THE JOURNAL OF Bro~ocrc.

a
CHEMISTRY
0 1990 by The American Society for Biochemistry

Vol. 265, No. 29, Issue of October 15, pp. 17792%17797,199O

and Molecular Biology, Inc.

Printed in U.S. A.

Molecular

Basis of Tyrosinase-negative

A SINGLE
BASE
SUBSTITUTION

MUTATION
IN THE
AT POSITION
59*

TYROSINASE

Oculocutaneous

GENE

CAUSING

ARGININE

Albinism

TO GLUTAMINE

(Received for publication,

Atsushi

TakedaS,

Yasushi

From the SDepartment

of Applied
Sendai, Miyugi
980, Japan

TomitaQ,

Jun MatsunagaQ,

Hachiro

Physiology

and

of Dermatology,

the SDepartment

Tyrosinase (EC 1.14.18.1) is a bifunctional

copper-containing enzyme responsible
for the conversion
of tyrosine to
dihydroxyphenylalanine
(DOPA)
and of DOPA to dopaquinone (l-3) and plays a central role in melanin biosynthesis.
Albinism
comprises a heterogenous
group of heritable disorders 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, tyrosinase-negative
OCA, inherited in an autosomal recessive fashion, is characterized
by a complete absence of melanin pigments in the skin, hair, and eyes, which leads to visual
disturbances
caused by optic neurologic defects and also predisposes 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
aduertisement
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).

Tohoku

and Shigeki
University

ShibaharaS?l

School

of Medicine,

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 tyrosinase gene of one tyrosinase-negative
OCA patient S. S., revealing 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,
arginine at position 59 (Arg 59) to glutamine.
The mutant gene
under a heterologous
promoter
is unable to direct the transient expression of tyrosinase activity, suggesting that tyrosinase containing
Gln at position 59 is unstable or catalytically 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 tyrosinase.
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
individual.
The family
history
of the patient
F. S. reveals
no consan-

guineous marriages.
Cloning and Sequencing
of Genomic
DNA Encoding
Human
Tyros&me-The
genomic
DNA library
of the patient
F. S. was constructed
in EMBL3
(7) using Mb01 partial
digests of the transformed
lymphocytes 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 Segment-Genomic
DNA,
extracted
from either
transformed
lymphocytes (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 amplification
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
recommended
by the manufacturer
(Perkin-Elmer
Cetus Instruments).
The two primers
(20-mer)
used for the PCR amplification
were: 5CTGCAGACCTTGTGAGGACT-3
(54/73)
and 5-GTATATCTAGCATATCTTAC-3,
designated
as Pl and P2, respectively
(see
Fig. 1A). The latter
sequence
is complementary
to the sequence
of

17792

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Tyrosinase-negative
oculocutaneous
albinism
(OCA)
is one of classical
inborn
errors
of metabolism,
characterized
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 patients
exon 1, which is invaluable
for screening 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 stability 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.

TagamiQ,

December 11, 1989)

Molecular

Basis

of Oculocutaneous

RESULTS

AND

DISCUSSION

Cloning and Sequencing Analysis of the Genomic DNA Segments Encoding Patients 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 tyrosinase 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-

17793

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 substitution 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 VitroWe wanted to know whether the tyrosinase gene is expressed
in patients 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
Procedures (Fig. lA). The presence of a-amanitin-sensitive
products 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), suggesting that its promoter is functional.
It is therefore conceivable that the tyrosinase
gene is expressed in the patients
melanocytes.
Unique Exon 1 Sequence and Diagnostic MspI Fragment of
the F. S. Tyrosinuse Gent-Recently,
Barton et al. (22) reported 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), containing 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-hybridizable 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). Moreover, 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

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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
using3P-end-labeled
primer
5-GAAGTTGCCAGAGCACT3 (complementary
to 351/367)
as described
(12, 13), with modifications 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)/ZaqI
fragment (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)/ZoqI
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 nucleasemapping
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 phosphatebuffered
saline containing
Ca and Mp,
and frozen in liquid nitrogen. Cells were then thawed,
lysed in 0.1% Triton
X-100 in phosphatebuffered
saline, and used for the assay of tyrosine
hydroxylase
(21).

Albinism

Molecular

17794

EXOn I
5-g

Basis of Oculocutaneous

Exon

Exon3

Albinism

Exon4

ExonS

ip

13

/
/
-

-lkb

PI
.

P2
.

SI probe

GATC

Wild

type

Mutant
Downloaded from http://www.jbc.org/ by guest on October 5, 2015

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

Intron

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.
Splice
number
Exon
5
Dosition
1

901/902

1118/1119

126611267

144811449

GTAAGATATGCTAGATATACGATG......
Gln255
TGGAAG
GTAATCTCTTTCTTTTCACTTTTA......
Gly328
TGACAG
GTTGGTTAATATTTCTTTATAAAT......
Ser377
ATTCAG
GTAAAGTTTACTTTCTTTCAGAGG......
TGGCAG

Asp438

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

..TTAACATGAGGGTGTTTTGTACAG

ATTGTC

..TTTTCATTTTTTTTTAATGAACAG

GATTTG

Ile256
Ey328
..TCTGAATAACCTTTTCCTCTGCAG

TATTTT

..CAATGGGATGTCTTTTTATTTCAG

ACCCAG

Ser377
Asp438

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 6.6kb 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.

Molecular

Basis of Oculocutaneous

BglII
r
Origin-

I23

23kb-

9.4

6.6

- - -

a
4

4.4 -

2.32.0-

868bp._ .-. - .61,

c.

---

868 b&l

Hpa II
IIt

257

2m7bp
.

al t.p

Mutant

Wild

type

Wild

type

0
-

832 bp

222

El

123456
FIG. 3. Pedigree
with
Filled
tially

and genotype
analysis
of the patient
F. S.
OCA.
Squares
represent male and a circle indicates female.
symbols
indicate the albino phenotype (homozygote) and parfilled 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).

17795

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 segments derived from his parents and sibling contained both
&a11 site and BstNI site, indicating that they are heterozygous 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. However, 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 (nucleotide 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 functional mouse tyrosinase (28, 29) always favors the sequence
deduced from the cDNA, pHT y 1.
Comparison of the nucleotide sequence of the F. S. tyrosinase 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-59We 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 amelanotic melanoma cells were transfected with the expression
plasmid pRHOHT2, containing the entire protein-coding region of the human tyrosinase cDNA under the rat heme
oxygenase gene promoter (18) or with the pRHOHTM2 containing 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 nucleasemapping 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-

Downloaded from http://www.jbc.org/ by guest on October 5, 2015

FIG. 2. Genomic
DNA
blotting
analysis
of the tyrosinase
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.
gene.

Albinism

Molecular

17796

Basis of Oculocutaneous

Albinism

pRRHOHT2

pRHOHTM2
pRHOHT0

C a)

123

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-I

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.1.
._.,..
;. \...l.,i:
,
-.rf:,.

.; .
.

48Rt.I
101
381
242
18
II?
IOR
7
tl?

b)
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.-$

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111

Downloaded from http://www.jbc.org/ by guest on October 5, 2015

Y
w
_

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
assayed for DOPA
oxidase or tyrosine
hydroxylase
activity
as described
under Experimental
DOPA
Experiment

oxidase

Tyrosine
Experiment

Experiment

%
pRHOHT2
pRHOHTM2
pRHOHT0

(wild type)
(mutant)
(mock)

Number
of positive
ND, not detectable.

1.2
None
None
cells

was counted

1
dpmfh.

0.9
None
None
(Fig.

plasmids
Procedures.

and

then

hydroxylase
Experiment

mg protem

2.4 X 10

3.0 x lo4

ND*
ND*

ND
NDh

4C).

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 protein 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. Considering 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 tyrosinase-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 tyrosinase activity (32), although here and in the previous report
(5) we provide evidence that the mutation affecting the catalytic activity of tyrosinase
leads to OCA-phenotype.
The
A. Takeda,
Y. Tomita,
J. Matsunaga,
hara, unpublished
observations.

H. Tagami,

and

S. Shiba-

Molecular

Basis of Oculocutaneous Albinism

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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physiological
significance
of tyrosinase-related
protein
remains to be investigated.
Implications
for the Arg to Gln Substitution
at Position 59The substitution
of Gln for Arg at position 59 has no apparent
effects on the predicted hydrophilicity
(33) or predicted secondary 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).
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 tyrosinaserelated 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 mammalian tyrosinase appear to be deleted in Neurospora tyrosinase, of which molecular weight is 46,000 (43). Tyrosinase
of
phylogenetically
lower organisms could utilize various structurally 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, including 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 catalytic activity of tyrosinase is invaluable
for further characterization of its structure and reaction mechanisms.

17797

:
Molecular basis of tyrosinase-negative
oculocutaneous albinism. A single base
mutation in the tyrosinase gene causing
arginine to glutamine substitution at
position 59.
A Takeda, Y Tomita, J Matsunaga, H Tagami
and S Shibahara
J. Biol. Chem. 1990, 265:17792-17797.

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