-After preparative gel electropho- resis, proteins were electroblotted in a Bio-Rad Trans-blot apparatus onto a polyvinylidene difluoride membrane for 30 min at 90 V and at ambient temperature, using cold transfer buffer (10 mM 3-[cyclo- hexylaminol-1-propanesulfonic acid, pH 11.0, 10% methanol). After transfer, the proteins were stained with Coomassie Blue. Dry mem- branes were stored at -20 "C until used.

Amino-terminal sequencing of the two protein bands was per- formed on an Applied Biosystems 475 instrument at the UCLA Protein Microsequencing Facility.

For sequencing of internal peptides, electroblotted proteins were digested in situ with V-8 protease (Boehringer Mannheim) according to Aebersold et al. (22). The resulting peptide fragments were sepa- rated by reverse phase high pressure liquid chromatography, then sequenced using an Applied Biosystems model 477A/120A instru- ment, essentially according to the manufacturer's instructions. Resi- due assignments were made by manual inspection of chromatograms.

Cloning and Characterization of cDNA-Total RNA was isolated from canine testis by acidic phenol-chloroform extraction in guani- dinium isothiocyanate (23) followed by sedimentation through CsCl (24); poly(A)+ RNA was selected by passage over a column of poly(dT) (Collaborative Research) as described (24) and provided to Stratagene (La Jolla, CA) for preparation of a cDNA library in lambda ZAP using oligo(dT) priming. A unique antisense 57-base oligonucleotide corresponding to the longest peptide sequence was designed on the basis of human codon usage (25), after a check of the 19 canine sequences in GenBank showed only minor disparity from the usage of human codons. Glutamic acid was assumed to be at the amino terminus of the sequenced peptide, based on the cleavage specificity of V-8 protease (26). The oligonucleotide, of the sequence, 5' GAAT-

GGGAGATGTACTC, was gel-purified and 5'-end-labeled with [y- "'PIATP (ICN) and T4 polynucleotide kinase. The radioactive oli- gonucleotide was used to screen the canine testis library by plaque hybridization (27); about one million plaques were screened. The filters were prehybridized, hybridized, and washed essentially as described (28). A strongly hybridizing clone was plaque-purified, and the cDNA insert of 1.7 kb was rescued in pBluescript SK(-) (Stra- tagene) as recommended by the supplier. This 1.7-kb insert was isolated, labeled with [cI-~'P]~CTP by random priming (MultiPrime kit, Amersham Corp.), and used as a probe to screen an additional 2 X lo6 plaques from the library. Two hybridizing clones were isolated, and the cDNA inserts, of 1.8 and 2.1 kb, were each rescued in pBluescript SK(-).

Nested deletions from the 5'-end of the 1.7-kb cDNA insert and from the 3'-end of the 2.1-kb cDNA insert were generated using the exonuclease III/mung bean nuclease deletion kit (Stratagene) accord- ing to the supplier's instructions. Purified plasmid cDNA from the resulting subclones was sequenced by the dideoxy chain termination method (29) using universal primers (Promega), [ W ~ ~ S I ~ A T P (Amer- sham Corp.), and Sequenase (U. S. Biochemical Corp.) as recom- mended by the supplier of the enzyme. In addition, the 1.7- and 1.8- kb cDNAs were completely sequenced using internal primers (syn-

Portions of this manuscript (including part of "Experimental Procedures," Table I, and Fig. 1) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

GGCAGGATGTATGGGGAGGAGCCAGCGCCCTTCTTGTGCA-

thesized by the phosphoramidite method on automated instruments). The many compressions were resolved using 7-deaza-dGTP or dITP (U. S. Biochemical Corp.).

Standard molecular techniques (27) were used throughout unless otherwise specified.

Cell Culture-Cos-1 cells (American Type Culture Collection) were cultured at 37 "C and 5% CO' in Dulbecco's minimal essential medium supplemented with 5% fetal bovine serum (Irvine Scientific), pyru- vate, non-essential amino acids, and antibiotics. Canine fibroblasts were derived from skin biopsies of dogs homozygous for a-L-iduroni- dase deficiency and from unaffected controls. Normal canine fibro- blast strain GM 3978 was obtained from the Coriell Institute for Medical Research. The fibroblasts were cultured at 35 "C and 5% CO, in Eagle's minimal essential medium supplemented with 15% fetal bovine serum, pyruvate, non-essential amino acids, and antibi- otics.

Transfection of Cos-1 Cells-A full-length a-L-iduronidase cDNA (pcIdu) was constructed in pBluescript I1 KS(+) by ligating the upstream portion of the 2.1-kb cDNA clone to the downstream portion of the 1.8-kb cDNA clone, using the Sac1 restriction site at nucleotide 802. The resulting 2.2-kb a-L-iduronidase cDNA was sub- cloned into the expression vector pSVL (Pharmacia LKB Biotech- nology Inc.). This construct (pSVcldu) contains the insert at the XbaI and SmaI sites and is driven by the SV40 late promoter.

The lipofection procedure (30) was used to introduce pSVcIdu or pSVL into cells; the plasmid pXGH5 (Nichols Institute), containing the growth hormone gene, was co-transfected as control for transfec- tion efficiency. Subconfluent Cos-1 cells in 100-mm plates were transfected using LipofectinTM (Bethesda Research Laboratories) as recommended by the supplier. After 9 h in the presence of lipofectin/ DNA in serum-free medium, the cells were washed and incubated 38 h in the growth medium (above) containing 10 mM NH,Cl. Aliquots of the medium were removed for assay of growth hormone and a - ~ - iduronidase activity; the cells were harvested by trypsinization, and extracts prepared by freeze-thawing and sonication were used for determination of protein content and a-L-iduronidase activity.

Northern Analysis-Total RNA was isolated from canine fibro- blasts by acidic phenol-chloroform extraction in guanidinium isothi- ocyanate (23). An aliquot was treated with RNase-free RQl DNase (Promega) prior to gel electrophoresis in agarose containing 2 M formaldehyde (27); 13 fig of DNase-treated or 25 pg of untreated total RNA was applied to each lane. Nucleic acids were transferred to nylon membrane (Nytran, Schleicher and Schuell) as recommended by the supplier. A 1.7-kb restriction fragment was isolated from the 2.1-kb cDNA by cleavage with SfiI (Stratagene) and used as a probe in preference to the entire 2.1-kb cDNA in order to avoid nonspecific binding attributable to the GC-rich 5'-untranslated region. A 2.7-kb cDNA probe encodingcanine kidney Na'/K'-ATPase (31) was kindly provided by Dr. Robert Farley (University of Southern California). Both probes were labeled with [a3'P]dCTP by random priming (Mul- tiprime kit, Amersham Corp.). Membranes were prehybridized and hybridized as described (32), washed twice at 65 'C for 20 min in 0.1 X SSC, 2% sodium dodecyl sulfate, and subjected to autoradiography.

RESULTS

Partial Amino Acid Sequence of a-L-Iduronidase-The pu- rification of a-L-iduronidase to apparent homogeneity and the properties of the enzyme are described in the supplemental material. The most highly purified preparations comprise two electrophoretically separable proteins of approximately 68 and 63 kDa (Fig. 1). Table I1 presents the amino-terminal sequences of the two electrophoretically separated proteins and of peptides derived from them by V-8 proteolysis. The 68- and 63-kDa species have internal peptide sequences in common but differ at their amino termini, suggesting that they represent two forms of the same protein, one of which has been derived from the other by proteolytic cleavage. This suggestion is consistent with the similar V-8 peptide maps of the two proteins (not shown).

Nucleotide and Predicted Amino Acid Sequences of cDNA- The three cDNA clones (1.7, 1.8, and 2.1 kb) were sequenced in their entirety; their relationship is shown in Fig. 2. The two shorter clones were missing the 5'-end; however, they both contained a 114-base pair segment that was absent from

6572 cDNA Encoding a-L-Iduronidase TABLE I1

Partial amino acid sequence of a-L-iduronidase Superscripts refer to the position of the peptides in the translated

cDNA sequence shown in Fig. 3; underlined residues differ from those predicted by the sequence of cDNA.

Intact proteins 68 kDa 26XAPXLVLV 63 kDa ‘OGXXYXFTLLD

Peptides generated by V-8

From 68-kDa protein only 95LITARE From 63-kDa protein only 257YISLHKKGAGSSEYILE From both 68- and 63-kDa lz3NQLLPGFE

proteolysis

proteins 486KQRLGRPVFPTAE 582mFSADG

1.8 kb

2.1 kb

, I \ ,

0 500 1000 1500 2000 I I I I I I I I I I I I I I I I I I I I I , I

FIG. 2. Alignment of the three partial cDNAs encoding a - ~ - iduronidase. The stippled bars represent coding regions, the black bars represent untranslated regions, the downward spike represents the deleted segment, and the wauy lines represent poly(A) tails. The length of the cDNAs is given below in base pairs, beginning with A of the initiating methionine.

the longer clone. The composite sequence of 2.2 kb, shown in Fig. 3, contains an open reading frame encoding 655 amino acids. It contains all the sequenced peptides shown in Table I1 in positions 95-100, 123-130, 257-274, 486-498, and 582- 588. Glutamic acid, a preferred cleavage site of V-8 protease, precedes each of these sequences except for 257-274 which is preceded by aspartic acid, an alternate cleavage site. The amino-terminal sequences of the two electrophoretically sep- arated proteins can also be found in the sequence deduced from cDNA. Glutamic acid at position 26 defines the NH2 terminus of the larger protein, and leucine at position 106 defines that of the smaller one. Five discrepancies between the amino acid sequences deduced from the cDNA (Fig. 3) and obtained by protein sequence analysis (Table 11) were observed. His-112 was missed because of the low amount of sample analyzed. Ile-269, Glu-273, and Trp-486 were in fact observed during sequencing but were mistakenly interpreted as secondary signals. Phe-274 was a protein sequencing error.

Expression of Canine a-L-Iduronidase in Cos-1 Cells-A full-length cDNA of 2.2 kb (pcIdu) was constructed as de- scribed under “Experimental Procedures” and inserted into the expression vector pSVL. The resulting plasmid, pSVcIdu, programmed the synthesis of enzymatically active a-L-iduron- idase in transfected Cos-1 cells (Table 111). There was an 8- and 22-fold increase in intracellular and secreted enzyme activity, respectively, over that of cells that had been mock- transfected (no vector) or transfected with the pSVL vector alone. By contrast, Cos-1 cells transfected with pSVL con- taining the 2.1-kb cDNA showed no increase in activity (data not shown).

Absence of Normal a-L-Iduronidase mRNA in Fibroblasts of MPS I Dogs-Total RNA from control canine fibroblasts contained a 2.2-kb species that hybridized to a-L-iduronidase cDNA (Fig. 4, left panel). This species was absent from total RNA derived from fibroblasts of an a-L-iduronidase-deficient

dog; instead, there was a small amount of a 2.8-kb species (Fig. 4, left panel, arrowhead). Hybridization to cDNA encod- ing canine Na+/K’-ATPase (Fig. 4, right panel) showed that the RNA was intact. The same qualitative and quantitative differences were observed between RNA of normal canine fibroblast strain GM 3978 and that derived from a second affected dog from the same colony (not shown).

DISCUSSION

A full-length 2.2-kb cDNA encoding a-L-iduronidase was constructed from two incomplete cDNAs that had been iso- lated from a canine testis cDNA library. Its authenticity was demonstrated by the dramatic increase in a-L-iduronidase activity when it was expressed in Cos-1 cells. Availability of an expressible cDNA paves the way for studies of the struc- ture, function, and regulation of the enzyme, as well as for its use in replacement therapy of canine MPS I.

The a-L-iduronidase cDNA is rich in guanine and cytosine: 70% G and C for the entire sequence, 92% for the 5”untrans- lated sequence, and 70% for the coding sequence. The nucleo- tide sequence near the first AUG codon, CGCCCGGCC- AUGC, is similar but not identical to the consensus sequence for initiation of translation, G C C G C C A / G C C ~ G (33). The sequence 24 base pairs upstream of the poly(A) cleavage site is CTTAAA rather than the consensus polyadenylation signal, AATAAA.

The first methionine is followed by a typical eukaryotic signal sequence, with a cleavage site predicted after alanine 25 (34). Thus the amino terminus of the larger protein seen in polyacrylamide gel electrophoresis, glutamic acid 26, is at the predicted signal peptidase cleavage site. The amino ter- minus of the smaller protein is at leucine 106. The molecular size of the corresponding polypeptides, deduced from the cDNA sequence, is 70.5 and 61.6 kDa; since they are glyco- sylated at one or more of the six potential N-glycosylation sites, both proteins must be somewhat larger than the 68 and 63 kDa estimated from their electrophoretic migration in denaturing polyacrylamide gels. Inaccuracy of the electropho- retic determinations probably accounts for differences be- tween observed and predicted sizes.

The existence of a larger species of a-L-iduronidase which is proteolytically cleaved to a smaller one is in agreement with results of biosynthetic studies of human (9, 10) and canine fibroblasts (11). Two electrophoretically separable proteins, similar in size to those seen in our preparations, have been reported for purified porcine liver a-L-iduronidase (8). Since numerous measurements by non-denaturing methods such as gel filtration and sedimentation have shown an apparent molecular mass of 60-85 kDa (2, 3, 5, 7, 8), the enzyme probably exists as a monomer. A previously reported subunit size of 30 kDa for the human kidney enzyme (2) may have resulted from proteolysis in vitro. An additional protein of approximately 59 kDa, which has not been analyzed, was seen in immunoaffinity-purified enzyme (Fig. l ) , especially before passage over heparin-agarose. We have not observed the five additional molecular species of a-L-iduronidase reported in immunopurified preparations from various human tissues (7).

The protein sequence is enriched in arginine residues, which are responsible for the basic nature of the protein (predicted PI, 8.08 and 8.29 for the larger and smaller species, respec- tively). Arginine-rich domains may explain the selective ad- sorption of a-L-iduronidase to heparin-Sepharose. Arginine residues had also been implicated in the receptor-mediated endocytosis of a-L-iduronidase because binding and uptake could be reduced by treatment of the enzyme with butane- dione, an arginine-modifying reagent (35).

cDNA Encoding a-L-Iduronidase 6573

FIG. 3. Nucleotide and deduced amino acid sequences of cDNA en- coding a-L-iduronidase. Nucleotides and amino acids are numbered starting with the first methionine in the open reading frame; negative numbers repre- sent 5”untranslated sequence. The six potential N-glycosylation sites are underlined by a heavy line and the poly- adenylation signal by a thin l ine. The amino termini of the large and small species of a-L-iduronidase are marked by arrows.

-66

1 1

76 26

151 51

226 7 6

3 0 1 101

3 7 6 126

4 5 1 151

526 176

601 2 0 1

6 7 6 226

7 5 1 251

826 2 7 6

YO1 3 0 1

976 326

1051 351

1126 376

1201 401

1276 426

1 3 5 1 4 5 1

1425 476

1501 501

1576 526

1651 551

1126 576

1801 601

1876 626

1 9 5 1 651

2026

2101

ccc cqc tcc cqc ccq qct ctc qcc ccq qcc cqc ccc aqc ccc cqc gcc cqc qac ccc cqc ccq qcc

ATG CGG CCC CCC GGC CCC CGC GCC CCC GGG CTG GCG CTG CTG GCC GCG CTG CTG GCG GCG CCC CGG GCC C T C GCA Met A r g Pro Pro G l y Pro A r q A l a Pro G l y Leu Ala Leu Leu Ala A l a Leu Leu A l a A l a Pro Arg A l a L e u A l a

GAG GCC CCG CAC CTG GTG CTC GTG GAC GCG GCC CGC GCG CTG CGG CCC CTG CGG CCC T T C TGG AGG AGC ACC GGC G 1 u A l a Pro His Leu V a l Leu V a l A s p A l a Ala A r q A l a Leu Arq Pro Leu A r q Pro Phe T r p Arg Ser T h r G l y

T T C T G C CCC CCC CTG CCG CAC AGC CAG GCT GAC CGC T A T GAC C T C AGC TGG GAC CAG CAG C T C AAC CTG GCC T A T 4

P h e cys Pro Pro Leu P r o His Ser G l n A l a A s p A r q Tyr A s p Leu Ser Trp A s p G l n G l n Leu A m Leu Ala T y r

GTG GGT GCT G T C CCT CAC GGG GGC ATC GAG CAG GTC CGG ACC CAC TGG CTG CTG GAG CTC ATC ACG GCC AGG GAG V a l G l y Ala V a l Pro Hi6 Gly G l y I le 61u G l n V a l A r g T h r His T r p Leu Leu G l u Leu Ile Thr Ala Arq G1U

TCA GCT GGG CAA GGC CTG AGC TAC AAC T T C ACC CAC CTG GAT GGC TAC CTG GAT CTC CTC AGG GAG AAC CAG CTC Ser A l a G l y G l n G l y Leu S e r Tyr A m Phe Thr His Leu A s p G l y Tyr Leu A s p teu Leu A r q G 1 u A s n G l n Leu 4 CTC CCA GGT TTT GAG CTG ATG GGC AGC CCC TCC CAG CGC TTC ACT GAC TTC GAG GAC AAG CGG CAG GTG TTG GCG Leu Pro G l y Phe G l u Leu net G l y Ser Pro Ser G l n Arg Phe T h r A s p P h e Glu A s p L y s A r q G l n V a l Leu A l a

TGG AAG GAG CTG GTG TCC CTC CTG GCC AGG AGA TAC ATC GGG AGG T A T GGA CTC TCA TAC GTT TCC AAG TGG M C Trp L y s Glu Leu V a l Ser Leu Leu Ala Arq A r q T y r Ile G l y A r g Tyr G l y Leu Ser Tyr V a l Ser L y s T rp A m

T T C GAG ACG TGG M T GAG CCA GAC CAC CAC GAC T T C GAC M C GTG ACC ATG ACC CTG CAA GGC TTC CTG M C TAC P h e G l u Thr Trp A m G 1 u P r o A s p His His A s p P h e A s p A s n V a l Thr Met T h r Leu G l n G l y Phe Leu A S n T y r

TAT GAC GCC TGC TCC GAG GGT CTG CGT GCT GCC AGC CCG GCC CTG CGC TTT GGC GGC CCC GGG GAC TCT TTC CAC T y r A s p Ala C y 5 Ser G l U G l y Leu Arg Ala A l a Ser Pro Ala Leu A r q Phe G l y G l y Pro G l y A S P Ser P h e His

CCC TGG CCG CGC TCC CCC CTG TGC TGG GGC CTC CTG GAG CAT TGT CAC AAC GGC ACC M C T T C T T C A C C GGG GAG

I

I

P r o T r p Pro A r q Ser Pro Leu cys T r p Gly Leu Leu G l u His cys His A s n G l y Thr A s n P h e P h e T h r G l y G 1 U I

CTG GGG GTG CGC CTG GAC TAC ATC TCC CTC CAC AAG M G GGC GCG GGG AGC TCC ATC TAC ATC CTG GAA CAG GAG Leu G l y V a l Arg Leu A s p T y r Ile Ser L e u His Lye Lys G l y Ala G l y Ser Ser I le Tyr Ile Leu G l u G l n G1u

CAG GCC ACC GTG CAG CAG ATC CGA CGG CTC TTC CCC AAG TTC GCC GAC ACC CCC GTT TAC AAC GAC GAG GCG GAC G l n Ala T h r V a l G l n G l n Ile A r q A r q Leu P h e Pro L y s Phe A l a A s p T h r Pro Val Tyr A s n A s p G l u Ala A s p

CCG CTG GTG GGC TGG GCC CTG CCG CAG CCC TGG AGA GCC GAC GTG ACG TAC GCG GCC ATG GTG GTG AAG GTC GTG Pro L e u V a l G l y T r p A l a Leu Pro G l n Pro T r p A r g Ala A s p V a l Thr T y r A l a Ala Met V a l V a l L y s V a l V a l

GCG CAG CAC CAG ARC CCG CCC CGG GCC M C GGC AGC GCG GCC CTG CGC CCC GCG CTC CTG AGC AAC GAC M C GCC A l a G l n His G l n A s n Pro Pro A r q A l a Asn G l y S e r A l a A l a Leu A r q Pro A l a L e u L e u Ser Asn A s p ASn A l a

T T C C T G AGC TTC CAC CCG CAC CCG TTC ACG CAG CGC ACG CTC ACC GCG CGC TTC CAG GTC ARC GAC ACG GAG CCG Phe L e u Ser Phe His Pro His Pro Phe Thr G l n A r g Thr Leu Thr A l a Arg P h e G i n V a l A s n A s p Thr G l u Pro

CCG CAC GTG CAG CTG CTG CGC AAG CCG GTG C T C ACG GCC ATG GCG CTG CTG GCC CTG CTG GAC GGC CGG CAG CTG P r o His V a l G l n Leu Leu A r g L y s P r o V a l L e u T h r A l a Met A l a Leu Leu A l a Leu Leu A s p G l y A r q G l n L e u

TGG GCC GAG GTG TCG CGG GGC GGG ACG GTG CTG GAC AGC M C CAC ACG GTG GGC GTC CTG GCC AGC GCG CAC CTG T r p A l a G 1 U V a l Ser A r g G l y G l y T h r V a l Leu A S P Ser A s n His T h r V a l G l y V a l Leu A l a Ser A l a Hi6 Leu

CCG GCC GGG CCC CGG GAC GCC TGG CGC GCC ACC GTG CTG CTC TAC GCG AGC GAC GAC ACG CGC GCC CAC GCC GCC Pro A l a G l y Pro A r q A s p A l a T r p A r g Ala Thr Val Leu L e u T y r A l a S e r A s p A s p Thr A r q Ala His A l a A l a

CGC GCC GTG CCC GTG ACG CTG CGC CTG CTC GGG GTG CCG CGG GGC CCA GGG CTC GTC TAC GTC ACC CTG GCC CTG Arq A l a V a l P r o V a l Thr Leu A r g Leu Leu G l y V a l Pro Arg G l y Pro G l y L e u V a l T y r Val T h r Leu A l a Leu

A s p A s n Pro A r q C y s Ser Pro His G l y G l u T r p G l n A r g L e u G l y A r q Pro V a l Phe Pro T h r A l a G l u G l u Phe GAC AAC CCG CGC TGC AGC CCC CAC GGC GAG TGG CAG CGC CTG GGC CGG CCC GTC TTC CCC ACG GCG GAG GAG l T C

A r q A r q Met A r q Ala A l a G1U A s p Pro V a l A l a G1U A l a Pro A r g Pro P h e Pro A l a Ser G l y A r g Leu Thr Leu CGG CGC ATG CGC GCA GCC GAG GAC CCG GTG GCC GAG GCG CCG CGC CCC TTC CCC GCC AGC GGC CGC CTG ACG CTC

AGC GTG GAG CTG CGG CTG CCC TCG CTG CTG CTG CTG CAC GTG TGC GCG CGC CCG GAG M G CCG CCG GGA CCG GTG Ser V a l G l u Leu A r q Leu P r o Ser Leu Leu Leu Leu His V a l C y s Ala A r q Pro G l u L y s Pro Pro G l y Pro V a l

ACC CGG CTC CGT GCC CTG CCC TTG ACC CGT GGG CAG GTG CTT TTG GTG TGG TCG GAT GAG CGC GTG GGC TCC M G T h r A r q Leu A r q A l a Leu P I 0 Leu Thr A r q G l y G l n V a l Leu Leu V a l T T p Ser A s p G l u A r q V a l G l y Ser LyS

TGC CTG TGG ACC TAT GAG ATC CAG TTC TCC GCG GAT GGA GAA GTG TAC ACG CCC ATC AGC AGG AAG CCA TCC ACC

I

I

I

C y 0 Leu Trp T h r Tyr G l u Ile G l n P h e Ser A l a A s p G l y G l u V a l T y r Thr Pro I le Ser A r q L y s Pro Ser Thr

T T T AAC CTG T T T G T G T T C AGC CCA GAG TCA GCC GTC ACC TCT GGC TCC TAC CGG GTT CGA GCG GTG GAC TAC TGG Phe A s n Leu P h e V a l Phe Ser Pro G 1 u Ser A l a V a l Thr Ser G l y Ser Tyr Arq V a l Arg A l a V a l A s p Tyr T r p

Ala A r q P r o G l y Pro Phe Ser Thr Arg V a l His Tyr V a l G l u V a l Pro A l a Pro S e r G l y Pro Pro A r q Pro Ser GCC CGA CCG GGC CCC TTC TCG ACC CGC GTG CAC TAC GTG GAG GTC CCT GCA CCG TCA GGG CCG CCG CGG CCC AGT

A s p C y s Glu A r q C y s ter GAC TGT GAG CGG TGC TGA Cct cgq aqc cqc gqa ggC cag qcg ctg aqa tgg gCC tcc aaq gaa ggC CCt Cqt CC9

ccg 999 atq gac gca qcc Cqt ccq tgg gct ccg ctc ctt etg tgc tgt CtC act ccc tta tgC =tt tgC qct

cac caa aaa ... - TABLE I11

Expression of a-L-iduronidase activity by transfected Cos-1 cells

Transfection vector a-L-Iduronidase activity

Intracellular Secreted

unitslmg cell protein

pSVL None 1.4 1.3

1.4 1.2 pSVcIdu 12 29

A Pearson FASTA search of GenBank (36) showed that the amino acid sequence of a-L-iduronidase has 26% identity (and an additional 29% similarity) to the P-xylosidase encoded by the xynB gene of the thermophilic microorganism, Caldo- cellum saccharolyticum (37) for a stretch of 210 residues near the amino termini of both proteins. Because of the similarity between the glycoside substrates (identical except that a - ~ - idopyranoside uronic acid has a carboxyl group linked to C-5 where P-D-xylopyranoside has a hydrogen atom), it is possible that the two enzymes have undergone convergent evolution

on the basis of function. No comparable similarity was found between a-L-iduronidase and other sequences currently in GenBank.3

The 2.1-kb cDNA clone was thought to contain the entire a-L-iduronidase sequence (20) but was subsequently found not to support the synthesis of active enzyme in transfected cells. Comparison of its sequence with that of two other clones derived from the same canine library as well as with a full- length human a-L-iduronidase cDNA clone (38) showed a deletion of 114 nucleotides (nucleotides 970-1083 in Fig. 3). Reverse transcription and PCR amplification of canine fibro- blast mRNA showed that most (>80%) of the a-L-iduronidase mRNA corresponded to the 2.2-kb cDNA but that a small amount of mRNA with the deletion was also pre~ent.~ The deletion of 114 nucleotides could arise from use of a cryptic acceptor splice site within exon 7.‘ In the course of cloning

Search of sequences in GenBank as of November 2,1991. E. Kakkis and E. F. Neufeld, unpublished results. K. P. Menon, P. T. Tieu, and E. F. Neufeld, unpublished results.

6574 cDNA Encoding a-I;-Iduronidase

9.5- 7.5 - 4.4 -

2.4 - 1 .4 -

IDUase ATPase

FIG. 4. Northern blot analysis of RNA from control and a- L-iduronidase-deficient canine fibroblasts. Total RNA derived from control or MPS I fibroblasts was subjected to electrophoresis in the indicated lanes. The filters were probed with cDNA encoding a- L-iduronidase (ZDUme, left panel) or Na+/K+-ATPase (right panel), as described under "Experimental Procedures." The numbers indicate molecular size standards. The arrowhead points to the 2.8-kb species of a-L-iduronidase RNA in cells of the MPS I dog.

cDNAs encoding lysosomal enzymes, e.g. @-glucuronidase (39), @-galactosidase (40), a-N-acetylgalactosaminidase (41), other investigators have isolated cDNAs with deletions or insertions, which were presumably derived from alternatively spliced mRNAs and which did not support the synthesis of active enzyme. Whether the alternative transcripts have some other function or represent splicing errors is not known.

The 2.1-kb cDNA clone also contained a single nucleotide difference from the other clones that would have resulted in asparagine instead of aspartic acid at position 412 (Fig. 3). Since the genomic DNA encodes aspartic acid: this change is probably a cloning artifact that arose during construction of the library. Whether it contributes to the non-functionality of the 2.1-kb clone is not known, as both the base change and the deletion were repaired simultaneously in constructing the full-length cDNA. A stretch of 37 nucleotides at the 5' end of the 2.1-kb cDNA (Fig. 3) may likewise represent a cloning artifact, since it was not found in the gene.5 However, this 5' untranslated sequence, which was retained in the full-length cDNA construct, did not interfere with expression of active a-L-iduronidase.

The existence of a-L-iduronidase mRNA in canine MPS I fibroblasts as a 2.8-kb species, in contrast to the 2.2-kb normal mRNA, might have been caused by insertion of a 0.6-kb fragment into the mutant gene. However, Southern analysis of EcoRI/BamHI double digests revealed hybridizing frag- ments of 8.5 and 9.0 kb for both the normal and mutant DNAs? Since these two fragments include all the exonic sequences of the a-L-iduronidase gene, insertion into the gene appears ruled out. A larger than normal mRNA could also result from utilization of alternate initiation, splicing, or polyadenylation site if the mutation obliterated the normal site. We are characterizing the normal and mutant genes in order to identify the canine mutation. In addition, we are using the canine cDNA as a starting point for the isolation, characterization, and mutation analysis of the human gene encoding a-L-iduronidase.

Acknowledgments-We thank Joanie Loh for assistance in cDNA sequence analysis, Nancy Uhrhammer for assistance in early molec- ular studies, Janice Bleibaum and Dr. Audree Fowler of the UCLA protein microsequencing facility for N-terminal sequencing, Tamara Bauer (Caltech) for peptide mapping and sequencing, Dr. Robert Farley (University of Southern California) for cDNA encoding canine

kidney Na+/K+-ATPase, Dr. Dohn Glitz and Anna Matynia for synthesis of oligonucleotides, and Larry Tabata for illustrations.

Addendum-After revision of this manuscript was completed, the sequence of human a-L-iduronidase was reported by Scott et al. (42). Both the nucleotide sequence and the deduced protein sequence of the human a-L-iduronidase cDNA (38, 42) show 82% identity with their canine counterparts.

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cDNA Encoding 0-L-Iduronidase 6575

Cloning and Characterization o f cDNA Encoding Canine e-L-lduronidsw Supplemental Material to:

Lnri J. Stoltzfus. Deatrir Lna-Pmcda. Samuel M. M<nkcwin. Kaurhiki P. Mcnon. Bonnie Dlatt. mRNA Deficicnry 10 Mucopolywceharidosis I Dog

Lucllla Hnlper. David B. Teplow. Rokn M. Shull and Ehrakth F. Ncufeld

Std 1 2 Std 3 - -= .. - 68

- 63

Homagenatc rupernatan~ 3)o 100 I Con A-Sephsrore eluate 250 75

26 610

Hepann-Sepharme eluate 170 51 3 . m 130 23

Hydroxylapaute eluate 9 2 6 11.100 433

540 pool I pool II 10 29 14.100 pool 111 30 9.1 4.700 180

WLC gel filtrate fraction I 9 26 124000 4.m fraction I I fracll"" 111 28 8.4 72403 2.m

7 2.1 64,700 2500

ALTERNATIVE PROCEDURE

I Con A-Sepharore eluate Homcgenatc supernatant 21 100

17 RI 34

Antibody.Sepharme eluate 9 43 840

200.000 5.900 25

Heparin-agarore eluate 3 14 239.000 7.W