Cloning of human gene encoding prostaglandin endoperoxide synthase and primary structure of the enzyme

Vol. 165, No. 2, 1989

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 888-894

December 15, 1989

OF H U M A N GENE E N C O D I N G P R O S T A G L A N D I N

CLONING

SYNTHASE AND PRIMARY

Chieko

Department Research

ENDOPEROXIDE

S T R U C T U R E OF T H E E N Z Y M E

Yokoyama

and

Tadashi

of Pharmacology, National Institute, Fujishiro-dai,

Tanabe*

Cardiovascular Suita, Osaka

Center Japan

565,

Received October 27, 1989

SUMMARY: The complete amino acid sequence of human prostaglandin endoperoxide synthase (EC 1.14.99.1, cyclooxygenase) was deduced by cloning and sequence analysis of human genomic DNA coding for the enzyme. The isolated clones covered approximately 40 kilobase pairs of human gene and the protein coding region of the enzyme was distributed into eleven exons, which encoded 599 amino acid residues with a calculated molecular weight of 68,548. Human prostaglandin endoperoxide synthase exhibited 91 % amino acid identity with the sheep enzyme. ®~9~9Ao~d~micpress, ~nc.

Prostaglandin fatty PG

acid

(PG)

cyclooxygenase,

biosynthesis

steroidal

endoperoxide

synthase

has

cDNA

(7-9).

acids

with

been

a

sequence

whom

0006-291~89

of

the

(i0),

all

mature

showed

has

cloned

determined

signal

cloned

was

been

a

enzyme

neither

and

enzyme

protein

is

Though

reported.

correspondence

cDNA

nucleotide Several

should

$1.50

Copyr~ht© 1~9 ~ Aca~micPress,~c. AHr~h~ ~repro~c~onina~rmreserved.

as

primary

66,175.

888

be

of for

in

fatty

by

nonand

sheep

PG

structure

of

the

sequence

of

the

of

The

deduced

the

enzyme the

human

sequence

nor

addressed.

4).

aspirin

composed

groups

the acid

(3,

encoding

the

form

enzyme

inhibited

such

nucleotide

of

its

is

the

precursor

peptide.

key

1.14.99.1,

activity

cDNA

from

weight

the

hydroperoxidase

Recently,

molecular

also

24-residue

*To

PGG

(EC

exhibits

drugs

6).

The

is

enzyme

anti-inflammatory (5,

structure

and

synthase

synthase) The

activity

indomethacin

been

2).

activity

cyclooxygenase

enzyme

PGH

(i,

cyclooxygenase The

endoperoxide

have

576

amino

amino

acid

including enzyme the shown

a has

primary that

Vol. 165, No. 2, 1989

hormones, of

PG

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

growth

factors

endoperoxide

which

is

in

by

epidermal factor

(ii,

suppress growth

in

12,

(EGF)

smooth

muscle

the

associated

mouse

the

other

hand,

enhanced

B transforming

cells

from

with

in

levels

type

cAMP,

enzyme On

RNA

synthesis

(11-15). is

16).

and

the

cells

of

messemger

factor

cultured

cultured

levels

cells

induce

stimulation,

RNA

MC3T3EI

corticosteroids

in

B-adrenergic

messenger

osteoblastic

interleukin-I

synthase

produced

increase

and

rat

by growth

thoracic

aortas

(15). In

this

paper,

characterization and from

the

of

primary

nucleotide

we human

report gene

isolation

encoding

structure

of

the

sequences

of

ii

human

PG

and

partial

endoperoxide

enzyme,

which

synthase, was

deduced

exons.

MATERIALS AND METHODS

[~-32p]dCTP 110 TBq/mmol) purchased from Amersham International. Restriction endonucleases, T4 DNA l i g a s e , exonuclase III a n d mung b e a n n u c l e a s e were obtained from Toyobo Co., the large fragment of E. coli DNA p o l y m e r a s e I (Klenow fragment) from Takara Shuzo Co., 7-Deaza-Sequenase kit (version 2.0) from United States Biochemical Co. and BluescriptII vector from Stratagene. T h e h u m a n g e n o m i c DNA l i b r a r y i n EMBL3 ( J a p a n e s e peripheral blood) generated by partial digestion with Sau3AI was kindly provided by Dr. Sakaki (Kyushu University, Fukuoka, Japan). The library was screened with restriction fragments of cDNA f o r s h e e p PG e n d o p e r o x i d e synthase as described3~previously (7). The double stranded probes were labeled with [a- aP]dCTP by random priming method (17). Screening of the library was performed with standard procedures (18). Isolated genomic DNA clones were characterized by restriction mapping and suitable restriction fragments were subcloned into pBluescriptII SK+ vector for further restriction mapping and sequence analysis. Restriction fragments carrying exon sequences were identified by Southern hybridization analysis u s i n g t h e s h e e p cDNA f r a g m e n t s as probes. For sequence analysis of subclones with long inserts, a series of overlapping DNA s e q u e n c e s were prepared using the exonuclease III/mung bean nuclease system. Nucleotide sequence was determined from the double stranded plasmid using the dideoxy chain-termination method (19) as described (20).

RESULTS

We sheep the

have

PG pPESI02

previously

endoperoxide (nucleotides

AND

DISCUSSION

reported synthase

the (7).

111-239),

889

isolation Among deleted

of

the

cDNA

sheep

fragment

clones cDNA of

for

clones, pPES40

by

Vol. 165, No. 2, 1989

Bal

31

nuclease

(nucleotides

(nucleotides

758-1442)

mixture

was

library

in

the

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

used

largest

hybridized

to

the

to

the

cDNA

SmaI

the

fragment

The

"gene

of

(1.4

the

hybridization

the

clones

IhPESI3

shown

to

Because

xhPES20 K

E KS

I

, I I(

----HI

PSE

i I

as

probe.

One

and

of

4

E

II

abc d e f g h

I

ATG

the

of

the

6-kb

K p_nI

Southern

IhPESI3 sheep

two

blot did

not

cDNA,

we

I

S

S

B

.~1 ]( I, I..~

I

i

further

5'-terminal

by

xhPES29

I

of

of

the

the

not

'1

BEEE

.. I.

I Jr,

I

with

xhPES27

t k

I

done

of

did

the

was

exons

and

-iii-i0)

sequence

fragment

xhPESl5

IhPESI3

covering

fragments

EcoRI,

hybridized

terminal

carry

any

3'-downstream

i

of

4.l-kb

and

weakly

characterized

with

pPES40

(nucleotides

amino

was

was

also

fragment

SalI

pPESI02,

insert

approach of

IhPES20 clone

the

clone

and

with

of

genomic

characterized.

4.7-kb

fragment

the

kb)

analysis. to

IhPES13

isolate

walking"

clones,

of

their

human the

was

pPES31

and

the and

i),

fragments

To

harboring

of

(kb),

However,

of

[~-32p]dCTP

pairs

SmaI

fragment

isolated

(Fig.

6.5-kb

(pPESI02).

region

positive

were

cDNA

5'-terminal

fragment

hybridize

sheep

SmaI

screening

digestion

fragment.

hybridize

enzyme,

clones

by

the

pPES40

upstream

for

~hPESI3

respectively.

sheep

with

6.5-kilobase obtained

to

labeled

probe

Five

fragments,

pPES31,

a

insert,

Approximately

and

were

as

EMBL3.

116-701)

i

I

j

k TGA

,--, IOObp AATAAA

Fig. i. R e s t r i c t i o n m a p of the g e n o m i c c l o n e s w i t h the l o c a t i o n of exons. O n l y the r e l e v a n t r e s t r i c t i o n s i t e s are s h o w n in the map. A b b r e v i a t i o n s of t h e r e s t r i c t i o n sites are as follows : BamHI, B; EcoRI, E; KpnI, K; PstI, P; SmaI, S. T h e e x o n s are i n d i c a t e d by c l o s e d boxes. The connected e x o n s are i l l u s t r a t e d under the restriction map. The protein coding region is i n d i c a t e d b y o p e n boxes, the 3' u n t r a n s l a t i o n a l r e g i o n by a s o l i d line. T h e e x o n c o n t a i n i n g the t r a n s l a t i o n a l i n i t i a t i o n c o d o n is l a b e l e d a a n d f o l l o w i n g e x o n s a r e l a b e l e d b t h r o u g h k. The point of the t r a n s l a t i o n a l i n i t i a t i o n codon, ATG, t e r m i n a l codon, TGA, and polyadenylation signal, A A T A A A are i n d i c a t e d by a r r o w s . The SalI s i t e s in E M B L 3 w e r e u s e d for p r e p a r a t i o n of t h e inserts.

890

Vol. 165, No. 2, 1989

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

screened

the

fragments

(nucleotides

EcoRI

human

genomic 1002-1221,

(nucleotides

fragments

of

pPES28

of

~hPES29

were

genemic

DNA

clones

covered

as

exons

were

described

sequence

in

in

nucleotide

sequences

intron/exon

junctions

with

codon,

that

putative

of

sheep

of

as

the shown

was

assigned

codon

initiation

sites

by

appeared

from

residue

of

termination

The

amino

deduced

from

the

nucleotide

the

human

enzyme

form

of

with

a molecular

PG

acid

weight

endoperoxide

sheep

and

encoding

terminal

both

23-amino

sequence

of

9).

potential

Four

aspirin the sites

the

acetylation

sheep

enzyme

(amino

acetylation

is of

the

acid site

of

The

exhibited

91

nucleotide enzymes

acid signal

showed

a

as

site (7-9). residues

were

529)

the

found

(21). bp

codon,

TGA

%

in

the

sequences

for

103,

143,

409)

were

also

found

891

precursor residues of

human of

open

homology.

amino

not

synthase

that

the

the

typical

(data

acid

to

for

downstream

structure

a

the

A

the

amino

of

in

sequence

sequence

identity

88.5

and

translational

the

reading The

amino

characteristic sheep

glycosylation

Similar 67,

regions

727-732

shows

shown

Determined

surrounding

primary

polypeptide peptide

i.

that

599

%

coding

endoperoxide

sequences

asparagine-linked

(residue

PG

four

gene.

protein

Kozak

of

68,548.

clones,

fragments

The

suggested

composed

PstI-

those

human

consensus

at

human

sequence

synthase

enzyme

frames

sequence

i,

comparing

by

AATAAA,

shown).

of

2.

the

described

the

kb

sequence

signal,

first

Fig.

Fig.

Fig.

with

and

1433-]922)

coding

polyadenylation the

in

nucleotide

agrees

PstI

Two

in

The

shown

in

of

restriction

protein

are

The

40

METHODS"

exons

pPES33,

probe.

suitable

AND

cDNA.

initiation

eukaryotic

from

a

shown

approximately

Ii

ATG

as

As

mixture

(nucleotides

cDNA

obtained.

"MATERIALS

a of

EcoRI

sheep

sequenced

appeared

initiation

and

the

and

with

1223-1310)

1311-1432)

XhPES27

The

library

enzyme

sites acid

(7-

and

the

sequence

of

glycosylation and

the

aspirin

in

the

primary

Vol. 165, No. 2, 1989

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

5 ' ~ - - C C G C G C C A T G A G C C gt g a g t g c g a H S R

..........

ca tc tgccagGGAGTCTCTTGCTCCGGTTCTTGCTG S L L L R F L L

II

T T G C T G C T C C T G C T C C C G C C G C T C C C C G T C C T G C T C G C G G A C C C A G G G G C G C C C A C G C C A G g t a g g c g g c c. . . .

L

L

......

L

L

L

P

P

L

P

V

L

L

A

D

P

G

A

P

T

P

V

32

t c a c c c a c agTGAATCCCTGTTGTTACTATCCATGCCAGCACCAGGGCATCTGTGTCCGCTTCGGCCTT N P C C Y Y P C Q H Q G I C V R F G L

51

GACCGCTACCAGTGTGACTGCACCCGCACGGGCTATTCCGGCCCCAACTGCACCATCCgtgagctggg .......

D

R

Y

Q

C

D

C

T

R

T

G

Y

S

G

P

N

C

T

I

P

71

• " "gcccctgcagCTGGCCTGTGGACCTGGCTCCGGAATTCACTGCGGCCCAGCCCCTCTTTCACCCACTTCCTG

O

L

W T

W L

R

N

S

L

R

P

S

P

S

F

T

H

F

L

91

V

L

116

.......... c t c t c t gcagTGCGCTCCAACCTTATCCCCAGTCCCCCCACCTACAACTCT R S N L I P S P P T Y N

S

131

K

156

t a a a & t gg g . . . . . . . . . . c c c c a a c c a g G G A A G A A G C A G T T G C C A G K K Q L P

171

CTCACTCACGGGCGCTGGTTCTGGGAGTTTGTCAATGCCACCTTCATCCGAGAGATGCTCATGCTCCTGGTACTC L T H G R W F W E F V N A T F I R E M L M L L ACAGgtgggtgtgg T V

GCACATGACTACATCAGCTGGGAGTCTTTCTCCAACGTGAGCTATTACACTCGTATTCTGCCCTCTGTGCCTAAA A H D Y I S W E S F S N V S Y Y T R I L P S V GATTGCCCCACACCCATGGGAACCAAAGg D C P T P M G T K

P

GATGCCCAGCTCCTGGCCCGCCGCTTCCTGCTCAGGAGGAAGTTCATACCTGACCCCCAAGGCACCAACCTCATG D A Q L L A R R F L L R R K F I P D P Q G T N

L

M

196

TTTGCCTTCTTTGCACAACACTTCACCCACCAGTTCTTCAAAACTTCTGGCAAGATGGGTCCTGGCTTCACCAAG F A F F A Q H F T H Q F F K T S G K M G P G F

T

K

221

GCCTTGGGCCATGGGg A L G H G

t g a g t ac ct

.

.

.

.

.

.

.

.

.

.

ct g t c c a c a g G T A G A C C T C G G C C A C A T T T A T G G A G A C A A T V D L G H I Y G D

N

CTGGAGCGTCAGTATCAACTGCGGCTCTTTAAGGATGGGAAACTCAAGTACCAGgtagtgctgg .......... L E R Q Y Q L R L F K D G K L K Y Q

236 c 254

a t c c c acagGTGCTGGATGGAGAAATGTACCCGCCCTCGGTAGAAGA~GCGCCTGTGTTGATGCACTACCCCCGA V L D G E H Y P P S V E E A P V L M H Y P R 276 GGCATCCCGCCCCAGAGCCAGATGGCTGTGGGCCAGGAGGTGTTTGGGCTGCTTCCTGGGCTCATGCTGTATGCC G I P P Q S Q M A V G Q E V F G L L P G L M L Y A 301 ACGCTCTGGCTACGTGAGCACAACCGTGTGTGTGACCTGCTGAAGGCTGAGCACCCCACCTGGGGCGATGAGCAG T L W L R E H N R V C D L L K A E H P T W G D E Q 326 CTTTTCCAGACGACCCGCCTCATCCTCATAGgt g a g g a c L F Q T T R L I L I G

tc ..........

ccctgcccagGGGAGACCATCAAG E T I K

341

ATTGTCATCGAGGAGTACGTGCAGCAGCTGAGTGGCTATTTCCTGCAGCTGAAATTTGACCCAGAGCTGCTGTTC I V I E E Y V Q Q L S G Y F L Q L K F D P E L L F 366 GGTGTCCAGTTCCAATACCGCAACCGCATTGCCACGGAGTTCAACCATCTCTACCACTGGCACCCCCTCATGCCT G V Q F Q Y R N R I A T E F N H L Y H W H P L M P 391 GACTCCTTCAAGGTGGGCTCCCAGGAGTACAGCTACGAGCAGTTCTTGTTCAACACCTCCATGTTGGTGGACTAT D S F K V G S Q E Y S Y E Q F L F N T S M L V D Y 416 GGGGTTGAGGCCCTGGTGGATGCCTTCTCTCGCCAGATTGCTGGCCGGgt aagcccc G V E A L V D A F S R Q I A G R

t ..........

ctctcgg

cagATCGGTGGGGGCAGGAACATGGACCACCACATCCTGCATGTGGCTGTGGATGTCATCAGGGAGTCTCGGGAG I G G G R N M D H H I L H V A V D V I R E S R E

432 456

ATGCGGCTGCAGCCCTTCAATGAGTACCGCAAGAGGTTTGGCATGAAACCCTACACCTCCTTCCAGGAGCTCGTA M R L Q P F N E Y R K R F G M K P Y T S F Q E L V 481 Ggtgagcagct G

..........

ctccttgtagGAGAGAAGGAGATGGCAGCAGAGTTGGAGGAATTGTATGGAGAC E K E M A A E L E E L Y G D

496

ATTGATGCGTTGGAGTTCTACCCTGGACTGCTTCTTGAAAAGTGCCATCCAAACTCTATCTTTGGGGAGAGTATG I D A L E F Y P O L L L E K C H P N S I F G E S M

521

ATAGAGATTGGGGCTCCCTTTTCCCTCAAGGGTCTCCTAGGGAATCCCATCTGTTCTCCGGAGTACTGGAAGCCG I E I G A P F S L K G L L G N P I C S P E Y W K P

546

AGCACATTTGGCGGCGAGGTGGGCTTTAACATTGTCAAGACGGCCACACTGAAGAAGCTGGTCTGCCTCAACACC S T F G G E V G F N I V K T A T L K K L V C L N T

571

AAGACCTGTCCCTACGTTTCCTTCCGTGTGCCGGATGCCAGTCAGGATGATGGGCCTGCTGTGGAGCGACCATCC K T C P Y V S F R V P D A S Q D D G P A V E R P S

596

ACAGAGCTCTGAGGGGCAGGAAAG--- 3 ' T E L *

599

Fig. 2~ Protein coding region of the nucleotide sequence of human PG endoperoxide synthase gene and deduced amino acid s e q u e n c e of the e n z y m e . Exon sequences a r e g i v e n in u p p e r c a s e letters. T h e d e d u c e d a m i n o a c i d s are s h o w n u n d e r the n u c l e o t i d e s e q u e n c e a n d n u m b e r e d b e g i n n i n g w i t h the t r a n s l a t i o n a l initiation methionine. I n t r o n s are g i v e n in l o w e r c a s e l e t t e r s a n d I0 b a s e s are p r e s e n t e d at b o t h the d o n o r a n d a c c e p t o r sites. T h e 5' a n d 3 ~ untranslation&l r e g i o n s of e x o n s a a n d k (see Fig. i) are s h o w n by l i m i t e d n u m b e r of r e s i d u e s .

892

Vol. 165, No. 2, 1989

structure amino

of

acid

that

of

the

human

the

exon

the

human

sequence EGF

is

This

has

of

the

enzyme

(22).

TATA

have

box-like

these

are

not

that

the

enzyme

exist

by

in

ATG

is

element the

notion

this

have

region.

Recently, by

cAMP

(12,

14),

endoperoxide

of

of the

domain evolution

5'-

CCAAT

and

functions

of

synthase

it

reported

has

been and

suppressed that

responsive gene.

In

the

cAMP

element

may

order

transcription

the from of ONO on

the

gene

are

now

in

studies

on

its

We thank Dr. T. Watanabe for a critical reading of manuscript. This work has been supported in part by grants the Ministry of Health, and Welfare and the Ministry Education, Science and Culture of Japan, and by grants from Medical Research Foundation, Yamanouchi Foundation of Research Metabolic Disorders, Japan.

of

gene,

and

to

5'-

region

synthase

sheep

endoperoxide

16)

the

of

several

suggesting

hormone

region

PG

PG

region

sequence

However,

of

steroid

mechanisms

the

found

of

the

EGF-like

3-kb

and

domain

of

the

approximately

(13,

the

that

the

induced

5'-flanking

sequence

the

resemble

entire

during

present.

and/or

the

to

EGF-like the

of

the

flanking

of

the

codon

corticosteroids

by

with

transcription

at

the

encoded

part

shown

exon-shuffling

in

the

was

by

start

known

investigate regulation

occured

in

gene

responsive

support

terminal

that

33-71)

sequences

sequences

directly

noteworthy

analyzed

the

aminQ

enzyme

identical

may

enzyme

of

is

completely fact

The

sheep

(residues

the

upstream

the

It

of

We

enzyme.

of

(22).

enzyme c

enzyme.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

progress.

ACKNOWLEDGMENTS

REFERENCES

i. 2. 3. 4.

Pace-Asciak, C.R., and Smith, W.L.(1983) in The Enzymes, Vol. 16, pp.543-603, (Boyer, P.D;, Ed.) Academic Press, New York. Needleman, P., Turk, J., Jakschik, B.A., Morrison, A.R., and Lefkowith, J.B. (1986) Annu. Rev. Biochem. 55, 69-102. Miyamoto, T., Ogino, N., Yamamoto, S., and Hayaishi, O. (1976) J. Biol. Chem. 251, 2629-2636. Ohki, S., Ogino, N., Yamamoto, S., and Hayaishi, O. (1979) J. Biol. Chem. 254, 829-836. 893

Vol. 165, No. 2, 1989

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

5.

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