BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 173, No. 2,1990
Pages 748-755
December 14,1990
MOLECULAR CLONING AND SEQUENCE ANALYSIS OF FERROCHELATASE + Yoshitsugu
]Third Hygiene,
cDNA
N a k a h a s h i l, S h i g e r u T a k e t a n i 2*, M a s a h i r o Kyoichi I n o u e 1 and R i k i o T o k u n a g a 2
Department of Internal M e d i c i n e and Kansai Medical University, Moriguchi,
HUMAN
ENCODING
Okuda 2,
2Department of O s ak a 570, JAPAN
Received November 8, 1990
SUMMARY: The cDNA e n c o d i n g human f e r r o c h e l a t a s e [EC 4 . 9 9 . 1. 1] was i s o l a t e d f r o m a human p l a c e n t a cDNA l i b r a r y in bacteriophage A g t | l by s c r e e n i n g with a radiolabeled fragment o f mouse f e r r o chelatase cDNA. The cDNA had an open r e a d i n g f r a m e o f 1269 b a s e pairs (bp) e n c o d i n g a protein o f 423 a m i n o a c i d residues (Mr. 47,833) with alternative putative polyadenytation signals in the 3' non-coding r e g i o n s and p o l y (A) t a i l s . Amino a c i d s e q u e n c i n g showed t h a t the mature protein consists o f 369 a m i n o a c i d r e s i d u e s (Mr. 42, 158) w i t h a putative leader s e q u e n c e o f 54 a m i n o acid residues. The human enzyme showed an 88~ i d e n t i t y t o mouse enzyme and 46~ t o y e a s t enzyme. Northern blot analysis showed t wo mRNAs o f a b o u t 2500 and 1600 bp f o r f e r r o c h e l a t a s e i n K562 and HepG2 c e l l s . As f u l l - l e n g t h cDNA f o r human f e r r o c h e l a t a s e is now a v a i l a b l e , molecular lesions related to erythropoietic protoporphyria can be c h a r a c t e r i z e d . ©1990AcademicPress,Inc.
The insertion
final
step
of
ferrous
catatyzed
by
enzyme
located
is
the
and
this
ent
homogeneity,
in
the ion
enzyme, in
enzyme f r o m
heme into
biosynthetic protoporphyrin
ferrochetatase
the
inner
various
molecular
pathway
mass
of
+ This sequence data will appear Nucleotide Sequence Databases D00726.
of
the
had been p u r i f i e d 40
to
in the under
42
kbp,
kilobase
0006-291X/90 $1.50 Copyright © 1990 by Academic Press, lnc, All rights of reproduction in any form reserved.
pairs;
748
kDa,
the
reaction (]).
This
mitochondria
kDa
to
(3-6).
EMBL / G enBa n k / D D BJ accession number
* Correspondence: Shigeru Taketani, Department of Kansai Medical University, Moriguchi, Osaka 570, (FAX No. 0 6 - 9 9 2 - 0 6 0 9 ) . Abbreviations:
a
[EC 4 . 9 9 . 1. ]]
membrane
sources
I X,
is
Hygiene, Japan
kilodattons.
(2)
apparLabbe-
Vol. 173, No. 2, 1990
Bois
(7)
and
and
murine
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
our
group
(8)
ferrochelatase,
A genetic
excretion
of
protoporphyrin
structure
of
human
the
protein
for
and
information
leading
to
prenatal
diagnosis
and
chelatase
and
the
(9).
However,
cDNA
should
human
enzyme
structures
of
infants
the
is
cloning
yeast
expression,
the
nature
of
diseases
and
aid
carrier
treatment.
We
sequence
deduced
amino
of acid
results
known and
facilitate
concerning
and
for
accumulation
little
the
nucleotide
the
excessive
Molecular
molecular
complete
with
in
ferrochelatase.
gene
provide
isolated
deficiency
protoporphyria
sequence
just
respectively.
ferrochelatase
erythropoietic
tide
have
in
report
a cDNA e n c o d i n g sequence
of
the
and of
the
nucleo-
studies and
can
genetic
here
in
on also
mutants
detection, the
isolation
human
ferro-
enzyme.
M A T E R I A L S AND METHODS Materials Human e r y t h r o l e u k e m i a K562 c e l l s w e r e g r o w n i n RPMI1640 medium supplemented with 10~ f e t a l calf serum (GIBCO C o . ) and h e p a t o m a HepG2 c e l l s were grown in Dulbecco's modified Eagle's medium with ION f e t a l calf serum. A human p l a c e n t a cDNA l i b r a r y in bacteriophage Xgtll was o b t a i n e d f r o m Dr. Shintaro Suzuki, University of Southern California School of Medicine, LA. Mouse ferrochelatase cDNA was i s o l a t e d f r o m a mouse e r y t h r o l e u k e m i a cell cDNA l i b r a r y in xgtll, as d e s c r i b e d (8). Restriction endonucleases and o t h e r n u c l e i c acid modifying enzymes were purchased f r o m T o y o b o Co. and T a k a r a S h u z o Co. Radioactive nucleotides and n y l o n m e m b r a n e s w e r e f r o m A m e r s h a m Co. All other reagents used were of analytical grade. Isolation o f Human F e r r o c h e l a t a s e cDNA The human p l a c e n t a cDNA library was s c r e e n e d using the EcoRI-Apal fragment o f t h e mouse ferrochelatase cDNA ( 1 0 2 0 - b p l o n g ) (8). Detailed procedures for the screening o f cDNA w e r e t h o s e described by M a n i a t i s et al. (10). Determination of Nuoleotide S e q u e n c e The i n s e r t s were subcloned into t h e EcoRI s i t e o f pUC p l a s m i d vectors and s e q u e n c e d by t h e dideoxynucleotide chain termination method (11). Determination of Amino-terminal Regions of Amino Acid Sequences Ferrochelatase from bovine kidney and human l i v e r was p u r i f i e d , as d e s c r i b e d (12). The p u r i f i e d e n z y m e was a n a l y z e d to determine the amino-terminal regions of the amino acid s e q u e n c e s as f o l lows: the enzyme was analyzed by sodium dodeoyl sulfatepolyacrylamide gel electrophoresis and t r a n s f e r r e d t o an I m m o b i lon P membrane (Millipore), using the method of Matsudaira (13). After the proteins on t h e f i l t e r had b e e n s t a i n e d , parts of the filter corresponding to the position o f t h e enzyme (Mr. 42,000) w e r e c u t and d i r e c t l y sequenced (8). Northern Blot Analysis Total RNAs w e r e p r e p a r e d using guanidine thiocyanate f r o m K562 c e l l s and HepG2 c e l l s (14). Poly (A) RNAs
749
V o l . 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
were selected by oligo(dT)-cellulose chromatography and were denatured with I M formamide, electrophoresed i n a 1~ a g a r o s e get and transferred to a nylon filter (15). Hybridization was carried out using as a probe the insert of &HF2-1. Conditions of hybridization and washing were as described (15).
RESULTS isolation
of
The the
human
in
isolated
cDNA
and AHF2-1, their
Human
x
were
2).
enytation
library mouse
1.6
cDNAs
shown the
Fig.
with as
plaques inserts
designed
of
in
cDNA,
the
were
relationship
and
screened
positive of
and
kbp,
are
was
ferrochelatase
Sizes
The
strategies
Agt]l
Two
plaques.
and
of
in
Methods.
respectively.
the
were of
as
two
de-
the
2HF1-2
clones
and
1.
Deduced
Amino
Acid
and
~HF2-1,
Sequence
Ferrochelatase of
3HF1-2
beginning
encoding
10 5
1.7
inserts
sequenced.
and
1
cDNA
of
and
Sequence
The
tail
fragment
sequencing
Nucieotide
cDNA
Materials
from
cloned
Ferrochelatase
placenta
radiolabeled
scribed
for
Human
AND D i S C U S S i O N
AHF2-1
clones, an
position
amino contained
signal
two
contained
at
partial
the
(position
acid a
AHF1-2
1711
2415
and
sequence 1576
bp
bp
insert the
of
insert
1530-1535)
and
with
a
nucieotide
a
(A)
tail
polyadbegin-
m z (J
U3 O'3 UJ
LU
kll
1
1000
I
2OOO (bp)
A HF2-1
A HF1-2
Fig. 1. Restriction maps and s e q u e n c i n g s t r a t e g y for ferrochel a t a s e cDNAs. The u p p e r d i a g r a m shows t h e p r o t e i n - c o d i n g regions of the clones, as b o x e s . The 3 ' ends o f t h e c l o n e s a r e shown by arrowheads. The d i r e c t i o n s and e x t e n t o f s e q u e n c e d e t e r m i n e d are indicated by a r r o w s .
750
(A)
(Figs.
a putative
poly
poly
sequence
ferrochelatase with
were
1
Vol. 173, No. 2, 1990
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
-29
GGGCGGGTCGGGCCGAGGCTGCCCAGGCA
1 1
ATGCGT TCA•T•G••G•AAA•AT•G•T••GG•••TG•G•G••G•GGGcG••••G•T••G•GAT••G•TGG•AT••AGCAGCTGGAGGGTC Me t A r g S e r L e u G l y A l a A s n M e l c A l a A l a A l a L e u A r g A l a A i a G l y V a l Leu L e u A r g A s p P r o L e u A l a S e r S e r S e r f r p A r g V a l
31
TGTCAGCCATGGAGGTGGAAGTCAGGTGCAGCTGCAGCGGCCG TCACCACAGAAACAGCCCAGCATGCCCAGGGTGCAAAACCTCAAGT T CysG1 n P r o T r p A r g I r p L y s S e r G l y A l a A l a A l a A l a A l a V a l T h r T h r G l u f h r A l a G t n H i s A l a G I n G I ~ A I a L ~ s P r o G 1 r i v a l
181 61
CAACCGCAGAAGAGGAAGCCGAAAACTGGAATATTAA TGCTAAACATGGGAGGCCC] GAAACTCTl GGAGATGI TCACGACTTCCTTCTG G1 nP r o 6 1 n L y s A r g L y s P r o L y s I h r G l y I l e L e u M e fcLeuAsnMe I ; G l y G l y P r o G l u ] h r L e u G l y A s p V a l H i s A s p P h e L e u L e u
271 91
AGACTCTTCTTGGACCAAGACCTCATGACACTT CCTAT TCAGAATAAGC TGGCACCATTCAICGCCAAACGCCGAACCCCCAAGATTCAA A r g L e u P h e L e u A s p G l n A s p L e u M e t T h r L e u P r o Il e G l nAsnLysLeuAlaP roPhe I l e A l a L y s A r g A r gf h r P r o L y s I l e G l n
361 121
GAGCAGTACCGCAGGA TTGGAGGCGGATCCCCCATCAAGATATGGACTTCCAAGCAGGGAGAGGGCATGGTGAAGCTGCTGGATGAATTG GIuG1 nTy r A r g A r g I l e G l y G l y G l y S e rP r o I l e L y s I l e T r p T h r S e r L y s G l n G l y G l u G l y M e t V a l LysLeuLeuAspGluLeu
451 151
TCCCCCAACACAGCCCCTCACAAATACTATAT TGGATTTCGGTACGICCATCCTTTAACAGAAGAAGCAATTGAAGAGATGGAGAGAGAT S e t P r o A s n T h r A 1 a P r o H i s L y s T y r T yr I l e G l y P h e A r g T y r V a l H i s Pr o L e u T h r G l u G l u A l a I l e G l u G l u M e t G l u A r g A s p
541 181
GGCCTAGAAAGGGCTATTGCTTTCACACAGTAI CCACAGTACAGCTGCTCCACCACAGGCAGCAGCTTAAATGCCATTTACAGATACTAT G i y L e u G t u A r g A l a l l e A l a P h e T h r G l nTyrProG1 n f y r S e r CysSe r Thr T h r G l y S e r S e r L e u A s n A l a ll e T y r A r g T y r l y r
631 211
AATCAAGTGGGACGGAAGCCCACGATGAAG TGGAGCACTATTGACAGGTGGCCCACACATCACC1CCTCATCCAGTGCTTTGCAGATCAT AsnGl n V a I G l y A r g L y s P r o T h r M e t L y e TrpSe r T h r I l e A s p A r g T rpProT h r H i s l l i s L e u L e u I l e G l n C y s P h e A l a A s p H i s
721 241
ATTCTAAAGGAACTGGACCATTTTCC~TTGAGAAGAGAAGCGAGGTGGTCATTCTGTTTTCTGC]CACTCACTGCCGATGTCTGTGGTC I l e L e u L y s G l u L e u A s p H i s P h e P r o L e u G l u L y s A r g S e r G l u V a l Va l I l e L e u P h e S e rA1 aH i s S e r LeuProMef~Se r V a l V a i
811
271
AACAGAGGCGACCCATATCCTCAGGAGG TAAGCGCCACTGTCCAAAAAGTCATGGAAAGGCIGGAGTACTGCAACCCCTACCGACTGGTG A s n A r g G l y A s p P r o T yr P r o G l n G l u V a l S e r A l aT h r V a I G l n L y s V a i M e t G l u A rgLeuGiuTy r C y s A s n P r o T y r A r g L e u V a l
901 301
TGGCAATCCAAGGTTGGTCCGATGCCCTGGTTGGGTCCTCAAACAGACGAA] CTATCAAAGGGCTTTGIGAGAGGGGGAGGAAGAATATC T rpGl n S e r L y s V a I G l y P r o M et ProT r p L e u G l y P r o G l n T h rAspGi uSe r I l e L y s G l y L e u C y s G l u A r g G l y A r g L y s A s n f l e
991 331
CTCTTGGTTCCGATAGCATTTACCAGTGACCAIATTGAAACGCT GTATGAGCTGGACATCGAGTACTCTCAAGTTTTAGCCAAGGAGTGT LeuLeuVaIProfleAlaPheThrSerAspHielleGluT h r L e u T y r G l u LeuAspI l e G l u Ty r Se r G l n V a l L e u A l a L y e G l u C y s
1081 361
GGAGTTGAAAACATCAGAAGAGCTGAGTCTCT TAATGGAAATCCATTGTTCTCTAAGGCCCTGGCCGACTTGGTGCATTCACACATCCAG GlyVal GluAsnI leArgArgAlaGiuSe r LeuAsnGlyAsnProLeu PheSerLysA1 aLeuAlaAspLeuValHisSerHisi leGin
1171 391
TCAAACGAGCTGTGT TCCAAGCAGCTGACCCTGAGCTGTCCGCTC TGTGTCAA TCC TGTCTGCAGGGAGACTAAATCCT ICTTCACCAGC S e t A s n G l u L e u C y s S e r L y s G l n L e u T h r L e u S e r C y s P r o L e u C y s V a t A s n P r o V a l C y s A r g G l u T h r L y s S e r PhePheT h r S e r
1261 421
CAGCAGCTGTGACCCCCGCCGGTGGACCCCGTGGCGTTAGGCAAATGCCCAACCTCCAGA TACCTCCGATGTGGAGAGGGTGTTAT TTAG GlnGtnLeu**~
1351 1441 1531 1621 1711 1801 1891 1981 2071 2161 2251 2341
AGATCAAGGAAGGAAGTCATCCT TCCTTGATATATATACAGCCTTTGGGTACAAATTGTGTGGTTTCTTGAGGATTGGACTCTTGATGGA TTTCTATTTTTATATAACTATACAGTAAGCATTTGIAT TTTCTCTCTCTAGGTATAAGTTAC TAGTTTGGAAIG[CCATCAGGACCTTI~ A I AA~r GAGGCTAAAAATTTGTCTTATGAGACACACCTATTTAAGCACAGATTTTGGC]TI AT1GCCCAAAACCCTCCCGAAAGGGTACG GAGAGTCCCCTCTGTGGGCTGGCAGTGTGAATGAGATCTGTTTAGTCTCGIGCATAIAGT TGCTGTTTTT TAAAIGAACACAGTTGAGTA TTTGAAGTGAATTTGAAAAAGAAATGTTACTTAATCTTTCCCIAAGCCCATGGGTTACAGAATGCTAGGGAGGCAATTTGGTTACCTGCA ATGGCTGCTT TTGCCAGCGAGGCCACCATTCATTGGTCATCTTGGTATTTGTGCTGTGAA; CTCACTTTCCTCAATGTAAAAAGGAATCA AGTATGGATTTCAGAGG TGCTCTTAGATTCCCCAT A C A C C C A A G G G I ~ G TGTACAAGTACAGTGTTCATGATACGTGCCTTGGT GGGAGTCCGTGGTGCCACAGGGAAGGGGCTCCCACTGCTTCTGGTCTCCAGGGACAGIGCTGCTGGAAAGGCTAGTGATGAGCTTCACCC TGGAGCTCCTCCCGGGACCT TGCAAGCCTCTCCATCCAGCATCTTCI CTATCTTAGTTGAATGCCTTCTTTCTGAACATTTGI TTTAAGA ATTATTTTATAAAGTCAACAATACTTTGCTTGAATTCTTTCTTAATTTACGATTTTT FATTATAAAAAAGTATAGTGATACAATGGGACA ]GTGAAGAATACAGAAAAGTAACCACTTTAATGCAATAACTGTTATCATAATATTGTAI TTCGIGGI AGTCCTTGCCTGTAGATATTTTT AATGCCAT TTAATGCCATTGTCACCTTGGATI T ATGAGTGAAAAGTGTTTCI AAAAA]A TAGAAATAATGTCAGAAAAAAAAAA
91
F i g . 2. S e q u e n c e s of human f e r r o c h e l a t a s e cDNAs. Underlined is the amino-terminal sequence estimated by h o m o l o g y t o t h o s e of purified mouse and b o v i n e f e r r o c h e l a t a s e . The a p p a r e n t polyadenylation signals (AATAAA) a r e boxed. (^). the sites of p o l y (A) addition.
ning
at
tained
position an
423
amino
the
two
inframe phobic peptide
open
1543. reading
acids clones
ATG
is
was
the
followed
human
(Fig.
nucleotide
frame
residues
sequence in
The
encoding
(Fig. same by 2,
2). in
sequence a
protein,
The
first
ferrochelatase.
We
751
con-
Mr.
47,833
with
sequence
regions.
specifying residue),
~ HF2-I
nucleotide
overlapped
codons
of
a
basic
defined
attempted
The
to
and as
a
determine
of
first hydrosignal the
VOI. 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
HF
MRSLGANMAAALRAAGVLLRDPLASSSWRVCQPWRNKSGAAAAAVTTETAQHAQGAKPQVQPQKRKPKTGILMLN
75
MF
MLSASANMAAALRAAGALLREPLVHGSSRACQPWRCQSGAAVAA-TTEKVHHAKTTKPQAQPERRKPKTGILMLN
74
YF
MLS ......
45
HF
HGGPETLGDVHDFLLRLFLDQDL--MTLPIQNKLAPFIAKRRTPKIQEQYRRIGGGSPIKIHTSKQGEGMVKLLD
MF
MGGPETLGEVQDFLQRLFLDRDL--MTLPIQNKLAPFIAKRRTPKIQE--RRIGGGSPIKHWTSKQGEGMVKLLD
145
YF
MGGPSKVEETYDFLYQLFADNDLIPISAKYQKTIAKYIAKFRTPKIEKQYREIGGGSPIRKWSEYQATEVCKILD
120
HF
ELSPNTAPHKYYIGFRYVHPLTEEAIEEMERDGLERAIAFTQYPQYSCSTTGSSLNAIYRYYNQVGRKPTHKNST
223
RTIRTQGSFLR
........
R-SQLTITRS
....
FSVT ....
FNMQNA ......
QKRSP-TGIVLMN
148
MF
ELSPATAPHKYYIGFRYVHPLTEEAIEEMERDGLERAIAFTQYPQYSCSTTGSSLNAIYRYYNEVGQKPTMKWST
220
YF
KICPETAPHKPYVAFRYAKPLTAETYKQHLKDGVKKAVAFSQYPHFSYSTTGSSINELWRQIKALDSERSISWSV
195
HF
IDRWPTHHLLIQCFADHILKELDHFPLEKRSEVVILFSAHSLPMSVVNRGDPYPQEVSATVQKVMERLEYCNPYR
298
MF
IDRWPTHPLLIQCFADHILKELNHFPEEKRSEVVILFSAHSLPMSVVNRGDPYPQEVGATVHKVMEKLGYPNPYR
295
YF
IDHHPTNEGLIKAFSENITKKLQEFPQPVRDKVVLLFSAHSLPMDVVNIGDAYPAEVAATVYNIMQKLKFKNPYR
270
HF
LVWQSKVGPMPWLGPQTDESIKGLCERGRKNILL--VPIAFTSDHIETLYELDIEYSQVLAKECGVENIRRAESL
371
MF
LVWQSKVGPVPWLGPQIDEAIKGLCERGRKNILL--VPIAFTSDHIETLYELDIEYSQVLAQKCGAENIRRAESL
368
YF
LVWQSQVGPKPWLGAQTAEIAEFL---GPKVDGLMFIPIAFTSDHIETLHEID---LGVIGESEYKDKFKRCESL
339
HF
NGNPLFSKALADLVHSH
IQSN ELCSKQLTLSCPL--CVN
PVCRETKSFFISQQL
423
MF
NGNPLFSKALADLVHSH
IQSNKLCSTQLSLNCPL--CVN
PVCRKT KSF FTSQQL
420
YF
NGNQTF I EGMADLVKSHLQSNQLYSNQLPLDFALGKSNDPVKDLSLVFGHH
393
EST
F i g . 3. H o m o l o g y i n t h e d e d u c e d a m i n o a c i d s e q u e n c e s o f human, mouse and yeast ferrochetatase. The s e q u e n c e s are shown by single-letter code. HF, human f e r r o c h e l a t a s e ; MF, mouse f e r r o cheiatase (8) ; YF, y e a s t ferrechelatase (7) ; *, identical amino a c i d s in t h e t h r e e s p e c i e s . amino-terminal it
was
blocked.
undertine)
tatase
The
was
sequences
amino
sequence
of
thus
acid
estimated
purified
ferroche]atase
(6).
ferrocheIatase
was
ferrochetatase and
enzymes
(7),
identity
to
the
The
enzyme
with
with The
the
acids
and
a 46~ 752
of
acid
identity
mouse
ferroche-
to
of
369
42, 158.
This
the
purified
human
sequence
of
mouse
and
mouse
ferrochelatase
2,
amino-terminat
humans
than
(Fig.
of
of
from
but
consisting
sequences
longer
Human
and
mass that
enzyme
the
protein
amino
enzyme
ferrochelatase
in
molecular
deduced
3).
amino
of
homologies
agreement
respectively. mouse
region
A mature
compared
(Fig. thirty
in
human
(X-A-K-P-Q-V-D-)
(8). had
was
purified
by
bovine
residues weight
the
amino-terminal
(T-T-K-P-O-A-Q-)
molecutar
three
of
yeast
consisted (8)
and
exhibited yeast
human
enzyme.
of yeast
an
88~ The
V o l . 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1
2
(kbp)
-28s 2.92.2-
-18s 1.5-
Fig. 4. RNA b l o t hybridization using ferrochelatase cDNA as a probe. P o l y (A) RNAs f r o m K562 c e i l s and HepG2 c e i l s were e ~ ctrophoresed on an a g a r o s e g e t c o n t a i n i n g formaldehyde. A 5Zp_ labeled insert o f XHF2-1 was used as a p r o b e . Lane 1, RNA (2 # g ) o f K562 c e l l s ; and l a n e 2, RNA (2 #g) o f HepG2 c e l l s .
carboxy-terminat conserved
than
regions the
at
same
around the
halves the
these
259
the
to
ferrochelatases
ha!ves.
267,
three
three
Blot
295
to
species,
regions
As
it
303
the
and
may
be
is
involved
in
of
poly
RNAs
are three
334 that
peptides
to
345
the
cata!ytic
better
were
sequence
reaction
of
Ana!ysis
RNA b ! o t celts
was
Fig.
4,
using
b!ot
two
and
1.6
kbp.
kbp
and
a
analysis,
hybridization
done
revealed
for
three
enzyme.
Northern
in
the
amino-termina!
position among
of
as
utilization
probe
hybridization
hybridization RNA
faint mRNA
from
band in
K562
bands
1. 6 and
in two
the
insert
for
cells kbp.
HepG2
of
K562
showed
to of
a
the
and
HepG2
As
shown
AHF2-1.
K562
ce!l
po!y at
positive data
on
(A) about
band the
at
RNA
contained
1.6
and
the
clones
of
AHF1-2
two
mRNAs
the
po!yadenylation 753
from
ferrochelatase
cells
lengths
alternative
of
From
corresponding
Variation of
the
ana!ysis
HepG2 at
ferrochelatase,
AHF2-I.
a
(A)
signals
2.5
is
due
found
RNA 2.5 2.5 blot kbp and to in
Vo1.173, No. 2 , 1 9 9 0
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AHF1-2
and A H F 2 - 1 .
at
3'-ending
region
signal
at
position
signal
of
~he
AATAAA
terminating Ferrochelatase similar
to
chetatase
The
apparent has
mRNA o f
those
seen
mRNA w i t h
been to
found,
1943
longer
HepG2
in
two
not
1938
the
polyadenyiation
although
which
may
be
ferroche]atase
celts
showed
K562 c e i l s . bands
signal
was
The
similar
of AHF1-2 there
used
is
for
a
the
mRNA m o l e c u l e .
two
bands
of
sizes
pattern
of
human
ferro-
to
of
mouse
enzyme
that
mRNA. The coding
the
values human cDNA of
encoding
sequence,
residues. and
cDNA
specifying
The m a t u r e predicted
obtained
protein
consists mass
(6).
The
ferrochelatase
lesions
related
for
369
to
erythropoietic
423
amino
of
proteinamino
acid
agrees
purified
facilitate
the
of
42,158
availability
wilt
has
protein of
of
electrophoretically
human
molecutar
ferrochelatase
a precursor
molecular
ferrochelatase for
human
the
residues
well
bovine
acid
with (4)
and
full-length
characterization protoporphyria.
ACKNOWLEDGMENTS: We t h a n k Dr. S. S u z u k i for a kind gift of a human p l a c e n t a cDNA l i b r a r y , M. O h a r a f o r critical c o m m e n t s and K. Y a s a k a f o r assistance with the amino acid sequencing. This w o r k was s u p p o r t e d in part by G r a n t s - i n - A i d for Scientific Research from the Ministry of Education, Science and C u l t u r e of J a p a n and by a g r a n t f r o m N i h o n P h a r m a c e u t i c a l Co.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
Porra, R. J. and J o n e s , O. T. G. (1963) B i o c h e m . J. 87, 186-190. J o n e s , M. S. and J o n e s , O. T. G. (1969) B i o c h e m . J. 113, 507-514. Taketani, S. and T o k u n a g a , R. (1981) J. B i o l . Chem. 256, 12748-12753. Taketani, S. and T o k u n a g a , R. (1982) E u r . J. B i o c h e m . 127, 443-447. Dailey, H. A. and F l e m i n g , J. E. (1983) J. B i o l . Chem. 258, 11453-11459. Mathews-Roth, M. M., D r o u i n , G. L. and D u f f y , L. (1987) Arch. Dermatol. 123, 4 2 9 - 4 3 0 . Labbe-Bois, R. (1990) J. B i o l . Chem. 265, 7 2 7 8 - 7 2 8 3 . Taketani, S., Nakahashi, Y . , O s u m i , T. and T o k u n a g a , R. ( 1 9 9 0 ) J. B i o l . Chem. i n p r e s s . Kappas, A., Sassa, S., Gatbraith, R. A. and N o r d m a n n , Y. ( 1 9 8 9 ) The M e t a b o l i c Basis of Inherited Disease, 6 t h Edn, (Scriver, C. R., B e a u d e t , A. L . , S l y , W. S. and V a l l e , D. eds.) pp1305-1365, McGraw-Hill, New Y o r k .
754
Vol. 173, No. 2, 1990
10.
11. 12. 13. 14. 15.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Maniatis, T., Fritsch, E. F. and Sambrook, J. (eds.) (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory P r e s s , C o l d S p r i n g H a r b o r , New Y o r k . Sanger, F., Nicklen, S. and C o u l s o n A. R. (1977) P r o c . N a t l . Acad. S c i . USA 74, 5 4 6 3 - 5 4 6 7 . Nakahashi, Y., Taketani, S., Sameshima, Y. and T o k u n a g a , R. (1990) B i o c h i m . Biophys. A c t a 1037, 3 2 1 - 3 2 7 . Matsudaira, P. (1987) J. B i o l . Chem. 262, 1 0 0 3 5 - 1 0 0 3 8 . Chomzynski, P. and S a c c h i , N. (1987) A n a l . B i o c h e m . 162, 156-159. Kamijo, K., T a k e t a n i , S., Y o k o t a , S., Osumi, T. and Hashimoto, T. (1990) J. B i o l . Chem. 265, 4 5 3 4 - 4 5 4 0 .
755
Vol. 173, No. 2,1990
Pages 748-755
December 14,1990
MOLECULAR CLONING AND SEQUENCE ANALYSIS OF FERROCHELATASE + Yoshitsugu
]Third Hygiene,
cDNA
N a k a h a s h i l, S h i g e r u T a k e t a n i 2*, M a s a h i r o Kyoichi I n o u e 1 and R i k i o T o k u n a g a 2
Department of Internal M e d i c i n e and Kansai Medical University, Moriguchi,
HUMAN
ENCODING
Okuda 2,
2Department of O s ak a 570, JAPAN
Received November 8, 1990
SUMMARY: The cDNA e n c o d i n g human f e r r o c h e l a t a s e [EC 4 . 9 9 . 1. 1] was i s o l a t e d f r o m a human p l a c e n t a cDNA l i b r a r y in bacteriophage A g t | l by s c r e e n i n g with a radiolabeled fragment o f mouse f e r r o chelatase cDNA. The cDNA had an open r e a d i n g f r a m e o f 1269 b a s e pairs (bp) e n c o d i n g a protein o f 423 a m i n o a c i d residues (Mr. 47,833) with alternative putative polyadenytation signals in the 3' non-coding r e g i o n s and p o l y (A) t a i l s . Amino a c i d s e q u e n c i n g showed t h a t the mature protein consists o f 369 a m i n o a c i d r e s i d u e s (Mr. 42, 158) w i t h a putative leader s e q u e n c e o f 54 a m i n o acid residues. The human enzyme showed an 88~ i d e n t i t y t o mouse enzyme and 46~ t o y e a s t enzyme. Northern blot analysis showed t wo mRNAs o f a b o u t 2500 and 1600 bp f o r f e r r o c h e l a t a s e i n K562 and HepG2 c e l l s . As f u l l - l e n g t h cDNA f o r human f e r r o c h e l a t a s e is now a v a i l a b l e , molecular lesions related to erythropoietic protoporphyria can be c h a r a c t e r i z e d . ©1990AcademicPress,Inc.
The insertion
final
step
of
ferrous
catatyzed
by
enzyme
located
is
the
and
this
ent
homogeneity,
in
the ion
enzyme, in
enzyme f r o m
heme into
biosynthetic protoporphyrin
ferrochetatase
the
inner
various
molecular
pathway
mass
of
+ This sequence data will appear Nucleotide Sequence Databases D00726.
of
the
had been p u r i f i e d 40
to
in the under
42
kbp,
kilobase
0006-291X/90 $1.50 Copyright © 1990 by Academic Press, lnc, All rights of reproduction in any form reserved.
pairs;
748
kDa,
the
reaction (]).
This
mitochondria
kDa
to
(3-6).
EMBL / G enBa n k / D D BJ accession number
* Correspondence: Shigeru Taketani, Department of Kansai Medical University, Moriguchi, Osaka 570, (FAX No. 0 6 - 9 9 2 - 0 6 0 9 ) . Abbreviations:
a
[EC 4 . 9 9 . 1. ]]
membrane
sources
I X,
is
Hygiene, Japan
kilodattons.
(2)
apparLabbe-
Vol. 173, No. 2, 1990
Bois
(7)
and
and
murine
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
our
group
(8)
ferrochelatase,
A genetic
excretion
of
protoporphyrin
structure
of
human
the
protein
for
and
information
leading
to
prenatal
diagnosis
and
chelatase
and
the
(9).
However,
cDNA
should
human
enzyme
structures
of
infants
the
is
cloning
yeast
expression,
the
nature
of
diseases
and
aid
carrier
treatment.
We
sequence
deduced
amino
of acid
results
known and
facilitate
concerning
and
for
accumulation
little
the
nucleotide
the
excessive
Molecular
molecular
complete
with
in
ferrochelatase.
gene
provide
isolated
deficiency
protoporphyria
sequence
just
respectively.
ferrochelatase
erythropoietic
tide
have
in
report
a cDNA e n c o d i n g sequence
of
the
and of
the
nucleo-
studies and
can
genetic
here
in
on also
mutants
detection, the
isolation
human
ferro-
enzyme.
M A T E R I A L S AND METHODS Materials Human e r y t h r o l e u k e m i a K562 c e l l s w e r e g r o w n i n RPMI1640 medium supplemented with 10~ f e t a l calf serum (GIBCO C o . ) and h e p a t o m a HepG2 c e l l s were grown in Dulbecco's modified Eagle's medium with ION f e t a l calf serum. A human p l a c e n t a cDNA l i b r a r y in bacteriophage Xgtll was o b t a i n e d f r o m Dr. Shintaro Suzuki, University of Southern California School of Medicine, LA. Mouse ferrochelatase cDNA was i s o l a t e d f r o m a mouse e r y t h r o l e u k e m i a cell cDNA l i b r a r y in xgtll, as d e s c r i b e d (8). Restriction endonucleases and o t h e r n u c l e i c acid modifying enzymes were purchased f r o m T o y o b o Co. and T a k a r a S h u z o Co. Radioactive nucleotides and n y l o n m e m b r a n e s w e r e f r o m A m e r s h a m Co. All other reagents used were of analytical grade. Isolation o f Human F e r r o c h e l a t a s e cDNA The human p l a c e n t a cDNA library was s c r e e n e d using the EcoRI-Apal fragment o f t h e mouse ferrochelatase cDNA ( 1 0 2 0 - b p l o n g ) (8). Detailed procedures for the screening o f cDNA w e r e t h o s e described by M a n i a t i s et al. (10). Determination of Nuoleotide S e q u e n c e The i n s e r t s were subcloned into t h e EcoRI s i t e o f pUC p l a s m i d vectors and s e q u e n c e d by t h e dideoxynucleotide chain termination method (11). Determination of Amino-terminal Regions of Amino Acid Sequences Ferrochelatase from bovine kidney and human l i v e r was p u r i f i e d , as d e s c r i b e d (12). The p u r i f i e d e n z y m e was a n a l y z e d to determine the amino-terminal regions of the amino acid s e q u e n c e s as f o l lows: the enzyme was analyzed by sodium dodeoyl sulfatepolyacrylamide gel electrophoresis and t r a n s f e r r e d t o an I m m o b i lon P membrane (Millipore), using the method of Matsudaira (13). After the proteins on t h e f i l t e r had b e e n s t a i n e d , parts of the filter corresponding to the position o f t h e enzyme (Mr. 42,000) w e r e c u t and d i r e c t l y sequenced (8). Northern Blot Analysis Total RNAs w e r e p r e p a r e d using guanidine thiocyanate f r o m K562 c e l l s and HepG2 c e l l s (14). Poly (A) RNAs
749
V o l . 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
were selected by oligo(dT)-cellulose chromatography and were denatured with I M formamide, electrophoresed i n a 1~ a g a r o s e get and transferred to a nylon filter (15). Hybridization was carried out using as a probe the insert of &HF2-1. Conditions of hybridization and washing were as described (15).
RESULTS isolation
of
The the
human
in
isolated
cDNA
and AHF2-1, their
Human
x
were
2).
enytation
library mouse
1.6
cDNAs
shown the
Fig.
with as
plaques inserts
designed
of
in
cDNA,
the
were
relationship
and
screened
positive of
and
kbp,
are
was
ferrochelatase
Sizes
The
strategies
Agt]l
Two
plaques.
and
of
in
Methods.
respectively.
the
were of
as
two
de-
the
2HF1-2
clones
and
1.
Deduced
Amino
Acid
and
~HF2-1,
Sequence
Ferrochelatase of
3HF1-2
beginning
encoding
10 5
1.7
inserts
sequenced.
and
1
cDNA
of
and
Sequence
The
tail
fragment
sequencing
Nucieotide
cDNA
Materials
from
cloned
Ferrochelatase
placenta
radiolabeled
scribed
for
Human
AND D i S C U S S i O N
AHF2-1
clones, an
position
amino contained
signal
two
contained
at
partial
the
(position
acid a
AHF1-2
1711
2415
and
sequence 1576
bp
bp
insert the
of
insert
1530-1535)
and
with
a
nucieotide
a
(A)
tail
polyadbegin-
m z (J
U3 O'3 UJ
LU
kll
1
1000
I
2OOO (bp)
A HF2-1
A HF1-2
Fig. 1. Restriction maps and s e q u e n c i n g s t r a t e g y for ferrochel a t a s e cDNAs. The u p p e r d i a g r a m shows t h e p r o t e i n - c o d i n g regions of the clones, as b o x e s . The 3 ' ends o f t h e c l o n e s a r e shown by arrowheads. The d i r e c t i o n s and e x t e n t o f s e q u e n c e d e t e r m i n e d are indicated by a r r o w s .
750
(A)
(Figs.
a putative
poly
poly
sequence
ferrochelatase with
were
1
Vol. 173, No. 2, 1990
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
-29
GGGCGGGTCGGGCCGAGGCTGCCCAGGCA
1 1
ATGCGT TCA•T•G••G•AAA•AT•G•T••GG•••TG•G•G••G•GGGcG••••G•T••G•GAT••G•TGG•AT••AGCAGCTGGAGGGTC Me t A r g S e r L e u G l y A l a A s n M e l c A l a A l a A l a L e u A r g A l a A i a G l y V a l Leu L e u A r g A s p P r o L e u A l a S e r S e r S e r f r p A r g V a l
31
TGTCAGCCATGGAGGTGGAAGTCAGGTGCAGCTGCAGCGGCCG TCACCACAGAAACAGCCCAGCATGCCCAGGGTGCAAAACCTCAAGT T CysG1 n P r o T r p A r g I r p L y s S e r G l y A l a A l a A l a A l a A l a V a l T h r T h r G l u f h r A l a G t n H i s A l a G I n G I ~ A I a L ~ s P r o G 1 r i v a l
181 61
CAACCGCAGAAGAGGAAGCCGAAAACTGGAATATTAA TGCTAAACATGGGAGGCCC] GAAACTCTl GGAGATGI TCACGACTTCCTTCTG G1 nP r o 6 1 n L y s A r g L y s P r o L y s I h r G l y I l e L e u M e fcLeuAsnMe I ; G l y G l y P r o G l u ] h r L e u G l y A s p V a l H i s A s p P h e L e u L e u
271 91
AGACTCTTCTTGGACCAAGACCTCATGACACTT CCTAT TCAGAATAAGC TGGCACCATTCAICGCCAAACGCCGAACCCCCAAGATTCAA A r g L e u P h e L e u A s p G l n A s p L e u M e t T h r L e u P r o Il e G l nAsnLysLeuAlaP roPhe I l e A l a L y s A r g A r gf h r P r o L y s I l e G l n
361 121
GAGCAGTACCGCAGGA TTGGAGGCGGATCCCCCATCAAGATATGGACTTCCAAGCAGGGAGAGGGCATGGTGAAGCTGCTGGATGAATTG GIuG1 nTy r A r g A r g I l e G l y G l y G l y S e rP r o I l e L y s I l e T r p T h r S e r L y s G l n G l y G l u G l y M e t V a l LysLeuLeuAspGluLeu
451 151
TCCCCCAACACAGCCCCTCACAAATACTATAT TGGATTTCGGTACGICCATCCTTTAACAGAAGAAGCAATTGAAGAGATGGAGAGAGAT S e t P r o A s n T h r A 1 a P r o H i s L y s T y r T yr I l e G l y P h e A r g T y r V a l H i s Pr o L e u T h r G l u G l u A l a I l e G l u G l u M e t G l u A r g A s p
541 181
GGCCTAGAAAGGGCTATTGCTTTCACACAGTAI CCACAGTACAGCTGCTCCACCACAGGCAGCAGCTTAAATGCCATTTACAGATACTAT G i y L e u G t u A r g A l a l l e A l a P h e T h r G l nTyrProG1 n f y r S e r CysSe r Thr T h r G l y S e r S e r L e u A s n A l a ll e T y r A r g T y r l y r
631 211
AATCAAGTGGGACGGAAGCCCACGATGAAG TGGAGCACTATTGACAGGTGGCCCACACATCACC1CCTCATCCAGTGCTTTGCAGATCAT AsnGl n V a I G l y A r g L y s P r o T h r M e t L y e TrpSe r T h r I l e A s p A r g T rpProT h r H i s l l i s L e u L e u I l e G l n C y s P h e A l a A s p H i s
721 241
ATTCTAAAGGAACTGGACCATTTTCC~TTGAGAAGAGAAGCGAGGTGGTCATTCTGTTTTCTGC]CACTCACTGCCGATGTCTGTGGTC I l e L e u L y s G l u L e u A s p H i s P h e P r o L e u G l u L y s A r g S e r G l u V a l Va l I l e L e u P h e S e rA1 aH i s S e r LeuProMef~Se r V a l V a i
811
271
AACAGAGGCGACCCATATCCTCAGGAGG TAAGCGCCACTGTCCAAAAAGTCATGGAAAGGCIGGAGTACTGCAACCCCTACCGACTGGTG A s n A r g G l y A s p P r o T yr P r o G l n G l u V a l S e r A l aT h r V a I G l n L y s V a i M e t G l u A rgLeuGiuTy r C y s A s n P r o T y r A r g L e u V a l
901 301
TGGCAATCCAAGGTTGGTCCGATGCCCTGGTTGGGTCCTCAAACAGACGAA] CTATCAAAGGGCTTTGIGAGAGGGGGAGGAAGAATATC T rpGl n S e r L y s V a I G l y P r o M et ProT r p L e u G l y P r o G l n T h rAspGi uSe r I l e L y s G l y L e u C y s G l u A r g G l y A r g L y s A s n f l e
991 331
CTCTTGGTTCCGATAGCATTTACCAGTGACCAIATTGAAACGCT GTATGAGCTGGACATCGAGTACTCTCAAGTTTTAGCCAAGGAGTGT LeuLeuVaIProfleAlaPheThrSerAspHielleGluT h r L e u T y r G l u LeuAspI l e G l u Ty r Se r G l n V a l L e u A l a L y e G l u C y s
1081 361
GGAGTTGAAAACATCAGAAGAGCTGAGTCTCT TAATGGAAATCCATTGTTCTCTAAGGCCCTGGCCGACTTGGTGCATTCACACATCCAG GlyVal GluAsnI leArgArgAlaGiuSe r LeuAsnGlyAsnProLeu PheSerLysA1 aLeuAlaAspLeuValHisSerHisi leGin
1171 391
TCAAACGAGCTGTGT TCCAAGCAGCTGACCCTGAGCTGTCCGCTC TGTGTCAA TCC TGTCTGCAGGGAGACTAAATCCT ICTTCACCAGC S e t A s n G l u L e u C y s S e r L y s G l n L e u T h r L e u S e r C y s P r o L e u C y s V a t A s n P r o V a l C y s A r g G l u T h r L y s S e r PhePheT h r S e r
1261 421
CAGCAGCTGTGACCCCCGCCGGTGGACCCCGTGGCGTTAGGCAAATGCCCAACCTCCAGA TACCTCCGATGTGGAGAGGGTGTTAT TTAG GlnGtnLeu**~
1351 1441 1531 1621 1711 1801 1891 1981 2071 2161 2251 2341
AGATCAAGGAAGGAAGTCATCCT TCCTTGATATATATACAGCCTTTGGGTACAAATTGTGTGGTTTCTTGAGGATTGGACTCTTGATGGA TTTCTATTTTTATATAACTATACAGTAAGCATTTGIAT TTTCTCTCTCTAGGTATAAGTTAC TAGTTTGGAAIG[CCATCAGGACCTTI~ A I AA~r GAGGCTAAAAATTTGTCTTATGAGACACACCTATTTAAGCACAGATTTTGGC]TI AT1GCCCAAAACCCTCCCGAAAGGGTACG GAGAGTCCCCTCTGTGGGCTGGCAGTGTGAATGAGATCTGTTTAGTCTCGIGCATAIAGT TGCTGTTTTT TAAAIGAACACAGTTGAGTA TTTGAAGTGAATTTGAAAAAGAAATGTTACTTAATCTTTCCCIAAGCCCATGGGTTACAGAATGCTAGGGAGGCAATTTGGTTACCTGCA ATGGCTGCTT TTGCCAGCGAGGCCACCATTCATTGGTCATCTTGGTATTTGTGCTGTGAA; CTCACTTTCCTCAATGTAAAAAGGAATCA AGTATGGATTTCAGAGG TGCTCTTAGATTCCCCAT A C A C C C A A G G G I ~ G TGTACAAGTACAGTGTTCATGATACGTGCCTTGGT GGGAGTCCGTGGTGCCACAGGGAAGGGGCTCCCACTGCTTCTGGTCTCCAGGGACAGIGCTGCTGGAAAGGCTAGTGATGAGCTTCACCC TGGAGCTCCTCCCGGGACCT TGCAAGCCTCTCCATCCAGCATCTTCI CTATCTTAGTTGAATGCCTTCTTTCTGAACATTTGI TTTAAGA ATTATTTTATAAAGTCAACAATACTTTGCTTGAATTCTTTCTTAATTTACGATTTTT FATTATAAAAAAGTATAGTGATACAATGGGACA ]GTGAAGAATACAGAAAAGTAACCACTTTAATGCAATAACTGTTATCATAATATTGTAI TTCGIGGI AGTCCTTGCCTGTAGATATTTTT AATGCCAT TTAATGCCATTGTCACCTTGGATI T ATGAGTGAAAAGTGTTTCI AAAAA]A TAGAAATAATGTCAGAAAAAAAAAA
91
F i g . 2. S e q u e n c e s of human f e r r o c h e l a t a s e cDNAs. Underlined is the amino-terminal sequence estimated by h o m o l o g y t o t h o s e of purified mouse and b o v i n e f e r r o c h e l a t a s e . The a p p a r e n t polyadenylation signals (AATAAA) a r e boxed. (^). the sites of p o l y (A) addition.
ning
at
tained
position an
423
amino
the
two
inframe phobic peptide
open
1543. reading
acids clones
ATG
is
was
the
followed
human
(Fig.
nucleotide
frame
residues
sequence in
The
encoding
(Fig. same by 2,
2). in
sequence a
protein,
The
first
ferrochelatase.
We
751
con-
Mr.
47,833
with
sequence
regions.
specifying residue),
~ HF2-I
nucleotide
overlapped
codons
of
a
basic
defined
attempted
The
to
and as
a
determine
of
first hydrosignal the
VOI. 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
HF
MRSLGANMAAALRAAGVLLRDPLASSSWRVCQPWRNKSGAAAAAVTTETAQHAQGAKPQVQPQKRKPKTGILMLN
75
MF
MLSASANMAAALRAAGALLREPLVHGSSRACQPWRCQSGAAVAA-TTEKVHHAKTTKPQAQPERRKPKTGILMLN
74
YF
MLS ......
45
HF
HGGPETLGDVHDFLLRLFLDQDL--MTLPIQNKLAPFIAKRRTPKIQEQYRRIGGGSPIKIHTSKQGEGMVKLLD
MF
MGGPETLGEVQDFLQRLFLDRDL--MTLPIQNKLAPFIAKRRTPKIQE--RRIGGGSPIKHWTSKQGEGMVKLLD
145
YF
MGGPSKVEETYDFLYQLFADNDLIPISAKYQKTIAKYIAKFRTPKIEKQYREIGGGSPIRKWSEYQATEVCKILD
120
HF
ELSPNTAPHKYYIGFRYVHPLTEEAIEEMERDGLERAIAFTQYPQYSCSTTGSSLNAIYRYYNQVGRKPTHKNST
223
RTIRTQGSFLR
........
R-SQLTITRS
....
FSVT ....
FNMQNA ......
QKRSP-TGIVLMN
148
MF
ELSPATAPHKYYIGFRYVHPLTEEAIEEMERDGLERAIAFTQYPQYSCSTTGSSLNAIYRYYNEVGQKPTMKWST
220
YF
KICPETAPHKPYVAFRYAKPLTAETYKQHLKDGVKKAVAFSQYPHFSYSTTGSSINELWRQIKALDSERSISWSV
195
HF
IDRWPTHHLLIQCFADHILKELDHFPLEKRSEVVILFSAHSLPMSVVNRGDPYPQEVSATVQKVMERLEYCNPYR
298
MF
IDRWPTHPLLIQCFADHILKELNHFPEEKRSEVVILFSAHSLPMSVVNRGDPYPQEVGATVHKVMEKLGYPNPYR
295
YF
IDHHPTNEGLIKAFSENITKKLQEFPQPVRDKVVLLFSAHSLPMDVVNIGDAYPAEVAATVYNIMQKLKFKNPYR
270
HF
LVWQSKVGPMPWLGPQTDESIKGLCERGRKNILL--VPIAFTSDHIETLYELDIEYSQVLAKECGVENIRRAESL
371
MF
LVWQSKVGPVPWLGPQIDEAIKGLCERGRKNILL--VPIAFTSDHIETLYELDIEYSQVLAQKCGAENIRRAESL
368
YF
LVWQSQVGPKPWLGAQTAEIAEFL---GPKVDGLMFIPIAFTSDHIETLHEID---LGVIGESEYKDKFKRCESL
339
HF
NGNPLFSKALADLVHSH
IQSN ELCSKQLTLSCPL--CVN
PVCRETKSFFISQQL
423
MF
NGNPLFSKALADLVHSH
IQSNKLCSTQLSLNCPL--CVN
PVCRKT KSF FTSQQL
420
YF
NGNQTF I EGMADLVKSHLQSNQLYSNQLPLDFALGKSNDPVKDLSLVFGHH
393
EST
F i g . 3. H o m o l o g y i n t h e d e d u c e d a m i n o a c i d s e q u e n c e s o f human, mouse and yeast ferrochetatase. The s e q u e n c e s are shown by single-letter code. HF, human f e r r o c h e l a t a s e ; MF, mouse f e r r o cheiatase (8) ; YF, y e a s t ferrechelatase (7) ; *, identical amino a c i d s in t h e t h r e e s p e c i e s . amino-terminal it
was
blocked.
undertine)
tatase
The
was
sequences
amino
sequence
of
thus
acid
estimated
purified
ferroche]atase
(6).
ferrocheIatase
was
ferrochetatase and
enzymes
(7),
identity
to
the
The
enzyme
with
with The
the
acids
and
a 46~ 752
of
acid
identity
mouse
ferroche-
to
of
369
42, 158.
This
the
purified
human
sequence
of
mouse
and
mouse
ferrochelatase
2,
amino-terminat
humans
than
(Fig.
of
of
from
but
consisting
sequences
longer
Human
and
mass that
enzyme
the
protein
amino
enzyme
ferrochelatase
in
molecular
deduced
3).
amino
of
homologies
agreement
respectively. mouse
region
A mature
compared
(Fig. thirty
in
human
(X-A-K-P-Q-V-D-)
(8). had
was
purified
by
bovine
residues weight
the
amino-terminal
(T-T-K-P-O-A-Q-)
molecutar
three
of
yeast
consisted (8)
and
exhibited yeast
human
enzyme.
of yeast
an
88~ The
V o l . 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1
2
(kbp)
-28s 2.92.2-
-18s 1.5-
Fig. 4. RNA b l o t hybridization using ferrochelatase cDNA as a probe. P o l y (A) RNAs f r o m K562 c e i l s and HepG2 c e i l s were e ~ ctrophoresed on an a g a r o s e g e t c o n t a i n i n g formaldehyde. A 5Zp_ labeled insert o f XHF2-1 was used as a p r o b e . Lane 1, RNA (2 # g ) o f K562 c e l l s ; and l a n e 2, RNA (2 #g) o f HepG2 c e l l s .
carboxy-terminat conserved
than
regions the
at
same
around the
halves the
these
259
the
to
ferrochelatases
ha!ves.
267,
three
three
Blot
295
to
species,
regions
As
it
303
the
and
may
be
is
involved
in
of
poly
RNAs
are three
334 that
peptides
to
345
the
cata!ytic
better
were
sequence
reaction
of
Ana!ysis
RNA b ! o t celts
was
Fig.
4,
using
b!ot
two
and
1.6
kbp.
kbp
and
a
analysis,
hybridization
done
revealed
for
three
enzyme.
Northern
in
the
amino-termina!
position among
of
as
utilization
probe
hybridization
hybridization RNA
faint mRNA
from
band in
K562
bands
1. 6 and
in two
the
insert
for
cells kbp.
HepG2
of
K562
showed
to of
a
the
and
HepG2
As
shown
AHF2-1.
K562
ce!l
po!y at
positive data
on
(A) about
band the
at
RNA
contained
1.6
and
the
clones
of
AHF1-2
two
mRNAs
the
po!yadenylation 753
from
ferrochelatase
cells
lengths
alternative
of
From
corresponding
Variation of
the
ana!ysis
HepG2 at
ferrochelatase,
AHF2-I.
a
(A)
signals
2.5
is
due
found
RNA 2.5 2.5 blot kbp and to in
Vo1.173, No. 2 , 1 9 9 0
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AHF1-2
and A H F 2 - 1 .
at
3'-ending
region
signal
at
position
signal
of
~he
AATAAA
terminating Ferrochelatase similar
to
chetatase
The
apparent has
mRNA o f
those
seen
mRNA w i t h
been to
found,
1943
longer
HepG2
in
two
not
1938
the
polyadenyiation
although
which
may
be
ferroche]atase
celts
showed
K562 c e i l s . bands
signal
was
The
similar
of AHF1-2 there
used
is
for
a
the
mRNA m o l e c u l e .
two
bands
of
sizes
pattern
of
human
ferro-
to
of
mouse
enzyme
that
mRNA. The coding
the
values human cDNA of
encoding
sequence,
residues. and
cDNA
specifying
The m a t u r e predicted
obtained
protein
consists mass
(6).
The
ferrochelatase
lesions
related
for
369
to
erythropoietic
423
amino
of
proteinamino
acid
agrees
purified
facilitate
the
of
42,158
availability
wilt
has
protein of
of
electrophoretically
human
molecutar
ferrochelatase
a precursor
molecular
ferrochelatase for
human
the
residues
well
bovine
acid
with (4)
and
full-length
characterization protoporphyria.
ACKNOWLEDGMENTS: We t h a n k Dr. S. S u z u k i for a kind gift of a human p l a c e n t a cDNA l i b r a r y , M. O h a r a f o r critical c o m m e n t s and K. Y a s a k a f o r assistance with the amino acid sequencing. This w o r k was s u p p o r t e d in part by G r a n t s - i n - A i d for Scientific Research from the Ministry of Education, Science and C u l t u r e of J a p a n and by a g r a n t f r o m N i h o n P h a r m a c e u t i c a l Co.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
Porra, R. J. and J o n e s , O. T. G. (1963) B i o c h e m . J. 87, 186-190. J o n e s , M. S. and J o n e s , O. T. G. (1969) B i o c h e m . J. 113, 507-514. Taketani, S. and T o k u n a g a , R. (1981) J. B i o l . Chem. 256, 12748-12753. Taketani, S. and T o k u n a g a , R. (1982) E u r . J. B i o c h e m . 127, 443-447. Dailey, H. A. and F l e m i n g , J. E. (1983) J. B i o l . Chem. 258, 11453-11459. Mathews-Roth, M. M., D r o u i n , G. L. and D u f f y , L. (1987) Arch. Dermatol. 123, 4 2 9 - 4 3 0 . Labbe-Bois, R. (1990) J. B i o l . Chem. 265, 7 2 7 8 - 7 2 8 3 . Taketani, S., Nakahashi, Y . , O s u m i , T. and T o k u n a g a , R. ( 1 9 9 0 ) J. B i o l . Chem. i n p r e s s . Kappas, A., Sassa, S., Gatbraith, R. A. and N o r d m a n n , Y. ( 1 9 8 9 ) The M e t a b o l i c Basis of Inherited Disease, 6 t h Edn, (Scriver, C. R., B e a u d e t , A. L . , S l y , W. S. and V a l l e , D. eds.) pp1305-1365, McGraw-Hill, New Y o r k .
754
Vol. 173, No. 2, 1990
10.
11. 12. 13. 14. 15.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Maniatis, T., Fritsch, E. F. and Sambrook, J. (eds.) (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory P r e s s , C o l d S p r i n g H a r b o r , New Y o r k . Sanger, F., Nicklen, S. and C o u l s o n A. R. (1977) P r o c . N a t l . Acad. S c i . USA 74, 5 4 6 3 - 5 4 6 7 . Nakahashi, Y., Taketani, S., Sameshima, Y. and T o k u n a g a , R. (1990) B i o c h i m . Biophys. A c t a 1037, 3 2 1 - 3 2 7 . Matsudaira, P. (1987) J. B i o l . Chem. 262, 1 0 0 3 5 - 1 0 0 3 8 . Chomzynski, P. and S a c c h i , N. (1987) A n a l . B i o c h e m . 162, 156-159. Kamijo, K., T a k e t a n i , S., Y o k o t a , S., Osumi, T. and Hashimoto, T. (1990) J. B i o l . Chem. 265, 4 5 3 4 - 4 5 4 0 .
755
