THE JOURNAL OF Bro~ocrc.a CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 265, No. 29, Issue of October 15, pp. 17792%17797,199O Printed in U.S. A. Molecular Basis of Tyrosinase-negative Oculocutaneous Albinism A SINGLE BASE MUTATION IN THE TYROSINASE GENE CAUSING ARGININE TO GLUTAMINE SUBSTITUTION AT POSITION 59* (Received for publication, December 11, 1989) Atsushi TakedaS, Yasushi TomitaQ, Jun MatsunagaQ, Hachiro TagamiQ, and Shigeki ShibaharaS?l From the SDepartment of Applied Physiology and the SDepartment of Dermatology, Tohoku University School of Medicine, Sendai, Miyugi 980, Japan Tyrosinase-negative oculocutaneous albinism (OCA) is one of classical inborn errors of metabolism, char- acterized by a complete lack of melanin pigments in the eyes and skin. We have isolated and characterized the tyrosinase gene of one child (F. S.) affected with tyrosinase-negative OCA. Sequence analysis reveals a single-base mutation in the exon 1 (a G to A transition at nucleotide residue 312), causing the Arg (CGG) to Gln (CAG) substitution at position 59. This base change eliminates one MspI site and creates a new BstNI site in the patient’s exon 1, which is invaluable for screen- ing other OCA patients and heterozygote carriers for this mutation. We are thus able to confirm that the patient F. S. is homozygous for this OCA allele. The family members of the patient F. S. are phenotypically normal, but are shown to be heterozygote carriers. Transfection of the mutant gene fails to give rise to detectable tyrosinase activity in transient expression assays, suggesting that the mutation affects the stabil- ity or the catalytic activity of the enzyme. We therefore propose that the albino phenotype of the patient F. S. is a consequence of the Arg to Gln substitution at position 59 caused by a point mutation in the tyrosinase gene. Tyrosinase (EC 1.14.18.1) is a bifunctional copper-contain- ing enzyme responsible for the conversion of tyrosine to dihydroxyphenylalanine (DOPA)’ and of DOPA to dopaqui- none (l-3) and plays a central role in melanin biosynthesis. Albinism comprises a heterogenous group of heritable disor- ders of melanin formation in the pigment cells (melanocytes) and is classified into two common forms: oculocutaneous albinism (OCA) and ocular albinism. Various types of OCA have been described in humans (4) and one of them, tyrosin- ase-negative OCA, inherited in an autosomal recessive fash- ion, is characterized by a complete absence of melanin pig- ments in the skin, hair, and eyes, which leads to visual disturbances caused by optic neurologic defects and also pre- disposes the patients to skin cancer. Since no tyrosinase * This work was supported in part by grants (to Y. T.) from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence should be addressed: Dept. of Applied Physiology, Tohoku University School of Medicine, Sendai, Miyagi 980, Japan. 1 The abbreviations used are: DOPA, dihydroxyphenylalanine; OCA, oculocutaneous albinism; PCR, polymerase chain reaction; kb, kilobase pair(s). activity is detectable in the pigment cells of the patients affected with tyrosinase-negative OCA, this type of OCA has been suggested to involve mutations in the tyrosinase gene (4). Recently, we have isolated and characterized the tyrosin- ase gene of one tyrosinase-negative OCA patient S. S., re- vealing a single base insertion in the exon 2 that shifts the reading frame and introduces a nonsense mutation (5). We were thus able to provide direct evidence that the mutation in the tyrosinase gene could lead to albino phenotype (5). In this report, we demonstrate that the tyrosinase gene of the other patient F. S. carries a G to A transition at nucleotide residue 312, leading to a single amino acid substitution, argi- nine at position 59 (Arg 59) to glutamine. The mutant gene under a heterologous promoter is unable to direct the tran- sient expression of tyrosinase activity, suggesting that tyro- sinase containing Gln at position 59 is unstable or catalyti- cally inactive. We therefore propose that this mutation is responsible for the OCA-phenotype of the patient F. S. We also discuss potential roles of Arg-59 in the function of tyro- sinase. EXPERIMENTAL PROCEDURES Preparation of Genomic DNA-Peripheral lymphocytes, collected from three Japanese patients (F. S., M. T., and S. S.) affected with tyrosinase-negative OCA, were transformed using Epstein-Barr virus (6) and used as sources of genomic DNA. Genomic DNA of parents and sibling of F. S. were prepared from peripheral blood. Control DNA was prepared from the placenta of phenotypically normal in- dividual. The family history of the patient F. S. reveals no consan- guineous marriages. Cloning and Sequencing of Genomic DNA Encoding Human Tyro- s&me-The genomic DNA library of the patient F. S. was constructed in EMBL3 (7) using Mb01 partial digests of the transformed lympho- cytes DNA. The library was screened for DNA segments encoding tyrosinase using the human tyrosinase cDNA as a hybridization probe. The probe used was the SalI(PstI)/XbaI(NdeI) fragment (59/ 1892) containing a full-length human tyrosinase cDNA, derived from the expression plasmid pRHOHT2 (8), and labeled with [a-32P]dCTP by the random priming method (9). The numbers in parentheses, shown together with restriction enzymes, indicate the 5’-terminal nucleotide generated by cleavage. Both sites for PstI and NdeI were eliminated during the construction of the expression plasmid pRHOHT2 and shown within parentheses (8). Nucleotide sequences were determined by the method of Maxam and Gilbert (10). Direct Sequencing of a PCR-amplified Human Genomic DNA Seg- ment-Genomic DNA, extracted from either transformed lympho- cytes (patient F. S.) or placenta (phenotypically normal individual), was subjected to 30 cycles of polymerase chain reaction (PCR) (11, 12) of nucleotides spanning exon 1 of the tyrosinase gene. An ampli- fication cycle was 1 min at 94 “C for denaturing, 2 min at 55 “C for annealing, and 3 min at 72 “C for extension under conditions rec- ommended by the manufacturer (Perkin-Elmer Cetus Instruments). The two primers (20-mer) used for the PCR amplification were: 5’- CTGCAGACCTTGTGAGGACT-3’ (54/73) and 5’-GTATATCT- AGCATATCTTAC-3’, designated as Pl and P2, respectively (see Fig. 1A). The latter sequence is complementary to the sequence of 17792 by guest on October 5, 2015 http://www.jbc.org/ Downloaded from
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THE JOURNAL OF Bro~ocrc.a CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 265, No. 29, Issue of October 15, pp. 17792%17797,199O Printed in U.S. A.
Molecular Basis of Tyrosinase-negative Oculocutaneous Albinism A SINGLE BASE MUTATION IN THE TYROSINASE GENE CAUSING ARGININE TO GLUTAMINE SUBSTITUTION AT POSITION 59*
(Received for publication, December 11, 1989)
Atsushi TakedaS, Yasushi TomitaQ, Jun MatsunagaQ, Hachiro TagamiQ, and Shigeki ShibaharaS?l From the SDepartment of Applied Physiology and the SDepartment of Dermatology, Tohoku University School of Medicine, Sendai, Miyugi 980, Japan
Tyrosinase-negative oculocutaneous albinism (OCA) is one of classical inborn errors of metabolism, char- acterized by a complete lack of melanin pigments in the eyes and skin. We have isolated and characterized the tyrosinase gene of one child (F. S.) affected with tyrosinase-negative OCA. Sequence analysis reveals a single-base mutation in the exon 1 (a G to A transition at nucleotide residue 312), causing the Arg (CGG) to Gln (CAG) substitution at position 59. This base change eliminates one MspI site and creates a new BstNI site in the patient’s exon 1, which is invaluable for screen- ing other OCA patients and heterozygote carriers for this mutation. We are thus able to confirm that the patient F. S. is homozygous for this OCA allele. The family members of the patient F. S. are phenotypically normal, but are shown to be heterozygote carriers. Transfection of the mutant gene fails to give rise to detectable tyrosinase activity in transient expression assays, suggesting that the mutation affects the stabil- ity or the catalytic activity of the enzyme. We therefore propose that the albino phenotype of the patient F. S. is a consequence of the Arg to Gln substitution at position 59 caused by a point mutation in the tyrosinase gene.
Tyrosinase (EC 1.14.18.1) is a bifunctional copper-contain- ing enzyme responsible for the conversion of tyrosine to dihydroxyphenylalanine (DOPA)’ and of DOPA to dopaqui- none (l-3) and plays a central role in melanin biosynthesis. Albinism comprises a heterogenous group of heritable disor- ders of melanin formation in the pigment cells (melanocytes) and is classified into two common forms: oculocutaneous albinism (OCA) and ocular albinism. Various types of OCA have been described in humans (4) and one of them, tyrosin- ase-negative OCA, inherited in an autosomal recessive fash- ion, is characterized by a complete absence of melanin pig- ments in the skin, hair, and eyes, which leads to visual disturbances caused by optic neurologic defects and also pre- disposes the patients to skin cancer. Since no tyrosinase
* This work was supported in part by grants (to Y. T.) from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 To whom correspondence should be addressed: Dept. of Applied Physiology, Tohoku University School of Medicine, Sendai, Miyagi 980, Japan.
1 The abbreviations used are: DOPA, dihydroxyphenylalanine; OCA, oculocutaneous albinism; PCR, polymerase chain reaction; kb, kilobase pair(s).
activity is detectable in the pigment cells of the patients affected with tyrosinase-negative OCA, this type of OCA has been suggested to involve mutations in the tyrosinase gene (4). Recently, we have isolated and characterized the tyrosin- ase gene of one tyrosinase-negative OCA patient S. S., re- vealing a single base insertion in the exon 2 that shifts the reading frame and introduces a nonsense mutation (5). We were thus able to provide direct evidence that the mutation in the tyrosinase gene could lead to albino phenotype (5).
In this report, we demonstrate that the tyrosinase gene of the other patient F. S. carries a G to A transition at nucleotide residue 312, leading to a single amino acid substitution, argi- nine at position 59 (Arg 59) to glutamine. The mutant gene under a heterologous promoter is unable to direct the tran- sient expression of tyrosinase activity, suggesting that tyro- sinase containing Gln at position 59 is unstable or catalyti- cally inactive. We therefore propose that this mutation is responsible for the OCA-phenotype of the patient F. S. We also discuss potential roles of Arg-59 in the function of tyro- sinase.
EXPERIMENTAL PROCEDURES
Preparation of Genomic DNA-Peripheral lymphocytes, collected from three Japanese patients (F. S., M. T., and S. S.) affected with tyrosinase-negative OCA, were transformed using Epstein-Barr virus (6) and used as sources of genomic DNA. Genomic DNA of parents and sibling of F. S. were prepared from peripheral blood. Control DNA was prepared from the placenta of phenotypically normal in- dividual. The family history of the patient F. S. reveals no consan- guineous marriages.
Cloning and Sequencing of Genomic DNA Encoding Human Tyro- s&me-The genomic DNA library of the patient F. S. was constructed in EMBL3 (7) using Mb01 partial digests of the transformed lympho- cytes DNA. The library was screened for DNA segments encoding tyrosinase using the human tyrosinase cDNA as a hybridization probe. The probe used was the SalI(PstI)/XbaI(NdeI) fragment (59/ 1892) containing a full-length human tyrosinase cDNA, derived from the expression plasmid pRHOHT2 (8), and labeled with [a-32P]dCTP by the random priming method (9). The numbers in parentheses, shown together with restriction enzymes, indicate the 5’-terminal nucleotide generated by cleavage. Both sites for PstI and NdeI were eliminated during the construction of the expression plasmid pRHOHT2 and shown within parentheses (8). Nucleotide sequences were determined by the method of Maxam and Gilbert (10).
Direct Sequencing of a PCR-amplified Human Genomic DNA Seg- ment-Genomic DNA, extracted from either transformed lympho- cytes (patient F. S.) or placenta (phenotypically normal individual), was subjected to 30 cycles of polymerase chain reaction (PCR) (11, 12) of nucleotides spanning exon 1 of the tyrosinase gene. An ampli- fication cycle was 1 min at 94 “C for denaturing, 2 min at 55 “C for annealing, and 3 min at 72 “C for extension under conditions rec- ommended by the manufacturer (Perkin-Elmer Cetus Instruments). The two primers (20-mer) used for the PCR amplification were: 5’- CTGCAGACCTTGTGAGGACT-3’ (54/73) and 5’-GTATATCT- AGCATATCTTAC-3’, designated as Pl and P2, respectively (see Fig. 1A). The latter sequence is complementary to the sequence of
the 5’ end of the intron 1 (see Table I). The amplified fragments were then gel-purified and subjected to 30 cycles of asymmetric amplification (13) using the primer, Pl. Nucleotide sequence of the products was determined by dideoxynucleotide chain termination method using3’P-end-labeled primer 5’-GAAGTTGCCAGAGCACT- 3’ (complementary to 351/367) as described (12, 13), with modifica- tions for the use of Taq polymerase (14).
Cell-free Transcription-Whole cell extracts were prepared from mouse &16 melanoma cells and cell-free transcription was carried out as describedmeviouslv (15). The subcloned plasmid, SpAFT2, derived from the X AFT1 (see Fig: lA), contains the 5’-flancing region and the exon 1 of the tyrosinase gene and was used as a template DNA. Transcribed products were identified by Sl nuclease mapping analysis as described previously (16). The Sl probe used was the EcoRI/ Sau961 fragment (about 460 nucleotides of the 5’-flanking region/ 279) end-labeled with 32P at the Sau961 site.
Southern Blotting Analysis-Genomic DNA, extracted from either transformed lvmnhocvtes (three OCA patients: F. S., M. T. and S. S.) or placenta (~hen&ypic~lly normal individual), was digested with restriction enzymes and subjected to Southern blotting analysis as described (17). The exon l-specific probe, the SalI(PstI)/Z’aqI frag- ment (59/454), was prepared from the pRHOHT2 (8).
Genotype Analysis-Genomic DNA segment containing the exon 1 was amplified as described above. The amplified fragments were digested with HpaII or BstNI, and subjected to a 5% polyacrylamide gel electrophoresis.
Construction of Expression Plasmid Carrying the Mutation-The PstI/SalI fragment (59/linker), isolated from the h AFTl, carries the exon 1 and a part of the intron 1 of the tyrosinase gene of the patient (F. S.), and the single-stranded end of the PstI site was digested with Klenow enzyme, ligated to Sal1 linker, and sequentially digested with Sal1 and TmI. The resulting SalI(PstI)/Z’oqI fragment (59/454) was purified andused as the 5cpart of the fusion cDNA encoding the tvrosinase of the natient (F. S.). The PstI site, eliminated bv these procedures, was shown within a parenthesis. This fragment and the 3’-part of the cDNA, TuqI/XbaI(NdeI) fragment (454/1892) derived from the wild-type construct, pRHOHT2 (8), were ligated to the larger fragment of the pRHOgpt2 (18) linearized with Sal1 and XbaI. The expression plasmid, pRHOHTM2, thus obtained, contains the A residue at 312. The mock construct, pRHOHT0, contains the RsaI/ NdeI truncated tyrosinase cDNA fragment (102/1892) as described previously (8).
Functional Analysis of the Albino Tyrosinase cDNA-Mouse K1735 amelanotic melanoma cells were transfected as described previously (19). After glycerol shock, transfected cells were incubated for 20 h at 37 “C. Cells were then treated for 1 h at 42 “C, incubated for additional 2 h at 37 “C and used for preparation of total RNA. The transient expression of each construct was confirmed by Sl nuclease- mapping analysis. The Sl probe was same as that used for cell-free transcription (see Fig. lA). Alternatively, transfected cells were treated for 1 h at 42 “C and incubated for additional 16 h at 37 “C. Cells were fixed with cold (-20 “C) acetone for 10 s and incubated for 6 h at 37 “C with 5 mM DOPA solution in 0.1 M DhosDhate buffer, pH 7.4 (20). Tyrosinase (DOPA oxidase)-positive cells-were micro: scopically identified. For the assay of tyrosine hydroxylase activity, the rest of transfected cells was harvested with 0.02% EDTA solution in phosphate-buffered saline, washed two times with phosphate- buffered saline containing Ca” and Mp, and frozen in liquid nitro- gen. Cells were then thawed, lysed in 0.1% Triton X-100 in phosphate- buffered saline, and used for the assay of tyrosine hydroxylase (21).
RESULTS AND DISCUSSION
Cloning and Sequencing Analysis of the Genomic DNA Seg- ments Encoding Patient’s Tyrosinase-More than 10 phage clones were isolated from the genomic DNA library of the patient F. S. and five of them were subjected to sequencing analysis (Fig. L4). The X AFTl, harboring the 5’-flanking region and the exon 1, overlapped with the X AFT5, containing a part of the exon 1 and the exon 2, although other clones were not overlapping (Fig. 1A). The size of the human tyro- sinase gene is thus expected to be greater than 70 kb and organized in five exons.
The nucleotide sequence of the 5’-flanking region, all exon/ intron junctions and all exons of the cloned F. S. tyrosinase gene, was determined. We thus identified a single base mu-
tation, a G to A transition, at nucleotide residue 312 (Fig. 1A), which was also verified by direct sequencing of the genomic DNA segment (Fig. 1B). This base change alters a CGG codon to a CAG codon, causing the Arg to Gln substi- tution at position 59. The sequences around the exonlintron junctions of the F. S. tyrosinase gene are summarized in Table I, since these sequences are identical to those of the wild-type gene and are of significance for designing PCR primers used for exon amplification.
Promoter Function of the F. S. Tyrosinase Gene in Vitro- We wanted to know whether the tyrosinase gene is expressed in patient’s melanocytes. However, it is practically impossible in Japan to excise skin strips for the isolation of melanocytes from the patient F. S., because such an operation is liable to cause hypertrophic scar in the excised area. Alternatively, we established the cell-free transcription system derived from mouse melanoma cells and explored the promoter function of the F. S. tyrosinase gene in vitro, since the tyrosinase gene is specifically expressed in pigment cells and the transcripts from the cloned tyrosinase gene were not detectable in HeLa whole cell extracts (data not shown). The template DNA used was the cloned DNA segment carried by the X AFTl, which harbors the 5’-flanking region and the exon 1 (Fig. lA). The Sl probe used was described under “Experimental Proce- dures” (Fig. lA). The presence of a-amanitin-sensitive prod- ucts of about 280 nucleotides (shown by an open triangle in Fig. 1C) indicates that the tyrosinase gene of the patient F. S. was transcribed from the assigned initiation site (8), sug- gesting that its promoter is functional. It is therefore conceiv- able that the tyrosinase gene is expressed in the patient’s melanocytes.
Unique Exon 1 Sequence and Diagnostic MspI Fragment of the F. S. Tyrosinuse Gent-Recently, Barton et al. (22) re- ported that the tyrosinase cDNA hybridized to two sites on the human chromosome ll:llq14 + q21 on the long arm and 11~11.2 on the short arm. They indicated that the region on the long arm represents the tyrosinase locus and suggested that the sequence on the short arm represents a truncated pseudogene or a related gene cross-hybridizable to the BglII/ EcoRI fragment of the tyrosinase cDNA, Pmel 34 (23), con- taining mainly the exon 5 sequence. We therefore carried out genomic DNA blotting analysis using the full-length cDNA probe and detected five restriction fragments of about 2.1, 4.4, 5.1, 7.5, and 8.5 kb in PstI-digested DNA (24), which is consistent with the results reported by Barton et al. (22). Since two PstI fragments of 7.5 and 4.4 kb were shown to represent the hybridizable DNA segments located on the short arm (22), and there were no apparent differences in sizes and strength of hybridization signals between patients’ DNA and control DNA (24), the tyrosinase gene and its cross-hybrid- izable DNA segment are present in the genome of three OCA patients (F. S., M. T., and S. S.) as well as a healthy individual. The identity of the cloned exons was verified by sequence analysis, revealing the nucleotide sequence identical to that of the cDNA, coding for functional tyrosinase (8, 25). More- over, the cloned tyrosinase gene appears to retain the same sequence organization as in the genomic DNA of the patient F. S. and a healthy individual (a part of data shown in Fig. 2).
In order to confirm that the DNA segment encompassing the mutation at nucleotide residue 312 represents the true exon 1 of the tyrosinase gene and to exclude the possibilities of sequencing errors or cloning artifacts, we took advantage that the G to A transition at residue 312 eliminates the MspI (or HpaII) site (Fig. lA). Namely, using several restriction enzymes including MspI, we hybridized the genomic DNA
FIG. 1. Structural organization of the albino human tyrosinase gene. A, schematic representation of the albino tyrosinase gene. The direction of transcription is from left to right. Five phage clones, used for sequence analysis, are shown at. the top. Solid lines represent the genomic DNA segments carried by the isolated phage clones. Only relevant sites for EcoRI (E) and Hind111 (H) are shown. A part of the exon 1 sequence of the message strand containing a single base mutation is enlarged. The nucleotide residues shown are numbered from the transcription initiation site of the human tyrosinase gene (8,25). The protein-coding region is indicated by a closed box and the 5’-untranslated region is indicated by an open box. The Sl probe used in C is also shown. An asterisk indicates the site of “‘P-end labeling. B, direct sequencing of the genomic DNA segment containing a single base mutation. The sequence of the cDNA strand is shown. An asterisk indicates the nucleotide residue 312, where the mutant gene contains the T residue indicated by an arrowhead (the A residue on the message strand). C, cell-free tranScription. The subcloned plasmid, SpAFT2, was derived from the X AFT1 carrying the 5’-flanking region and the exon 1 of the tyrosinase gene and used as a template. The template DNA (40 rg/ml) was transcribed in mouse melanoma whole cell extracts, and the products were analyzed by Sl mapping. Lane I, transcripts produced in the absence of cr-amanitin; lane 2, transcripts in the presence of oc-amanitin (1 pg/ml). The open triangle indicates the expected protected fragments. Size markers are pUC8 DNA fragments generated by the digestion with HpaII and given in base pairs.
TABLE I Exon-intron organization of the human tyrosinuse gene
The nucleotide sequences of exons are shown in thick letters and those of introns are shown in thin letters. The nucleotide residues are numbered from the transcription initiation site (8, 25). The amino acids present in the exon/intron junctions are shown and the corresponding codons are underlined.
blots with the exon l-specific probe, showing a single band in DNA digested with three enzymes, BglII, PstI, and Hind111 (Fig. 2), indicating that the exon 1 is present as a unique sequence in human genome. There are no differences in the size and strength of hybridization signals among the genomic DNA of three OCA patients and a healthy individual, except for the MspI-digested DNA of the patient F. S., yielding a single band of 8.5 kb (indicated by an open triangle in Fig. 2). In contrast, the probe hybridized to two fragments of 1.9 and 6.6 kb in the MspI-digested DNA of other two patients (S. S. and M. T.) and a healthy individual. The molecular basis of OCA in these patients, S. S. and M. T., was shown to be the single base insertion between the nucleotide residues 1011 and 1012 of the tyrosinase gene, of which the exon 1 contains
no mutations at residue 312 (5). These results indicate that the exon 1 of the F. S. tyrosinase gene lacks the MspI site, which divides the 8.5-kb MspI fragment into the 1.9- and 6.6- kb fragments detected in the wild-type gene, confirming the presence of base change at nucleotide residue 312 in the F. S. tyrosinase gene.
Genotype Analysis of PCR-amplified Genomic DNA-The G to A transition at residue 312 eliminates the HpaII (or MspI) site and creates a new recognition site for BstNI (Fig. lA). The amplified DNA segments derived from the patient F. S. did contain the BstNI site instead of the HpaII site, whereas the segments derived from the phenotypically normal person and another patient S. S. contained the HpaII site and no BstNI site (Fig. 3). These results indicate that the patient F.
FIG. 2. Genomic DNA blotting analysis of the tyrosinase gene. Each lane contained 10 pg of genomic DNA digested with restriction enzymes indicated. Sources of genomic DNAs were the transformed lymphocytes from patients with tyrosinase-negative OCA (lone 1, F. S.; lane 2, M. T.; and lane 3, S. S.) and the placenta of phenotypically normal individual (lane 4). The hybridization probe was the exon l-specific fragment. Closed triangles indicate the positive signals representing the DNA fragments containing the exon 1 of the wild-type gene. The open triangle indicates the 8.5-kb fragment, specifying the MspI mutation.
868bp ._ .-‘. - .-
0
Mutant 868 b&l
61, c. --- -
Hpa II
IIt .
Wild type 2m7bp al t.p
257
222
El
- Wild type 832 bp
123456 FIG. 3. Pedigree and genotype analysis of the patient F. S.
with OCA. Squares represent male and a circle indicates female. Filled symbols indicate the albino phenotype (homozygote) and par- tially filled symbols indicate heterozygote carrier for OCA allele. Genomic DNAs were amplified for the DNA segment carrying the nucleotide residue 312. The G to A transition at 312 eliminates the HpaII site and creates the new BstNI site in the OCA allele. Shown are the digested patterns of the amplified DNA segments stained with ethidium bromide. Upper panel, digested with HpaII; lower panel, with BstNI. Amplified DNAs were derived from father of F. S. (lane I), mother of F. S. (lane 2), sibling of F. S. (lane 3), patient F.S. (lane 4), patient S. S. (lane 5), and normal individual (lane 6).
S. is homozygous for this mutation. Since the recognition sequence of BstNI is CC(A/T)GG, the F. S. tyrosinase gene must carry either A or T residue at 312, which is consistent with our sequencing data (Fig. 1, A and B).
We then studied the segregation of the allele bearing the A residue at 312 in his family. Since the family of F. S. is phenotypically normal, his parents are expected to be obligate carriers of this OCA trait. Indeed, the amplified DNA seg- ments derived from his parents and sibling contained both &a11 site and BstNI site, indicating that they are heterozy- gous for this allele (Fig. 3).
Arg to Gln Substitution at Position 59-We have assigned the histidine residue at position 1 as the amino-terminal end of human tyrosinase (25), which was recently verified by the report on the sequencing of the amino-terminal region of purified human tyrosinase (26). We were thus able to establish that human tyrosinase is composed of 511 amino acid residues excluding a signal peptide of 18 residues (8, 25).
Two human cDNAs, pHT y 1(25) and BBTY-1(27), coding for functional tyrosinase were cloned and sequenced. How- ever, there are two amino acid differences at positions 161 and 174 according to our numbering (25). Namely, the pHT y 1 contains the codons ATG (nucleotide residues 617-619) for Met at 161 and TCT (nucleotide residues 656-658) for Ser at 174 (25), whereas the other clone BBTY-1 contains the codons ATC for Ile at 161 and TAT for Tyr at 174 (27). Since both cDNAs are able to code for functional tyrosinase, these base changes may represent polymorphism. Incidentally, both base changes at nucleotide residues 619 and 657 are within the recognition sites for BanHI of BBTY-1 and Sau3AI of pHT y 1, respectively. Namely, the pHT y 1 contains GGATGC (nucleotide residues 615-620) and GATC (nucleo- tide residues 654-657), whereas the BBTY-1 contains GGATCC and GATA at corresponding positions (27). We were this able to confirm the absence of BumHI site and the presence of Sau3AI site in our tyrosinase cDNA and its gene (data not shown). The deduced amino acid sequence of func- tional mouse tyrosinase (28, 29) always favors the sequence deduced from the cDNA, pHT y 1.
Comparison of the nucleotide sequence of the F. S. tyrosin- ase gene with that of the functional wild-type cDNA (8, 25) reveals one base change: a G to A transition at nucleotide residue 312 of the tyrosinase gene, resulting in the substitution of Gln (CAG) for Arg (CGG) at 59. The Arg residue is also conserved at position 59 of functional mouse tyrosinase (28, 29). The significance of this substitution was then analyzed.
Functional Anulysis of the Tyrosinase Containing Gln-59- We investigated the consequence of the G to A transition at residue 312 in the tyrosinase gene on the catalytic activity of the tyrosinase using transient expression assay. Mouse amel- anotic melanoma cells were transfected with the expression plasmid pRHOHT2, containing the entire protein-coding re- gion of the human tyrosinase cDNA under the rat heme oxygenase gene promoter (18) or with the pRHOHTM2 con- taining the same insert as pRHOHT2, but with the G to A transition at residue 312 (Fig. 4A). The rat heme oxygenase gene promoter was chosen because of its inducibility by heat shock (18). Mouse amelanotic melanoma cells used contained no detectable levels of tyrosinase mRNA (data not shown) and of tyrosinase activity (Fig. 4C and Table II). The transient expression of each construct was confirmed by Sl nuclease- mapping analysis, showing the presence of similar amounts of RNA transcribed from the plasmid DNA introduced (Fig. 4B). About 1% of cells transfected with pRHOHT2 were melanin-deposited (DOPA oxidase-positive) (Fig. 4C and Table II), which is consistent with the efficiency of transient expression under similar conditions (5,8,19). Moreover, only cells transfected with the wild-type construct (pRHOHT2) showed detectable activity of tyrosine hydroxylase (Table II). In contrast, neither melanin-deposited cells nor tyrosine hy-
FIG. 4. Transient expression of the tyrosinase gene of patient F. S. A, schematic representation of the expression plasmids. Solid lines, 5’- and 3’.flanking regions of the rat heme oxygenase gene (18); closed box, heat shock element; stippled boxes, parts of exons 1 and 5 of the rat heme oxygenase gene; and open box, full-length tyrosinase cDNA. Both wild-type and mutant constructs are represented. The Sl probe was the same as that used in Fig. lC, and the expected product was shown as Sl. B, transient expression of each construct determined by Sl nuclease-mapping analysis. The protected fragments of about 220 nucleotides were indicated by an arrow. Lane 1, pRHOHT2; lane 2, pRHOHTM2; and lane 3, pRHOgpt2 (181, control plasmid. The size markers shown are pUC8 DNA fragments generated by the digestion with HpaII and given in base pairs. C, expression of DOPA oxidase activity. Shown are examples of the cells transfected with pRHOHT2 (a) or with pRHOHTM2 (b). Bars indicate 100 pm.
TABLE II Functional analysis of tyrosmase encoded by the wild-type or mutant gene
Mouse Kl735 amelanotic melanoma cells were transfected with the indicated expression plasmids and then assayed for DOPA oxidase or tyrosine hydroxylase activity as described under “Experimental Procedures.”
” Number of positive cells was counted (Fig. 4C). ” ND, not detectable.
droxylase activity were detected in the cells transfected with the construct carrying the mutation (pRHOHTM2) or with the mock construct (pRHOHT0) (Fig. 4C and Table II). Moreover, prior to these experiments described, we have confirmed that the amounts of RNA transcribed from the expression plasmid carrying mouse tyrosinase cDNA, Tyrs-J (29), paralleled with those of immunoreactive tyrosinase pro- tein using anti-mouse tyrosinase antibodies (30) (data not shown). It is therefore unlikely that translation of the RNA transcribed only from the mutant construct, pRHOHTM2, was specifically blocked in transient expression assay. Con- sidering all these observations, we propose that the mutated gene of the patient F. S. is unable to code for functional tyrosinase.
Since the anti-mouse tyrosinase antibodies used (30) failed to react with human tyrosinase, we tried to produce polyclonal antibodies against three synthetic peptides of deduced human tyrosinase (amino acid residues 4-16, 35-49, and 496-511). However, our initial trials were unsuccessful and another
trials are currently underway by using new synthetic peptides of other regions.
We initially proposed that a pigment cell-specific mouse cDNA, pMT4, encodes tyrosinase (19). However, the pMT4 was shown to map to the brown (b) locus (31) and was not able to direct the transient expression of tyrosinase activity (28).’ The protein, encoded by the pMT4, was therefore termed as tyrosinase-related protein (31). The homology in amino acid sequences between mouse tyrosinase and tyrosin- ase-related protein is about 40% (28), suggesting that both proteins may be derived from the common ancestral gene. Recently, polyclonal antibody against synthetic peptides of tyrosinase-related protein was shown to partially inhibit ty- rosinase activity (32), although here and in the previous report (5) we provide evidence that the mutation affecting the cata- lytic activity of tyrosinase leads to OCA-phenotype. The
’ A. Takeda, Y. Tomita, J. Matsunaga, H. Tagami, and S. Shiba- hara, unpublished observations.
physiological significance of tyrosinase-related protein re- mains to be investigated.
Implications for the Arg to Gln Substitution at Position 59- The substitution of Gln for Arg at position 59 has no apparent effects on the predicted hydrophilicity (33) or predicted sec- ondary structure (34) of the F. S. tyrosinase (data not shown), although it is conceivable that this substitution removes a positive charge (35), which may be crucial for interaction with anionic substrates or cofactors (reviewed in Ref. 36). The presence of arginine residues essential for catalytic activity has been reported in more than a hundred enzymes (36, 37), and some enzymes were shown to lose or change their catalytic activity by the replacement of arginine with neutral residues such as glutamine (35, 38, 39). Charged residues are also known to be important for the folding stability in some proteins (40).
7.
8.
9.
10.
11.
12.
(1978) Cancer Res. 38,253-256 Frischauf, A.-M., Lehrach, H., Poustka, A., and Murray, N. (1983)
J. Mol. Biol. 170, 827-842
The tertiary structure of the mammalian tyrosinase has not yet been ascertained, and our knowledge of the relationship between structure and function of this enzyme is limited at present. However, the two potential copper-binding sites have been predicted, based on the sequence homology in binuclear copper proteins throughout evolution (41, 42). The Arg-59 is not aligned in these putative copper-binding sites, but is conserved in mouse tyrosinase (28,29) as well as in tyrosinase- related protein at the corresponding position (19), suggesting that it constitutes an important structural element. It is of particular interest that the amino acid residues l-72 of mam- malian tyrosinase appear to be deleted in Neurospora tyrosin- ase, of which molecular weight is 46,000 (43). Tyrosinase of phylogenetically lower organisms could utilize various struc- turally related molecules as substrates, whereas mammalian tyrosinase utilizes only the l-form of tyrosine or DOPA as substrates and has a restricted requirement for I-DOPA as a cofactor (42). It is therefore reasonable to speculate that the amino-terminal 72 residues of mammalian tyrosinase, includ- ing Arg-59, may be crucial for defining substrate specificity. Such an assumption is in part consistent with the facts that the arginine residues serve as substrate sites for the enzymes acting on amino acid substrates, providing a positive charge for interaction with carboxyl groups (reviewed in Ref. 36). Alternatively, the Arg-59 may have an indirect role on the conformation of the active site or play an important role for enzyme stability. Analysis of mutation(s) affecting the cata- lytic activity of tyrosinase is invaluable for further character- ization of its structure and reaction mechanisms.
Acknowledgments-We thank Drs. N. Ito, K. Sonoda, S. Nagao, S. Kondo, Y. Sato, and S. Iijima for the introduction of the patients to us and Dr. H. Tohda for the supply of Epstein-Barr Virus. We also thank Dr. H. Yamamoto and Prof. T. Takeuchi for immunological analysis of the transfected cells with anti-mouse tyrosinase antibody. A. T. is grateful to Prof. K. Kogure for encouragement.
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