Isolation and expression in Escherichia coli of a cDNA clone encoding human β-glucuronidase

105

Gene, 34 (1985) 105-110 Elsevier GENE

1173

Isolation and expression in Escherichia coli of a cDNA clone encoding human j-glucuronidase (Recombinant DNA; lysosomal hydrolase; mucopolysaccharidosis

Kevin S. Guise”, Robert G. Korneluk”,

type VII; lac promoter; b-galactosidase)

John WayeaVb, Anne-Marie

Lamhonwaha,

Frank Quana*b, Robin

Roger E. Ganschow ‘, William S. Sly d and Roy A. Gravel”vb*

Palmer’,

DResearch Instituteof The Hospitalfor Sick Children, Toronto, Ontario MSG 1X8, Tel. (416) 5986352; bDepartment ofMedical Genetics, Universityof Toronto, Toronto, Ontario MSS IA8 (Canada) Tel. (416) 9786116; ‘Division of Molecular Genetics, Children’s Hospital Research Foundation, Cincinnati, OH 45229, Tel. (513) 529-4742, and dDepartment of Biochemistry, St. Louis University, St. Louis, MO 63104 (U.S.A.) Tel. (314) 454-6093 (Received

July 2nd, 1984)

(Revision

received

and accepted

September

13th, 1984)

SUMMARY

Mucopolysaccharidosis type VII is a lysosomal storage disease resulting from a deficiency of fl-glucuronidase (BG) activity. To facilitate the investigation of mutation in the disease and provide molecular diagnostic tools for affected families, we have isolated human BG cDNA clones. The SV40-transformed human fibroblast cDNA library of Okayama and Berg [Mol. Cell. Biol. 3 (1982) 280-2891 was screened with a fragment of a murine BG cDNA clone (pGUS-1). The 17 human cDNA clones (pHUG) isolated were identical by restriction mapping,, varying only in length. The pHUG clones show 80% DNA sequence homology with pGUS-1 in a 198-bp PvuII-S.stI restriction fragment. Both pGUS-1 and the pHUG clones contained an open reading frame (ORF) throughout the sequenced region with a predicted amino acid sequence homology of 73 % . Expression in Escherichia coli of a 1150-bp fragment of pHUG-1 subcloned in pUC9 resulted in an isopropylthio-/I-galactoside (IPTG)-inducible 35-kDal fusion protein which was specifically immunoprecipitated by goat anti-human BG immunoglobulin G (IgG). This evidence provides direct confirmation that the pHUG cDNA clones correspond to human BG. -

INTRODUCTION

Lysosomes of virtually all mammalian tissues studied to date contain BG (B-D-glucuronide glu-

* To

whom reprint

Abbreviations:

requests

Ap, ampicillin;

pairs; Cm, chloramphenicol; propylthio-j%galactoside; ORF, open reading phoresis;

should be sent at the first address.

kb, kilobase

frame(s);

SDS, sodium

0378-l 119/84/$03.00

BG, j3glucuronidase; IgG, immunoglobulin;

0

PAGE,

dodecyl

pairs;

bp, base IPTG,

LB, Luria

polyacrylamide

gel electro-

sulfate.

1984 Elsevier

iso-

Broth;

Science Publishers

curonosohydrolase, EC 3.2.1.3 1) a homotetrameric acid hydrolase. In several mammals as well as in man, evidence points toward a single structural gene encoding a 69-75-kDal polypeptide which is glycosylated in the mature enzyme (Paigen, 1979). The human genetic disease mucopolysaccharidosis type VII, is a clinically heterogeneous disorder resulting from a deficiency of BG (Sly et al., 1973; Beaudet et al., 1975; Gehler et al., 1974). Material crossreacting with BG antibody in fibroblasts from four patients suggested the involvement of the structural gene in the disorder (Bell et al., 1977).

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It has become clear in recent years that the most

effective approach to the study of mutation in inherited metabolic disorders must involve the isolation of the relevant gene. Most methods to achieve this objective suffer from the low quantities of mRNA and protein usually specified by these genes. Because many gene sequences are expected to be higly conserved between species, an alternative approach to their isolation is the use of the cloned gene or cDNA from another species as a probe. This opportunity was available in the case of human BG since a murine cDNA clone (pGUS-1) was recently isolated (Palmer et al., 1983). This murine clone was isolated by hybrid selection techniques from a cDNA library established from androgen induced murine kidney mRNA. We have used pGUS-1 to screen a human cDNA library constructed from SV40-transformed human fibroblasts (Okayama and Berg, 1982; 1983). Here we report the isolation and partial characterization of cDNA clones coding for human BG. These clones will be important for the ex~ination of the genetic basis of mucopolysacch~dosis type VII and to provide molecular probes for the diagnosis of this disease.

MATERIALS

AND METHODS

(a) Colony screening The l-32-kb ~~~I-~~~dIII fragment of pGUS-1, the murine BG cDNA clone (Pahner et al., 1983), was eluted from a 1.2% agarose gel by the method of Gervitz et al. (1980). One pg of the isolated fragment was nick translated with [a-32P]dCTP (3000 Ci/mmol, New England Nuclear) and purified by Sephadex G-50 spun column chromatography (Maniatis et al., 1982). The SV40-transformed human libroblast cDNA library, generously supplied by Drs. Okayama and Berg, was plated at a density of lo4 colonies/l00 mm2 petri dish, on LB plates cont~mg 100 pg Ap/ml. Autoclaved nitrocellulose filers were applied to each plate, incubated overnight at 37 “C, amplified by overnight incubation on LB plates with Ap (100 pg/ml) and spectinomycin (300 pg/ml), and processed according to Grunstein and Hogness (1975).

~ehyb~dization and hyb~d~ation were done in a Seal-a-Meal bags, in batches of 24-30 ftlters, overnight at 37”C, in 40-50 ml of each solution (Willard et al., 1983). Nick-translated probe was boiled for 10 min and added to the hybridization bags at a concentration of 5 x 106-1 x lo7 cpm/ml of hybridization solution. Filters were washed at 65 ’ C (Schmeckpeper et al., 1981). (b) ~aracte~zati~

of clones

Rapid plasmid DNA preparations were made by the boiling method of Holmes and Quigley (198 1). Purified plasmid DNA was prepared by CsCl density gradient centrifugation after Triton X-100 lysis (Maniatis et al., 1982). Restriction mapping was by standard methods (Maniatis et al., 1982), with preliminary restriction data on pGUS-1 provided by D’Amore, M.A., Gallagher, P.M. and Ganschow, R.E. (unpub~shed). (c) Nu~leotide DNA sequence analysis Restriction fragments from both pGUS-1 and the pHUG clones were eluted from agarose gels and cloned into the M13mplO and mpll phages (Messing and Vieira, 1982). The single stranded templates were prepared and subjected to dideoxy sequencing (Sanger et al., 1977; Sanger and Coulsen, 1978), as per the New England Biolab protocol. (d) Expression in E. coli A 1150-bp SalI-BarnHI fragment of pHUG87 was isolated from polyacrylamide gels (Maniatis et al., 1982). This fragment was inserted into the polylinker in the amino terminal coding region of the 1ucZ gene of pUC9 (Messing and Vieira, 1982). Sl nuclease digestion was by standard methods (Maniatis et al., 1982) using 5-10 units of Sl nuclease per 20 pg of DNA for 10 min at room temperature. The reaction was terminated by phenol extraction and ethanol precipitation, and the linearized plasmid recircularized with T4 DNA ligase. pUC plasmids were transfected into E. coli JM83 and examined for lactose utilization on MacConkey indicator media. Expression of IPTG-inducible fusion proteins was done in E. coli D1210 (ZacZq) (Sader et al., 1980). One-ml cultures were grown in M9 medium in

107

A 66,, = 0.1 and amplified by incubating overnight in the presence of 20 pg Cm/ml (Maniatis et al., 1982). The cultures were microfuged and radiolabelled by resuspension and incubation in 1.0 ml M9 (lacking MgSO,) containing 100 #Zi of [s% jmethionine (1100 Ci/mmol, New England Nuclear) for 45 min. Induced cultures were radiola~~ed as above except that 1 mM IPTG was included during the last 3 h of Cm treatment and during labelhng (Neidhart et al., 1980). Cells were microfuged, resuspended in 1.Oml of precipitation buffer (20 mM Tris pH 7.5, 1.0% NP40, 1.0% sodium deoxycholate, 150 mM NaCl, 0.2% SDS; Lassam et al., 1979) and lysed by sonication. Cell debris was removed by 5 min centrifugation in a microfuge. Samples were stored at -20°C. Normal goat IgG was purchased from Rappel Labs. The immune IgG fraction was prepared from antiserum raised in goat against human placental BG (Brot et al., 1978). Equivalent concentrations of the two IgG fractions were cleared by preincubation with excess unlabelled E. cd’ D1210 extracts three times prior to use in immunoprecipitation experiments. Radiolabelled extract (100 ~1) was incubated overnight at 4°C with constant rotation with the equivalent of 20 ~1 anti-BG IgG or control IgG and 100 jil of a 10% suspension of ~~~~~y~~c~cusprotein A-Sepharose beads (Ph~acia) in a total volume of 1 ml. The next day, the beads were collected, washed three times with 100 mM Tris - HCI pH 7.5,250 mM LiCl,, 0.1% 2-mercaptoethanol, and suspended in 50 ~1 of loading buffer (Laemmli, 1970). The beads were boiled for 3 min to release immune complexes, centrifuged and the supematants were analyzed by SDS-PAGE on 12% polyacrylamide slab gels (Laemmli, 1970). pHUG

RESULTS

(a) Characterization of cDNA clones The 1.38-kb &&Hind111 fragment of the murine BG cDNA clone, pGUS-1 was used to screen the human fibroblast cDNA library. 17 probe-binding clones were isolated by screening approx. lo6 of the estimated 1.4 x 10” independently derived recombinants of the library. All 17 clones were identical by restriction enzyme analysis and differed only in length. The eight longest clones proved to be indistinguishable in length, approx. 1.95 kb, as determined by agarose gel electrophoresis. Two of the clones, pHUG 87 and pHUG 86.1, were chosen for further study. Relatedness of the mouse and human cDNA clones was initially assessed by extensive restriction mapping of pGUS-1 and pHUG87. These clones were found to share a significant number of restriction sites (7/16, see Fig. 1). One highly homologous region contained three coincident restriction sites, PvuII-BamHI-SstI, in a span of approx. 190 bp. This fragment was selected for sequencing to provide a direct comparison of pGUS-1 and a pHUG clone. The mouse and human fragments are each 198 bp in length and show a DNA sequence homology of 80 %. Both sequences contain a single ORF with an amino acid sequence homology of 73% (Fig. 2). (b) Expression of pUC9 fusion protein To confii that pHUG87 coded for BG, the 1150-bp SalI-BamHI fragment was subcloned into the expression vector pUC9. The resultant plasmid, pJW1, was expected to encode a fusion polypeptide

87 (p> 5-

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F

C

T

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B

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Fig. 1. Restriction maps of pGUS-1 and pHUG87. Enzymes represented are: A, AvuI; B, BumHI; C, CM; D, A@; F, BglII; G, BglI; H, HindIII; L, SalI; N, BunII; P, PstI; S, Sill; ‘I’,SstII; and V, PuuII. (P) indicates the Psri site of the pCD vector (Okayama and Berg, 1983). Enzymes not cieaving either pGUS-1 or pHUG87 are BunI, Be& BsfEII, EcoRI, KpnI, MuI, Pvuf, SmaI, X&I, and XhoI. Lower-case letters indicate fragments subcloned into Ml3 for sequencing: a, pHUG PvuII-&I; b, pGUS-1 PvuII-SstI; c, pHUG SalI-BarnHI.

108

pHUG86.1 pcus-1

1

pHUG86.1 pGUS-1

52

pHUG86.1

103

pHWG86.1 pGus-1

154

pGUS-I

Gln Leu Ala Thr Gln Phe Glu Asn Trp Tyr Lys Lys Tyr Gln Lys Pro Ile CAG CTG GCC ACC CAG TTT GAG AAC TGG TAT AAG AAG TAT CAG MG CCC ATT AAT G cc G Am Ser Thr His

51

Ile Gin Ser Glu Tyr Gly Ala Glu Thr Ilr Ala Gly Phe His Gin Asp Pro ATI!CAG AGC GAG TAT GGA GCA GAA ACG ATT GCA GGG TTT CAC CAG GAT CCA C CGA CC AC G C G Asp Ala Pro Ile Glu

102

Pro Leu Met Phe Thr Glu Glu Tyr Gln Lys Ser Leu Leu Glu Cln Tyr His. CCT CTG ATG TI'CACT GAA GAG TAC CAG AAA AGT CTG CTA GAG CAG TAC CAT GC G G GGC GT G AT Ser Ala Val Asn AW

153

Leu Gly Leu Asp Gln Lys Arq Arg Lys Tyr Val Val Gly Glu Leu CTG GGT CTG GAT CAA AAA CGC AGA AAA TAT GTG G'l.T GGA GAG CTC TCA T G TAG C C Ser Val Lys Glu

198

Fig. 2.DNA sequence and predicted amino acid sequence comparison of the PvuII-&I restriction fragment from murine (pGUS-1) and human (pHUG-86.1) BG clones. Those nucleotides and amino acids of murine BG which differ from human BG are shown below the human sequences.

of 350 amino acidsif+the insert contained an ORF in phase with the lacZ gene. pJW 1 was introduced into E. coii D1210 (Zuclq) and [35S]methioninelabelled protein extracts were prepared from both uninduced and IPTG-induced cultures. These were immunoprecipitated with goat anti-human BG IgG (Fig. 3, lanes 1 and 2). No inducible plasmidencoded protein was detected To place the SatI-EarnHI fragment in phase with the 1ucZ gene, pJW1 was digested with Sal1 and a mild S 1 nuclease digestion was conducted to remove the Sal1 cohesive ends. Under these conditions, minimal digestion of double-stranded sequences was anticipated. Following digestion, the DNA was religated and treated with Sal1 to prevent reisolation of pJW1. E. co& D1210 was transformed with the religated plasmid, and 18 SalI - clones were selected (PJW2 to 19). All of the recombinants had lost the ErrI and Hind111 sites of the polylinker, indicating that the S 1 treatment had removed at least 10 bp double-stranded sequence around the Sal1 site. The heterogeneity of the Sl nuclease digestion was expected to shift some of the clones into phase with the laclz gene. One of the clones, pJW18, showed a 35-kDal polypeptide upon immunoprecipitation of an extract of induced culture (Fig. 3, lane 7). This protein band was not observed when either uninduced extracts were treated with specific antibody (lane 6) or induc-

ed extracts were treated with normal goat IgG (lane 8). A protein of 35 kDaI approximates the expected size of a fusion polypeptide encoded by most or all of the MI-BumHI fragments. The absence of this protein in extracts of induced pUC9 cells (not shown) or the uninduced cells containing pJW18 indicates that the protein is a product of the luc promoter-initiated fusion transcript. Finally an extract of induced pJW 16, another of the S 1 nuclease generated isolates, was negative for the 35-kDal polypeptide (lanes 3 and 4) and was apparently still out of phase. This isolate provides an induced plasmid control that most closely mimics pJW18. We conclude that pJWl8 encodes a protein fragment corresponding to about halfthe mature size of human BG.

DISCUSSION

Palmer et al. (1983) have demonstrated that pGUS-1 encodes a portion of the murine BG mRNA by hybrid selection and specific immunoprecipitation using goat anti-mouse BG antisera. CatteraIl and Leary (1983) have also isolated BG cDNA clones from androgen-induced mouse kidneys. We have used a fragment of pGUS-1 to isolate human BG cDNAs (pHUG). Two inde-

109

pendent lines of evidence establish that the pHUG clones described in this report correspond to human BG. First, the mouse pGUS-1 probe detected a single class of 17 related human isolates which prov-

Fig. 3. SDS-PAGE I analysis of immunoprecipitates from [3sS]methionine-la~lled extracts of E. coii D1210 cultures harbouring pJW plasmids. IPTG-induced and uninduced cultures were harvested, lysed and immunoprecipitated as described in the MATERIALS AND METHODS, section d. Immunoprecipitates were run on 12% polyacrylamide slab gels. Lane 1, pJW1, uninduced; lane 2, pJW1, induced; lane 3, pJWl6, uninduced; lane 4, pJWl4, induced; lane 6, pJW18, uninduced; lane 7, pJW18, induced; lane 8, pJW18, induced. Lanes l-7 contain samples immunoprecipitated with goat-anti-human BG IgG, while sample in lane 8 was immunoprecipitated with normal goat IgG.

ed to be homologous to the mouse clone upon comparison of restriction maps and sequence of corresponding 198-bp cDNA fragments. Secondly, in the pUC9 expression experiment, a 35-kDal plasmidspecific inducible fusion protein was generated which was specifically immunoprecipitated by goat anti-human BG IgG. The sequence of the conserved 198-bp PvuII-SstI fragments from both pGUS-1 and pHUG 86.1 contain single ORFs. The high homology of the nucleotide and predicted amino acid sequences of the two clones suggests that this region encompasses part of the polypeptide coding sequence. The longest pHUG clones are approx. 1.95 kb long. Based on the M, of 70 x lo3 for the BG monomer, 1.9 kb of mRNA would be required for the coding sequence alone. Furthermore, the BG mRNA from mouse is estimated to be 2.6-2.8 kb long (Palmer et al., 1983 ; Catterall and Leary, 1983) and preliminary experiments using pHUG as a probe suggest that the human tibroblast mRNA is of similar length. Thus the eight longest pHUG clones are probably missing OS-l.0 kb of 5’ sequence. The isolation of cDNA clones for human BG provides molecular probes for the examination of the mutations causing mucopolysaccharidosis type VII. The structure and expression of the mutant genes can now be examined directly. In addition the identification ofrestriction fragment length polymorphisms linked to the BG gene will permit carrier detection and prenatal diagnosis for affected families.

We are grateful to P.M. Gallagher, H.F. Willard, M. dAmore, B. Williams, L.-C. Tsui, M. Breitman and L. Siminovitch for helpful discussion. K.S.G. is a recipient of a Hospital for Sick Children Foundation Foreign Postdoctoral Fellowship. These studies were supported by M.R.C. Grant PG-4 to R.A.G. REFERENCES Beaudet, A.L., DiFerranti, N.M., Ferry, G.D., Nichols, B.L. and Mull&., C.E.: Variation in the phenotypic expression of fi-glucuronidase deficiency. J. Pediat. 86 (1975) 388-394.

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Bell Jr., C.E., Sly, W.S. and Brot, F.E.: Human p-glucuronidase deficiency mucopolysaccharidosis, J. Clin. Invest. 598 (1977) 97-10.5. Brot, F.E., Bell Jr., C.E. and Sly, W.S.: Purification and properties of/?-glucuronidase from human placenta. Biochemistry 17 (1978) 385-391. Catterall, J.F. and Leary, S.L.: Detection of early changes in androg~-induced mouse renal ~-glucuronidase messenger ribonucleic acid using cloned complementary deoxyribonucleic acid. Biochemistry 22 (1983) 6049-6053. Gehler, J.M., Cantz, M., Tolksdorf, M., Spranger, J., Gilbert, E. and Drube, H.: Mucopolysaccharidosis VII: fl-glucuronidase deficiency. Humangenetik 23 (1974) 149-1.58. Gervi_tz, SC., Bacchetti, S., Rainbow, A.J. and Graham, F.L.: A rapid and efficient procedure for the purification of DNA from agarose gels. Anal. Biochem. 106 (1980) 492. Grunstein, M. and Hogness, D.: Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene, Proc. Natl. Acad. Sci. USA 72 (1975) 3961-3965. Holmes, D.S. and Quigley, M.: A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114 (1981) 193-197. Laemmli, U.V.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (1970) 680-685. Lassam, N.J., Bayley, ST. and Graham, F.L.: Tumor antigens of human Ad5 in transformed cells and in the cells infected with transformation defective host-range mutants. Cell 18 (1979) 781-791. Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. Messing, J. and Vieira, J.: A new pair of Ml3 vectors for selecting either DNA strand of double-digest restriction fragments. Gene 19 (1982) 269-276. Neidhardt, F.C., Writh, R., Smith, M.W. and Bogelen, R.V.:

Selective synthesis of plasmid-coded proteins by Escherichia coli during recovery from chloramphenicol treatment. J. Bac-

teriol. 143 (1980) 535-538. Okayama, H. and Berg, P.: High efficiency cloning of full length cDNA. Mol. Cell. Biol. 2 (1982) 161-170. Okayama, H. and Berg, P.: A cDNA cloning vector that permits expression of cDNA inserts in rna~~~ cells. Mol. Cell. Biol. 3 (1983) 280-289. Paigen, K.: Acid hydrofases as models of genetic control. Annu. Rev. Genet. 13 (1979) 417-466. Palmer, R., Gallagher, P.M., Boyko, W.L. and Ganschow, R.E.: Genetic control of levels of murine kidney glucuronidase mRNA in response to androgen. Proc. Natl. Acad. Sci. USA 80 (1983) 7596-7600. Sadler, J.R., Tecklenberg, M. and Betz, J.L.: Plasmids containing many tandem copies of a synthetic lactose operator. Gene 8 (1980) 279-300. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with ch~-te~ating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467. Sanger, F. and Coulson, A.R.: The use of thin acrylamide gels for DNA sequencing. FEBS Lett. 87 (1978) 107. Schmeckpeper, B.J., Willard, H.F. and Smith, K.D.: Isolation and characterization of cloned human DNA fragments carrying reiterated sequences common to both autosomes and the X chromosomes. Nucl. Acids Res. 9 (1981) 1853-1872. Sly, W.S., Quinton, B.A., McAlister, W.H. and Rimoin, D.L.: Beta glucuronidase deficiency: report of clinical radiologic and biochemical features of a new mucopolysaccharidosis. J. Pediat. 82 (1973) 249-257. Willard, H.F., Smith, K.D. and Sutherland, J.: Isolation and ch~acter~ation of a major tandem repeat family from the human X chromosome. Nucl. Acids Res. 11 (1983) 2017-2033. Communicated by R.W. Davies.