Download Molecular basis for the deficiency in humans of gulonolactone

Molecular basis for the deficiency in humans
of gulonolactone
oxidase, a key enzyme
for ascorbic acid biosynthesis13
Morimitsu
Nishikimi
ABSTRACT
bic
and
The
acid
is known
inability
an enzyme
that
vitamin.
Isolation
allowed
us to study
ofa
level
to L-gulono-y-lactone
covering
lactone
oxidase
human
the
analysis
oxidase
ofmutations
as a pseudogene
gene
on the genetic
Enzymatic
L-gulono-y-
indicated
has accumulated
being active
genome.
zyme
deficiency,
nucleotide
oxidase,
sequence,
aspects
Before
a
and
that
Am J
we will
ascorbic
acid,
en-
The
dealing
ent
pseudogene
in the
as its essentiality
as a nutrient
number
of exceptional
species
animals.
In other
vitamin
C, or L-ascorbic
demonstrated
that
as shown
(from
uronic
acid
and
two
steps
tion.
The
oxidase
the pathway
acid.
latter
and
thus
This
need
trait has been
most prevalent
carried
genetic
prone
carry
We have
deficiencies
fication
carried
been
enzyme
(8).
is missing
intake
arose
through
disorder,
generations.
because
of the
gluc-
synthesis
point
from
branches
consists
and
of
oxida-
by L-gulono-’y-lactone
of vitamin
during
guinea
pigs
L-ascorbic
C to prevent
the genetic
scurvy-prone
and this
In a sense,
all individuals
this is the
of scurvy-
characterization
of rat and
studies
12035-85.
defect
animals.
Printed
underlying
Starting
goat
liver
on the enzyme
in USA.
with
GLO
GLO
puri(3),
from
gene
level
biological
studies
in humans.
we
deficiencies
© 1991 American
basis
of the
GLO
enzymological
deficiency,
aspects
in vertebrates
The
resides
from
in the liver
animals:
and
reptiles
is located
enzyme
and
in the
kidney
Asano
from
rat liver
revealed
the
by which
000
is present
to the N(l)
position
ofa
histidyl
group
at the
C(8)
methyl
(9,
1 1). During
the
electrons
from
the substrate
reduced
flavin
is oxidized
2-oxo-L-gulono-y-lactone
the products;
the
former
L-ascorbic
above.
liver
(3)
the
method
the enzyme
was successfully
of
by use of appropriate
The
enzyme
weight
residue
by GLO,
the
di-
being
apoprotein
of the
linked
through
isoalloxazine
flavin
L-gulono-’y-lactone,
by molecular
adenine
enzyme,
ofthe
position
as de-
gel electro-
of flavin
of the
de-
preparations
molecular
molecule
molecule
catalysis
of birds
goat
sulfate-polyacrylamide
(3, 9). One
per
in both the
level of
species
rat and
purified
properties.
dodecyl
of
improving
microsomes
nucleotide
the
by
liver
of primitive
mentioned
from
of the
following
by sodium
is -5O
(9)
(10),
Characterization
phoresis
in certain
a
and/
it is pres-
in the
kidney
mammals
to homogeneity
chicken
and
is missing
scurvy-prone
GLO
of GLO.
is interesting
enzyme
of amphibians
tergents.
to form
evolution,
genetic
some
enzyme
purified
termined
trait.
studying
199l;54:
D-glucose
in the above-mentioned
deficiency
out immunochemical
Am J C/in Nutr
is catalyzed
molecular
deficiency
(7). The
as in the
solubilized
it was
starting
lactonization
a dietary
this
in these
and
step
is part
acid
possible
at the
oft-ascorbic-acid-synthesizing
The
Nakagawa
synthesize
from
the branch
viz,
and
of the metabolic
pathways
defect
in the liver of highly
evolved
species,
and
and liver of certain
species
at the intermediate
We
a limited
higher
l950s,
eg, humans,
other primates,
and
these animals
cannot
synthesize
enzyme
species
acid)
after
reactions,
oxidation
This
is synthesized
metabolic
part
can
In the
first portion
of L-ascorbic
The
of enzymatic
(GLO).
scurvy.
of the
animals
body.
to L-gulonic
one
exceptional
species,
(2); as a consequence,
acid
acid
D-glucose
L-gulonic
all higher
in their
1 (1). The
cycle,
D-glucose;
nearly
L-ascorbic
inasmuch
is restricted
to only
among
phylogenetically
acid,
in Figure
pathway
from
words,
discovered
the
of GLO
kidney
(8) as well
the vitamins
our
GLO
discuss
standpoint
evolution
among
with
distribution
mammals.
Introduction
C is unique
genetic
isolation
it became
of GLO
first briefly
species,
kidney
Vitamin
of the
accomplished
that
phylogenetic
Gulonolactone
of the
we summarize
basis
We
(5); as a result,
clone
199 l;54:l203S-8S.
WORDS
(4).
GLO
nature
article,
or the kidney
KEY
monkey
overlapping
rat
study
since it stopped
in the human
the
(6). In this
deficiency
genomic
of the
pig and
for rat-liver
to investigate
oxidase
as well as three
guinea
of a cDNA
of this
this
human
region
Sequence
L-gulono-’y-lactone
Nuir
underlying
ofa
coding
eDNA.
large number
it now exists
C/in
defect
oxidase
entire
biosynthesis
for rat L-gulono-”y-lactone
basic
in the
and
oxygen.
ring
accepts
two
the resulting
In this
process,
and hydrogen
peroxide
are formed
product
isomerizes
nonenzymatically
as
acid.
I From
the Institute
Mitake, Gifu, Japan.
2 Supported
in part
of Applied
Biochemistry,
by a Grant-in-Aid
(01580156)
Yagi
Memorial
Park,
for Scientific
Re-
search from the Ministry
of Education,
Science, and Culture of Japan.
3 Address
reprint requests to M Nishikimi,
Institute
of Applied
Biochemistry,
Yagi Memorial
Park, Mitake, Gifu 505-01, Japan.
Society
for Clinical
Nutrition
12035
Downloaded from ajcn.nutrition.org by guest on June 29, 2015
related
L-ascor-
of L-gulono--y-lactone
for the
led to isolation
clones
the
is required
the
to synthesize
to a lack
cDNA
and
Yagi
ofhumans
to be due
oxidase,
at the gene
Kunio
12045
NISHIKIMI
AND
YAGI
COOH
H-C-OH
H-C-OH
H-C-OH
H-C-OH
CH2OH
FIG
Cloning
of a cDNA
To elucidate
GLO
antibody
used
vector,
amino
amino
the
acid
acid
of rat GLO
culated
from
the
of the
expression
1 cells
with
SV4O
late
the
(M Nishikimi
The
that
GLO,
enzyme
tide
protein
sequence
ofthe
of 1320
ATG
co-ion
protein.
The
GLO
The
adenine
ular
weight
tail
nor
the
genetic
value
region,
the
a polyadenylation
Yet,
it was
defect
utilized
in GLO
fixation
antibody
and
latter
signal,
region
the cDNA
as a versatile
deficiency,
contains
tool
as will
isolation
RNA
any
preparation
was detectable
the conditions
signal
not
Yet,
humans
region
a
have
expressed
and
in the
next
question
GLO
occurs
clone
is not
the use ofthe
in our
study
of genomic
of gene
protein
is not
it is very
from
out Northern
related
RNA from
liver poly(A)
guinea
no
guinea
RNA
pigs
mRNA
human
to
that
do not
in their
liver
contains
mRNA,
in view
gene that was discovered
to a great extent
(see
products
of the GLO
indicated
animals,
to be addressed
rat GLO
if it
or not scurvyin their
liver
showed
that
whether
in the
aberrant
we have had no opportunity
to
of human
liver poly(A)
RNA.
scurvy-prone
DNAs
present
sequence
analysis
same
material
whether
mRNA
DNA
(13)
the
of detection.
us to carry
blot
levels
in the genomes
using
limit
no GLO-specific
mRNA
Udenfriend
ofGLO-specific
that the GLO
has deteriorated
measurement
protein
of that
or that
any
yet determined
should
con-
1/600
<
by
the
it is clear
amount
microsomes
and
with poly(A)
where
rat
(6). Thus,
detectable
the observation
human
genome
a 5’-
be discussed
and
that
monkey
ofcross-reacting
enabled
Northern
green
or
below
GLO
rat cDNA
genome.
We have
The
neither
was
animals
ofthe
anform
cross-reactive
microcomplement
Sato
the amount
that
and
technique
GLO
blot analyses
to examine
possess
GLO-specific
a strong
liver.
to that
a 3’-noncoding
that
Nishikimi
pig liver
for
guinea
scurvy-prone
an aberrant
African
protein
Later,
GLO-specific
mRNA,
because
test an undegraded
preparation
flavin
of the
guinea
microsomes.
indicated
in their
gave
that
microsomes
and
investigation
the Ouchterlony
and
reactive
showed
studies
possess
a molec-
contains
pig
indicated
liver
positive
signal
pig liver under
sulfate-polyacryl-
the cDNA
and
reading
weight
primates
deficiency,
a radioimmunoassay
GLO
bound
this
by using
guinea
in rat liver
established
poly(A)
mi-
of
immunologically
present
acid
in
In their
the question
ofwhether
pigs and monkeys,
have
found
test also
no
nucleofirst
other
contains
no detectable
protein
immunologically
with antirat
GLO
rabbit
antiserum.
Their
the
of 22 nucleotides
Because
of the
The
to form
is comparable
dodecyl
They
liver
and Southern
prone
animals
(3).
to the coding
of full length.
this
of GLO.
the
like
deficiency
introduction.
basis
(4) raised
as guinea
liver of the scurvy-prone
is present
at all.
(12). Thus
acids of the
molecular
molecular
These
the consensus
to have
the
Udenfriend
imals,
such
to GLO
in GLO
in the
of the
the
is removed
lacking
into
as that
an open
are
as mentioned
L-Ascorbic
approach
pSVL
amino
acid
by sodium
a covalently
Humans
in human
Moreover,
surrounding
presumed
sodium
the
1991).
The
a presumed
is thus
by
poly-A
below.
(FAD)
In fact,
nucleotides.
control
vector
to contain
with
by
of COS-
analysis.
well with
with
enzyme
determined
of 776
au-
described
by Kozak
encodes
440 amino
polypeptide
region
cal-
was the same
methionine
gel electrophoresis
noncoding
the
blot
sequence
agreed
of 5 1 267.
In addition
on
The
mature
dinucleotide
of rat GLO
amide
was found
amino-terminal
of 50 483.
that
The
CH2OH
in animals.
genetic
pigs,
tam
(5).
observations,
synthesized
(PCCATGG)
that the clone
the mature
under
of
corn-
with
gel electrophoresis.
cDNA
that
acid
presumed
from the deduced
that of rat GLO
determined
(ATCATGG)
tiation
sequence
we concluded
with
amino
expression
by Western
nucleotides.
33-
confirmed
unpublished
as demonstrated
The
amino
by transfection
placed
sulfate-polyacrylamide
frame
identical
the
further
eukaryotic
K Yagi,
the molecular
weight
sequence
agreed
with
dodecyl
was
the
(5).
its deduced
sequence
protein
was
in the
and
size ofthe
ofrat
cDNA
GLO
cDNA
promoter
acid
in an
encoding
agreement
amino
cloned
library
amino-terminal
was
and
a
to us,
isolated
and
in reasonable
of functional
was
2. The
enzyme
deduced
acid biosynthesis
GLO
available
a cDNA
cDNA
sequence,
was
the
to obtain
cDNA
GLO
in Figure
of the
amino
position
thenticity
cloned
shown
sequence
deduced
of rat
HO-C-H
L-Gulono--y’iactone
acid
of L-ascorbic
animal.
was
a rat-liver
1 1 . As a result,
ofthis
are
underlying
it is pivotal
rat GLO
to screen
sequence
sequence
sequence
pathway
L-ascorbic-acid-synthesizing
against
Agt
acid
nucleotide
acid
ofan
as a probe
expression
entire
mechanism
directed
HO-C-H
CH2OH
Molecular
humans
animals,
H-C
that
as discussed
is whether
ofthe
cDNA
various
the
animals
gave
answer
to this question
(6). In the hybridization
only DNAs
from L-ascorbic-acid-synthesizing
The
related
blot
an
is not
above.
animals.
Southern
at both
gene
gene
scurvy-prone
as a probe,
gene
the
of
in the
below).
to
With
analysis
unequivocal
analysis,
not
animals
(mouse,
Downloaded from ajcn.nutrition.org by guest on June 29, 2015
encoding
it was
molecular
in scurvy-prone
Because
1 . The metabolic
for rat GLO
the
deficiencies
L-Gulonic
0
HO-C
H-C
CH2OH
Acid
II
0
HO-C-H
HO-C-H
D-Glucuronic
D-Glucose
I
H-C-OH
COOH
HO-C
HO-C-H
HO-C-H
HO-C-H
H-C-OH
cDNA
HO-C-H
H-C-OH
HO-C-H
CO
GULONOLACTONE
OXIDASE
DEFICIENCY
IN
12055
MAN
-22
GATCCTCCTGATCACTGGAATC
1
ATGGTCCATGGGTACAAAGGGGTCCAGTTCCAAAATTGGGCAAAGACCTATGGTTGCAGTCCAGAGGTGTACTACCAGCCCACCTCCGTG
M
91
V
H
C
Y
K
C
V
Q
F
Q
N
W
A
K
T
Y
G
C
S
P
E
V
I
Y
Q
P
T
S
V
30
K
V
V
G
C
G
H
S
P
S
D
I
A
60
V
D
K
E
K
K
Q
I
T
V
E
A
C
90
S
N
L
G
A
V
S
D
V
T
V
A
G
120
T
Q
V
V
A
L
T
L
M
T
A
D
G
150
R
V
H
L
G
C
L
G
I
I
L
T
V
180
T
L
K
E
V
L
0
N
L
D
S
H
L
210
S
I
I
Y
Q
D
H
T
N
K
A
P
S
240
F
L
L
W
T
S
T
Y
L
P
C
L
V
270
S
S
N
L
S
H
K
I
F
T
Y
E
C
300
E
A
L
L
E
L
K
A
M
L
E
A
H
330
D
I
L
L
S
P
C
F
Q
R
0
5
C
360
0
Y
W
L
A
Y
E
T
I
N
K
K
F
390
E
E
M
Y
P
T
F
H
K
F
C
D
I
420
V
F
Y
GAGGAGGTCAGAGAGGTGCTGGCCCTGGCCCGGGAGCAGAAGAAGAAAGTGAAGGTGGTGGGTGGTGGCCACTCGCCTTCAGACATTGCC
E
181
E
V
R
E
V
L
A
L
A
R
E
Q
K
K
K
V
TGCACTGACGGTTTCATGATCCACATGGGCAAGATGAACCGGGTTCTCCAGGTGGACAAGGAGAAGAAGCAGATAACAGTGGAAGCCGGT
C
27 1
T
D
G
F
M
I
H
M
C
K
M
N
R
V
L
Q
ATCCTCCTGGCTGACCTGCACCCACAGCTGGATGAGCATGGCCTGGCCATGTCCAATCTGGGAGCAGTGTCTGATGTGACAGTTGCTGGT
I
3 61
L
L
A
D
L
H
P
Q
L
0
5
H
G
L
A
M
GTCATTGGATCCGGAACACATAACACAGGGATCAAGCACGGCATCCTGGCCACTCAGGTGGTGGCCCTGACCCTGATGACAGCTGATGGA
V
45 1
I
G
S
G
T
H
N
T
G
I
K
H
G
I
L
A
GAAGTTCTGGAATGTTCTGAGTCAAGAkaTGCAGATGTGTTCCAGGCTGCACGGGTGCACCTGGGTTGCCTGGGCATCATCCTCACCGTC
E
54 1
V
L
E
C
S
E
S
R
N
A
D
V
F
Q
A
A
ACCCTGCAGTGTGTGCCTCAGTTTCACCTTCAGGAGACATCCTTCCCTTCGACCCTCAAAGAGGTCCTTGACAACCTAGACAGCCACCTG
T
63 1
L
Q
C
V
P
Q
F
H
L
Q
E
T
S
F
P
S
AAGAGGTCTGACTACTTCCGCTTCCTCTGGTTTCCTCACACTGAGAACGTCAGCATCATCTACCAAGACCACACCJJCAAGGCCCCCTCC
K
721
R
S
E
Y
F
R
F
L
W
F
P
H
T
E
N
V
TCTGCATCTAACTGGTTTTGGGACTATGCCATCGGGTTCTACCTACTGGAGTTCTTGCTCTGGACCAGCACCTACCTGCCATGCCTCGTG
8 11
A
S
N
N
F
N
0
Y
A
I
G
F
Y
L
L
E
Downloaded from ajcn.nutrition.org by guest on June 29, 2015
S
GGCTGGATCAACCGCTTCTTCTTCTGGATGCTGTTCAACTGCAAGAAGGAGAGCAGCAJCCTCAGTCACAAGATCTTCACCTACGAGTGT
G
90 1
W
I
N
R
F
F
F
N
M
L
F
N
C
K
K
E
CGCTTCAAGCAGCATGTACAAGACTGGGCCATCCCTAGGGAGAAGACCAAGGAGGCCCTATGGAGCTAAAGGCCATGCTGGAGGCCCACC
R
99 1
F
K
Q
H
V
Q
0
W
A
I
P
R
E
K
T
K
CCCAAAGTGGTAGCCCACTACCCCGTAGAGGTGCGCTTCACCCGAGGCGATGACATTCTGCTGAGCCCCTGCTTCCAGAGGGACAGCTGC
P
10 81
K
V
V
A
H
Y
P
V
E
V
R
F
T
R
C
D
TACATGAACATCATTATGTACAGGCCCTATGGAAAGGACGTGCCTCGCCTAGACTACTGGCTGGCCTATGAGACCATCATGGAAGTTT
Y
1 171
N
N
I
I
M
Y
R
P
Y
G
K
D
V
P
R
L
GGAGGAAGACCCCACTGGGCAAAGGCCCACAATTGCACCCAGAAGGACTTTGAGGAAATGTACCCCACCTTTCACpJGTTCTGTGACATC
G
12 6 1
G
R
P
H
W
A
K
A
H
N
C
T
Q
K
0
F
CGTGAGAAGCTGGACCCCACTGGAATGTTCTTGAATTCGTACCTGGAGAAAGTCTTCTACTACCAGGAGTGGAACAAACCACCCTGA
R
E
K
L
D
P
T
C
M
F
L
N
S
Y
L
E
K
1351
CCCCTCACACTTCTGCTGCCCCCGGGGGTCTGGGGAGCAGAGAAGTGCCTCACAAGCACAATGGGAACTGACCTCTCCTCCTGACCACAA
1441
AGAAAGGCTGGGCTCTGGGCGGGTCCTCTCTGCCTTCGGCATCATTTCCCTTACATCCAGGCGAAGAAGTGGCCTCTCACTCAAATTCCT
1531
GTTAGCATTTCCATGGGTCACACATA)ACTGCAATCCTCTCAGGAGAAGGGGATCCCTGATACATCATATCTATCCAGACTAAGGATGT
1621
GGTTCTTCCTAGATTCTATGGCTCCACCAGGTATAGAGAGATTCCTGGGGCCTGCAGTTCTCCATCCCTCTTCAGGGGAGGGATCCCT
1711
TGGCGAGAGTTTGGCTCAGAGGTGGCATGAAGCATGCTCTGCTCTCTCTTACCCTTGAAGGTCCTTCGGATGCCCAGAGATGTCTGCTGG
1801
TCCTGGGCAAGCCATCATTCAAACGTCCAJCCTGGCCTTCTGTCTGCCATGGCCTGACCCTCGCAGTGTCTCTTCCAGATGTTTAG
1891
AGTGGAACTCGCTTCAACCTCTTAACCAGTTGCTGATCCCTGTGTTTCTCTCCCTTCTCCTTAGACTACTCTTGGAGGGGGATCCCAC
1981
CATCTCCTTGGCTTTCCCTGGGTATTGTTCTCCTCTTCCTCTTCACAAATATGATTTCAGTTTGATTTGTGGCCTTTCTGGAGTGTTCCT
2071
TGGACAACCAAGATGTTCCAGCTAC
FIG 2. The nucleotide
sequence
ofa cDNA encoding
rat liver GLO and its deduced amino acid sequence.
Revisions
were made from our previous
paper (5) as follows: a change from G to C at nucleotide
position
567 accompanying
a
change from glutamine
to histidine
at amino acid residue
I 89, and deletion
of the G at nucleotide
position
I and the
Cs at positions
1460 and 2097 of the previous
sequence.
The amino-terminal
amino acid sequence
determined
by the
sequence
analysis of purified rat liven GLO is underlined.
Adapted
from Koshizaka
et al (5).
dog,
pigs
cow,
gave
and chicken)
but also
positive
signals
(Fig
both humans
and
related
to rat GLO.
of Figure
were
3 that
far weaker
synthesizing
indicates
than
using
were
those
studied
a 5’-terminal
do
coding
have
for DNAs
and
the human
extent
The structures
pigs
pigs
GLO
a nucleotide
sequence
However,
it should be noted from inspection
the intensities
of the signals for human
DNA
mammals
that
to a great
guinea
DNAs
from humans
and guinea
3). Accordingly,
it is clear that
even
GLO
gene
since
the
ofthe
GLO-related
in some
fragment
gene
from
from
sequence
stopped
detail
the
L-ascorbic-acid-
chickens.
genes
and
encoding
the amino-terminal
(nucleotides
947-1293)
the amino
The
results
contain
acid
sequence
close
demonstrated
coding
carboxyl-tei-minal
that
sequences
parts
amino
acid sequence
of the same cDNA
enthe
to the
carboxyl
human
corresponding
of GLO,
and
terminus
and
guinea
to both
hence
pig
amino-
probably
the
finding
entire
has become
altered
To more quantitatively
analyze
the alteration
of the human
GLO gene, we attempted
to isolate both the rat and human
GLO genes. Screening
of partial EcoRI- and HaeIII-cut
rat ge-
genes ofhumans
(nucleotides
(6).
cDNA
a fragment
This
functioning.
by Southern
and
blot
-22-367)
and guinea
analysis
of the
by
rat
coding
sequence.
nomic
DNA
libraries
in Charon
overlapping
clones
covering
the
4A
entire
led to
coding
isolation
region
of three
of the rat
12065
NISHIKIMI
AND
0)
YAGI
carboxyl-terminal
tyrosine.
exon-related
C
C)
C
C)
.
.2
D)
0
0
.
C)
E
0
0
0
.E
I
The
of the
in Figure
relatively
-
tified
analysis
c
ization
probe,
even at lower
weak hybridization
resulting
(!
by Southern
in the
human
of the
four
single
23.0
sequences.
and
two
out
human
and
When
mology
S
was
4.4
insertion.
It was
junctions
sequenced
did
not
conform
splice-donor
site
sequences
of the
words,
the GLO
found
in the
rule:
the
of the
introns
were
GC
human
four
exon-related
were compared
the overall
ho-
At the amino
and,
accumulated
pressure
gene
what
a large
once
now
acid
level,
is more,
number
it ceased
exists
many
of mutations
to be active;
as a pseudogene
in other
in the
human
exon junction
coding
region
sequences
was found
The determined
identical
with
three
followed
the
to be divided
nucleotide
the sequence
differences.
Two
and C to T at position
codons
for glutamine
probably
are
due
Screening
may
during
homologous
to several
and
unpublished
K Yagi,
encompasses
that
the
encode
to A at position
polymorphism.
12 1 1), which
ofa
(G
regions
except
252
alters
genomic
from
other
the codon
also be a polymorphism
the cloning
procedures.
clone
regions
The
that
exons
DNA
ofthe
observations,
corresponding
phenylalanine
rat GLO
1991).
gene
(M Nishikimi
Perhaps,
this clone
to the six rat GLO
at position
202
gene
to the
and
of amino
site
and
(Ks)
per
acid
sub-
nonsynon-
two genes.
substitutions
The
obtained,
ofthe
K value estimated
between
The value of K calculated
to this
to the K value
one,
GLO
neutral
indicating
genes
been
nonsynonymous
by the selective
restricted
that
occurred
we can
make
theory
ofevolution.
to suppose
that the substitution
is far less in the rat GLO gene
have
the following
rate
It may
at nonsynon-
than
substitutions
pressure
the
at the
in the human
in the rat gene
during
evolution
whereas
the nonsynonymous
have occurred
as frequently
substitutions
in the human
gene
as the synonymous
substitutions
since
active.
this
to the
re-
and
of Miyata
number
ofthe
(18).
is comparable
on the basis
gene, because
the
sequences
4 by Nei
method
site (Ks) between
for superimposed
in the
GLO
or may
in EMBL3
(silent)
substitutions
as in other genes.
be reasonable
ymous
sites
for glutamine
nucleotide
genes
gene
substitutions
be an
of Figure
of the
the
synonymous
genes is similar
and an average
human
and rat genes is 0.823
GLO
and
genes.
was
nonsynonymous
ancestor
site
million
at neutral
sites
lineage
years
The
gene
the
we can date
ago,
assuming
estimated
number
occurred
as the
between
as 2.3 X iO
(19).
the
that
obtained
On this assumption,
at ‘-70
time of mammalian
at the synonymous
Therefore,
site
the common
of 0. 16, which
per
the primate
being
nonsynonymous
gene
value
of GLO
tutions
stopped
per
human
stitutions
library
contained
difference
data
(15)
K, respectively)
( 1 7) were 0.86 and 0. 1 6, respectively.
synonymous
substitutions
are not subjected
to selective
during
evolution,
their number
calculated
for various
and
Because
pressure
(IC
discussion
(14). The
by I 1 in-
sequences
ofthe
exon
of the rat GLO
cDNA
differences
of a human
in isolation
GT/AG
rule
into 12 exons
sequence
version
(amino-acid-altering)
of K and K corrected
In regard
unpublished
observathat all of the intron-
432) occur
in the third
position
of the
and threonine,
respectively,
and
most
to allelic
(A to G at position
for arginine,
that arose
K Yagi,
showed
genes
( 16) for calculating
for the GLO
(M Nishikimi
and
199 1). Sequence
analysis
the
per
synonymous
same rate
cDNA
of GLO
modified
stitutions
FIG 3. Southern
blot analysis ofgenomic
DNAs from various animals.
Genomic
DNAs from the indicated
animals
were digested with EcoRI
and analyzed
by Southern
blot hybridization.
The rat GLO eDNA fragment covering
the 5-noncoding
region and most of the coding region
(nucleotides
-22- 1293) was used as a probe. Size markers
are shown
on the left. Adapted
from Nishikimi
et al (6).
exons
gene
analyzed
ymous
values
suIted
also
GT/AG
in number,
four rat exons,
(“-‘70%);
(a
for one
to the
the species.
yet lower
evolution
Yasunaga
to that
artifact
between
GLO
Molecular
2.0
for
deletions
were not subtle
and two stop codons
were present
sequences.
These
results
led us to conclude
that
no selective
We
trons.
were
sequences
three
eight
was
under
Gojobori’s
tions,
human
were
genome.
S
GLO
as a hybrid-
This may be due to too
number
of substitutions
single-nucleotide
intron-exon
iden-
substitution
date
radiation.
By letting
the
sites be equal to that at the
of
between
could
be close
number
of sub-
rat and
human
the primates’
the
rate
.
site’
loss
of substi.
y
corresponds
substitution
nonsynonymous
for
to the
rate
Downloaded from ajcn.nutrition.org by guest on June 29, 2015
the human
rat cDNA
cor-
not
nucleotides
‘-80%
substitutions
in the human
6.6
were
three
the
the homology
determined
two regions
determined
regions,
a total of 548 nucleotides
with those of the corresponding
9.4
the
and
at the
GG.
using
for two deletions
gene
dinucleotides
other
stringency.
from a large
of four
were
rat exons
there
ofthe
GLO
4. The
regions,
one
sequences
ones
short
In the
exon-related
nucleotide
deletion)
that
blot
nucleotide
six expected
and the result
is shown
responding
to the two
c
C)
Cl)
regions
GULONOLACTONE
OXIDASE
DEFICIENCY
IN
MAN
12075
202
Human
F
LH
S
LD
?
HWXKSED
tCCtgCtgaggtCagTTCCTCCATAGCCTGGACAG-CATTGGAAGTCTGAGGACTTCTGCTTCCTCTGGTTCCCACA
***
**
Rat
t CCCt
Ct
***
****
*
*
***
*****
**
FC
*****
******
#{163}LWFPB
*****
*************
**
**
tgtaCCagGTCCTTGACAACCTAGACAGCCACCTGAAGAGGTCTGAGTACTTCCGCTTCCTCTGGTTTCCTCA
VLDNLDSHLKR$EYRFISWPPH
E
S
t
S
V
A
I
Q
H
0.
5
t
S
CAGTGAGAACGTCAGTGCCATCCACCAGGACCACACCAGCAGgCagCtgC
**
************
****
****
**********
*****
CACTGAGAACGTCAGCATCATCTACCAAGACCACACCAACAAGgt
T
Et4V$
I
Z
YQP
*
****
aaCtgC
HNK
264
I
F
I
Q C
I V
G
W
I
l
G
CCCCtgCagCATCTTCTTGCAGTGCCTTGTGGGCTGGATCAATGGCTTTTT’rTTCTGACTCCTGTTTGCCTGC
aCCCtgt
**
****
tgt
t CCt
**
******
**
*****
**************
****
**
*
*****
r
L
L
*
A
C
*****
****
YLpCLVGwiNRrrwMLrNc
QEN
SNL$
P
KX
FTHVCH
‘KQRVQHWA
I
-CAGGAGAACAGCAACCTCAGCCCCAAGATCTTCACCCACGTGTGCCACTTCAAGCAGCATGTCCAGCACTGGGCCAT
-
-
***
FrE’
CaCCggCagCACCTACCTGCCATGCCTCGTGGGCTGGATCAACCGCTTCTTCTTCTGGATGCTGTTCMCTGC
T
7
*
F
******
************
*
*************
***
***
*
***************
**
**********
K
RE
S
SNLS
H
I(
I
FT
Y
Downloaded from ajcn.nutrition.org by guest on June 29, 2015
AAGAAGGAGAGCAGCAACCTCAGTCACAAGATCTTCACCTACGAGTGTCGCTTCAAGCAGCATGTACAAGACTGGGCCAT
ECRFKQRVQDWAI
314
P
1k
KXTTEALLE
CCCCAGgggCtCt
***
L1A
tgggCtgag-tgCagAAAGAAGACCACGGAGGCCCTGCTGGAGCTGAAGGC
***
*********
CCCTAGgtaggag
****
*********
*********
********
*****
tgggCtgaCgggCagGGAGAAGACCAAGGAGGCCCTACTGGAGCTAAAGGC
PR
EXTKEALYE
VLARP
E
VV
S
MY
LKA
LVGVfl
?
EDD
I
LJS
CGTGCTGGAGGCCCACCCTGAGGTGGTGTCCCACTACCTGGTGGGGGTACGCTTCACCTG-GAGGATGACATCCTACTGA
*
****************
*
*****
*********
**
*
***
*********
*
*
********
**
****
CATGCTGGAGGCCCACCCCAAAGTGGTAGCCCACTACCCCGTAGAGGTGCGCTTCACCCGAGGCGATGACATTCTGCTGA
MLEAHP
KVVAHYPVEVR$’TRGDDI
PCrwDs
Ry
LLS
I
LN
N
LY
GCCCCTGCTTCCAGTGGGACAGCCGCTACCTGCATCAACCTGTACAGgtgaCag
**************
********
*****
*********
*********
*
GCCCCTGCTTCCAGAGGGACAGCTGCTACATGAACATCATTATGTACAGgt
PCFQRDSC
**
aaaag
YMN1
I
MY
399
V
tggt
**
tgCtt
P
CT
H
S
KNFE
KMHP
AF
P
KC
*
**********
Al
Ct
*******
*****
*
*
**
V
***
*
**
****
***
tgattgCttCCaCagGCCCACATTGCACCCGGAGGACTTTGAGGATGTACCCCACCTTTCACGTTCTGTGACAT
AHNCTQKD
B
*
It
L
E
P
T
FE
G
M
F
L
N
L
Y
EMYPT
t
E
FM
It
V
F
Y
KFCD
I
*
CTGAGAAGCTAGAACCCACTGGGATGTTCCTATTTGTATTTGGAGAGGTGTTCTACTGGTCAGA
*
*
RE
**
*****
K
**
IDP
********
******
TGMF
*
LN
****
***
S
*******
YLEKVFY
**
*******
***
**
*
FIG 4. Comparison
of four exons of the rat GLO gene with the human nucleotide
sequences
related to them. Exon
sequences
are shown by uppercase
letters, and intron sequences
by lowercase
letters. Identical
residues
in the rat and
human sequences
are indicated
by asterisks.
The deduced
amino acid sequences
for humans
and rats are shown above
and below the respective
sequences;
identical
amino acids between
the two species are shaded.
Numbers
above the
human
amino acid sequences
indicate
the residue numbers
of rat GLO shown in Figure 2. Question
marks in the
human amino acid sequences
represent
the positions
where there is a deletion of nucleotide(s)
in the human sequence:
and asterisks
indicate stop codons.
In the genomic
sequence
of the rat GLO gene, the A at nucleotide
position
12 1 1
ofthe eDNA shown in Figure 2 is G (arrowhead).
l208S
NISHIKIMI
sites
in the
human
onymous
substitution
tution
‘
with
gene,
in the
.
y
‘.
calculated
average
times
site
.
rates
consistent
the
GLO
substitution
the
than
calculate
the
and
rat
presumption
loss of the L-ascorbic-acid-synthesizing
thought
keys
to have
and
monkeys
Old
World
ofboth
L-ascorbic
able,
been
though
acid
before
monkeys
lineages
(20).
they
Thus,
are
(35-45
very
rough
years
to be unable
calculations
and
(19)
based
are
that
is 4-10
date
of the
in primates,
ofNew
million
the above
genes
in rodents
ability
are reported
substi-
GLO
As to the
the divergence
of syn-
10 X l0
Tanimura
rate
primates.
‘
synonymous
by Li and
in higher
rate
to be
human
substitution
that
the
gene
Interestingly,
for the
synonymous
higher
we can
rat GLO
it is
World
ago)
monbecause
to synthesize
seem
on
AND
reason-
a number
of
a
assumptions.
We thank the Japanese
Cancer
Resources
Bank for donating
an
EMBL3 human genomic DNA library that was constructed
by Yoshiyuki
Sakaki.
References
Biol Chem
l988;263:l6l9-2l.
6. Nishikimi
M, Koshizaka
T, Ozawa
and guinea pigs ofthe gene related
‘y-lactone oxidase. Arch Biochem
T, Yagi K. Occurrence
in humans
to their missing enzyme L-gulonoBiophys
1988;267:842-6.
7. Chatteijee
lB. Evolution
and the biosynthesis
l973;182: 127 1-2.
8. Chaudhuri
CR, Chatterjee
lB. i-Ascorbic
phylogenetic
trend. Science 1969;l64:435-6.
ofascorbic
acid
acid. Science
synthesis
in birds:
9. Kiuchi K, Nishikimi
M, Yagi K. Purification
and characterization
of i-gulonolactone
oxidase from chicken
kidney microsomes.
Biochemistry
l982;2 1:5076-82.
10. Nakagawa
H, Asano A. Ascorbate
synthesizing
system in rat liver
microsomes
I. Gulonolactone-reducible
pigment as a prosthetic group
ofgulonolactone
oxidase. J Biochem
(Tokyo)
1970;68:737-46.
1 1. Kenney WC, Edmondson
DE, Singer TP, Nakagawa
H, Asano A,
Sato R. Identification
ofthe covalently
bound flavin of i-gulono--ylactone oxidase. Biochem Biophys Res Commun
l976;7l:1
194-200.
12. Kozak M. Compilation
and analysis
of sequences
upstream
from
the translational
start site in eukaryotic
mRNA.
Nucleic Acids Res
l984;l2:857-72.
13. Sato P, Udenfriend
S. Scurvy-prone
animals,
including
man, monkey, and guinea pig, do not express the gene for gulonolactone
oxidase. Arch Biochem
Biophys
l978;l87:l58-62.
14. Mount SM. A catalogue
ofsplicejunction
sequences.
Nucleic Acids
Res 1982; 10:459-72.
15. Nei G, Gojobori
T. Simple methods
for estimating
the numbers
of
synonymous
and nonsynonymous
nucleotide
substitutions.
Mol Biol
Evol 1986;3:418-26.
16. Miyata T, Yasunaga
T. Molecular
evolution
of mRNA:
a method
for estimating
evolutionary
rates of synonymous
and amino acid
substitutions
from homologous
nucleotide
sequences
and its application. J Mol Evol l980;16:23-6.
17. iukes T, Canton CR. Evolution
of protein
molecules.
In: Munro
HN,
ed. Mammalian
protein metabolism
III. New York: Academic
Press, 1969:21-123.
18. Hayashida
H, Miyata T. Unusual
evolutionary
conservation
and
frequent
DNA segment
exchange
in class I gene of the major histocompatibility
complex.
Proc Natl Acad Sci USA 1983:80:2671-5.
19. Li W-H, Tanimura
M. The molecular
clock runs more slowly in
man than in apes and monkeys.
Nature
l987;326:93-6.
20.
Stone I. Studies ofa mammalian
enzyme system for producing
evolutionary
evidence
on man. Am J Phys Anthropol
l965;23:83-6.
Downloaded from ajcn.nutrition.org by guest on June 29, 2015
1 . Burns ii. Biosynthesis
ofL-ascorbic
acid: basic defect in scurvy. Am
I Med
l959;26:740-8.
2. Burns ii. Missing step in man, monkey and guinea pig required
for
the biosynthesis
of L-asconbic acid. Nature
1957; 180:553.
3. Nishikimi
M, Tolbert
B, Udenfriend
S. Purification
and characterization of i-gulono-’y-lactone
oxidase from rat and goat liven. Arch
Biochem
Biophys
1976;l75:427-35.
4. Nishikimi
M, Udenfriend
S. Immunologic
evidence
that the gene
for L-gulono-’y-lactone
oxidase is not expressed
in animals
subject
to scurvy. Proc Natl Acad Sci USA l976;73:2066-8.
5. Koshizaka
T, Nishikimi
M, Ozawa T, Yagi K. Isolation and sequence
analysis of a complementary
DNA encoding
rat liven i-gulono--ylactone
oxidase, a key enzyme
for L-ascorbic
acid biosynthesis.
J
YAGI