Vol.

168,

No.

3, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages 1059-1065

May 16,199O

STRUCTURE

OF GENE CODING

Masayuki Faculty

KOMADA,

of

Ichiro

KUDO*,

Pharmaceutical Hongo,

Received

FOR RAT GROUP II

and Keizo

Science,

Bunkyo-ku,

PHOSPHOLIPASE INOUE

University

Tokyo

113,

A2

of

Tokyo,

Japan

March 29, 1990

The gene coding for rat group II phospholipase A2 was isolated from a rat genomic library by using the cDNA for rat platelet phospholipase A2 as a hybridization probe. The rat group II phospholipase A gene about 3.5 kilobase pairs and spanned consisted of fi 4 e exons. Southern blot analysis revealed that a single copy of this gene exists in the rat haploid genome. A TATA-like sequence and two AP-2 binding site-like loci were found upstream from the tentatively identified transcription initiation site. O1990 Academic Press, Inc. Mammalian have

phospholipase

recently

porcine

been

rabbit These

they

are

with

purified

intestine

(4), ed.

A2 's

Cl),

platelet enzymes

from

rat

various

platelet

(5),

and

exhibit

different

molecular

cellular

(2),

human

masses rat

platelet

considerable

of

about

14 KDa

sources,

spleen (6),

(3), and

structural

such rat

as

liver

characterizhomology,

but

phospholipase A2 and are A Is, like the enzymes from 2 crotalid or viperid venom (7). Mammalian group II phospholipase A2 's have also been detected in extracellular space and purified from inflamed sites in some animals experimental and humans with diseases and it was suggested that these group II (8,9,10), phospholipase A2 's may be involved in the process of inflammation. classified

as

We have platelets

from pancreatic II" phospholipase

found in both secrete group

Phospholipase inflammation their

"group

in II

vivo and phospholipase

A2's detected may not, however,

characteristics

were We previously

in

in

vitro studies A2 upon stimulation

extracellular

space

*

To whom all

of

the

sites

rat (11). of

be derived from platelets, although quite similar to those of plateletisolated a cDNA clone (12), and in

derived enzymes. the present study, we have cloned and characterized rat group II phospholipase A2 in order to clarify expression

at

that

the gene the mechanism

for of

gene.

correspondence

should

be addressed. 0006-291x/90 $1.50 1059

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

BIOCHEMICAL

166, No. 3, 1990

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

MATERIALS AND METHODS Materials: Reagents were obtained as follows: [ =-32P]dCTP (3000 Ci/mmol) from ICN Radiochemicals; large fragment of E. coli DNA polymerase I and T4 DNA ligase from Takara Shuzo Co.; restriction enzymes from Takara Shuzo Co. and Toyobo Co. Southern blot analysis: Genomic DNA was extracted from rat spleen as described (13). The DNA (10 ug) was digested with a restriction enzyme, electrophoresed on a 1% agarose gel, and transferred to a nylon filter (Gene Screen Plus, as described by NEN, Boston) The 708-bp EcoRI fragment of the cDNA clone for rat Southern (14). platelet phospholipase A2, prPLA2-1 (12), was purified32by gel electrophoresis and labeled with random primers and [ a - P]dCTP according to the method of Feinberg and Vogelstein (15)8 and then used as a hybridization probe (spec&fic activity, 5 x 10 cpm/u g 1. Hybridization was carried out at 42 C for 16 h in 50% formamide, 5 x ssc, 1 x Denhardt's solution, 5% dextran sulfate, 20 mM phosphate buffer (pH 6.8), 0.1% SDS, 100 u g/ml denatured salmon sperm DNA, and 10 rig/ml denatured probe. The filter was washed in 0.3 x SSC, 0.1% SDS at 65OC for 30 min, and hybridized DNA fragments were detected by autoradiography. A rat genomic library in a Isolation of the genomic DNA clone: Sharon 4A vector (Clonetech, Palo Alto) was used for screening the rat group II phospholipase A2 gene, and 1 x lo0 plaques from the library were transferred to a nylon filter. Clones positive on the hybridization were rescreened and purified. Nucleotide sequence analysis: Phage DNAs were isolated from plaques of the positive clones, and restriction mapping was carried out. Restriction fragments were cloned into M13mp18 and mp19 phage vectors using E. coli strain JM109 as a host (16) and sequenced by the dideoxy chain termination method (17).

RESULTS Southern

blot

analysis

isolated

from

rat

enzymes

and the

zation which

of

spleen

rat was

DNA preparations

genomic digested

DNA:

Total

with

several

were analyzed

by the

ccl .lular

DNA

restriction blot-hybridi-

technique covered

with an EcoRI fragment of cDNA clone prPLA2-1, the structure entire gene but missed the 5' untranslated region, as a probe. The EcoRIor HindIII-digested genomic DNAs yielded a single positive band (Fig. l), suggesting that a single copy gene exists for rat group II phospholipase A2. The digests generated by BamHI showed three distinct positive bands (Fig. found

with 11, in accordance in the region coding for

the fact that two BamHI sites the enzyme (Fig. 2A).

were

Isolation and characterization of phage clones carrying rat group II phospholipase A2 gene: From the rat genomic library in Sharon 4A vector, group

II

1 x lo6 plaques phospholipase

were screened for clones containing A2 gene by the plaque hybridization

the

rat

method phospholipase A2

using the EcoRI fragment of cDNA for rat platelet isolated from the clone prPLA2-1 (12). Seven clones gave positive signals. They were rescreened and isolated. All seven clones showed 1060

Vol.

BIOCHEMICAL

168, No. 3, 1990

Fiq. 1. (lane l), hybridized prPLA2-1

AND BIOPHYSICAL

Southern blot analysis of rat HindIII-digests (lane 2), and for rat to the cDNA clone (12) as described in materials

RESEARCH COMMUNICATIONS

genomic DNA. BamHI-digests

ptatelet

and methods.

EcoRI-Digests (lane 3) were

phospholipase

A2r

the same restriction digestion patterns (data not shown) and each carried an 11 Kb insert. The insert contained five EcoRI fragments, one of which, 5.3 Kb fragment, hybridized to cDNA c1on.e prPLA2-1 PalI

t

A.

Poll

EcoRl

EcoRl

Pall

Samtil

Ball

BamHl

Ball

Kpnl

Sau3Al

--+--

B.

Kpnl

.-

--

C. gene

exonz

t

exon3

exon5 I 1’ : : t’

CDNA 4 SIgnal I 5’ non-coding

Fiq. 2. Restriction

Structure map: B,

peptide

+ mature

anzym

4 3’ non-coding

group II strategy;

phospholipase C, exon-intron

region

region

of rat sequencing

1061

A2 gene. structure.

A,

BIOCHEMICAL

Vol. 168, No. 3, 1990

(data as

not

shown).

shown in

Fig.

Sequence 2A,

that

AND BIOPHYSICAL RESEARCH COMMtJNlCATlONS

analysis the

was performed

entire

transcript

and it of

was found,

rat

group

II

phospholipase A2 gene was generated from a region spread over three EcoRI fragments (2.9 Kb, 0.4 Kb, and 5.3 Kb). The sequence of this

region

(Fig.

3)

and

the

strategy

for

sequencing

(Fig.

2B)

are

-284

AClCAAGGCCCGTGAGAACGCAGCClCACTAAGGlClGTCClCCAACCAG

1487

-214

~TGClGTGTGAClCAlGACTCTTCTTACAACCCTC7GGAGAAACGTGGAl

1537

-164

pCysCysVallhrHisAspCysCyslyrAsnArgLeuGluLysArgGlYc

-114

GlGGCACAAAGTlTClGACCTACAAClTClCClACCGAGGGGGCCAAA~

-64

ysClylhrLysPheLeulhrlyrLysPheSerTyrAr6GlyGlyClnlle

-14

lCClGClClAGlAAGAlACCCTCACATACCTGCCCGClllCllCACGGGG

37

SerCysSerl

87

1587

1637

ClCllGAGCACACACATGCAtGClGGGAAClllAClGGlGCAGGCllACT

1687

137

lACACAACCACGCCTGllAGCAGCACAGCACGCCCAAAGAlCTAGClCAG

1737

187

ClCGCTCGGlGCTAGCClAC~ATACGlGAGGGCCTCCGTlCCACCCTCAG

1787

237

CACTGTATGAAAlCCACAAAAlTlGCCATGACCTCAATCCCACTGCTCAl

1837

287

GlGCAGGCAGGAGGAlCACAAGllCAAGGCCATCTTCAGClACTlAGAGA

1887

337

AClCAAAGGCAGCCTAAGClAlAAACACCCTGlCCCClCACCCClGCTCC

1937

ga7

CTCCCCCCTCCCTCCTCCCCCTTCCCCCTCTCCCTCCCCTCCCCCCCAAA

1987

437

AAAACCClACAAGAGGGlGGClAGGGAlCGAGGCAAACClCTGGCAGCGC

2037

467

CAlCTGlGGCCAClGlCTGlCCCCAlCACAlCGlCAGATGGCGllClGCC

2087

537

TTCCCAGCAAGCAGACAGTCCCCACGAGCAGCCATGAGACAGTAGCCATC

2137

587

ACClCTGTGlCCGTllCCCCClAAllGCAGCAAACCAGCAClCClGCCGG

2167

637

hrAsnGlnAspSerCysAr6

687

AAACACCTCTGCCAGTCCCATAAAGClGCCGClGAATGTTTTGCCCGGAA

737

LysClnLeuCysGlnCysAspLysAlaAlaAlaGluCysPheAlaAr~As

787

CAAGAAAAGCTACAGlTTAAAGlACCAGlTClACClCAACAAGllllG~

837

nLysLysSerTyrSerteuLystyrG1nPhelyrLeuAsnLysPheCysL

887

AACGGAAGACGCCCAGllGClGAAAGAGACATCllCTGAAACAlCCAGA~

2237

2287

2337

ysClyLyslhrProSerCystSt 937

AlCCTClAACACClClCClAGCCCAACCAAGllCCCCAGlGAlCAAGAAA

2387

967

ACACCCClCTCCAACCClAGAAGCAGGCGGGCCCllCTGlClTCACCCAG

2437

1037

AACCAGCCGCTCAACCClGAlClllCCCCAACAClCCACAGCCTlGCAlC

2487

1087

CGCCCAClllCAClTTlCCClTGGCAlCCAACllCClGClGCGT~

2537

1137

lAAGAGAGlCClGACAGGClCTCCCAAGTAAAG~ATTCAlCAACAACCA

2587

CGlCTGTGlCTCAlAAClCGAAACGAGACAGATAlAAAATAlGCATGClC

2637

AAAGlATAGGCCTTGACGClGGGGAGGTGGClCAGlCCATAAAGTGClTG

2687

CCAAAAAAAAAAAAAAACAAAAAAAACAAAACA~~A~~~~~~A~G~~CAA

2737

CCCCCAGAACCCAGGGACAlCAAGGGCATlCllGlllGCAAlCCTAGAGl

2787

lCGGCAAACAAAGAAAGTGGACCCClGGGGClCAATGGCCAGCCAGGCTA

2837

GC~GAA~CAG~AAA~~C~~AGG~~AG~GAGAGA~CC~G~~~CCA~AAACA

2867

1167

1237

1267

Fig.

3.

and

its

GG~AA~ATGG~GCCCCAAGAAGA~A~C~GAG~~~GA~CCG~GG~~~CCA~

2937

1337

ACACAlATCCAlGCAlTlTCCTGGAlGCAlGGGCCCACAlGGAGAGACAT

2987

1387

ACATACACTTGATTACAAAAGAACAAGGTTClGGCTCAGlCCAGllGGGl

3037

1437

CTGACCCTTGGTACC

3052

Nucleotide sequence of :rat group II- phospholipase - A2 .gene sequence. Exons are unaerIlnea. deduced amino acid Nucleotide residues are numbered beginning with the tentatively identified transcription initiation site. ***; termination codon. 1062

Vol.

BIOCHEMICAL

168, No. 3, 1990

shown.

Sequence

2 gene and the gene consists

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

comparison

between

cDNA revealed

that

the

(Fig.

2C).

of

five

exons

the

rat

group

rat

II

group

phospholipase

II

A

phospholipase

A2

DISCUSSION The

gene

for

The

(6,181.

highly

human group

structure

homologous

to

five

exons,

and the

gene

(introns

2,3,4)

position different

of in

revealed

that

a

group

of

the

introns are

phospholipase

located

II

They

at

the

blot gene

consist

region

same positions, 5'

non-coding

analysis exists

the

case

phospholipase

cloned

A2 gene was

both

protein-coding the

copy as suggested in

been

phospholipase

at

Southern

has

A2

human gene.

located

The group

II

on the

single

A2,

A2 gene.

rat

that

intron 1, each case.

phospholipase lipase

of

II

of

of

each

though

the

region,

is

of rat genomic DNA for rat group II of the human phospho-

A2 has been detected

in

various cells and tissues of rat. Although many common properties have been found among the group II phospholipase A2's, some differences have also been reported (3,4,11). The present study supports This

the

idea

single

gene

that

all

for

rat

typical secretory signal. the enzyme is a secretory

are

translated

group

II

Therefore, protein

from

an identical

phospholipase it is strongly and its lipolytic

only expressed extracellularly. The possibility the primary translational product may be further The transcription determined

to

initiation codon to the canonical was located CAAT box

located

site 62

bp

of

this

upstream

gene.

carries

suggested activity

still remains modified.

a that is that

gene was tentatively from

the

translation

(Fig. 4). A TATTTAA sequence, which is identical TATA box often seen in eukaryotic promoters (19),

26 to

a CCAT sequence,

initiation

be

A2

20 bp upstream which

often

present

-30

. . . . .-20. .

from the

was moderately further

cap site.

homologous

upstream

(20),

There to

the

was also canonical

positioned

71 to

-10

GAGCTATTTAAGAGCATTGGGAG~ACAGG~~AAACAAGG~~GGCCC~~GA .

Fiq. 4. tentatively sequence: group II

Nucleotide identified V, CAAT-like phospholipase

sequence

of

5'-flanking

region.

0,

The

transcription initiation site; n , TATA-like sequence: -, sequence homologous to human A2 gene (6,18); '-,, AP-2 binding site-like

sequence. 1063

68

Vol.

166, No. 3, 1990

bp

upstream

from

TATA-like regulating A2

BIOCHEMICAL

the

cap

sequence is the expression

gene for

4 shows

the

site.

The

likely of the

following

a high

AND BIOPHYSICAL

homology

with

DNA segment

to play mammalian

reasons;

RESEARCH COMMUNICATIONS

including

the

an important role in group II phospholipase

1) the region underlined in Fig. found in the 5' untranslated

that

region of human group II phospholipase A2 gene (63 bases out of 83 are identical), 2) sequences homologous to the activator protein-2 (AP-2) binding site were found at two positions in this region, and similar segments are also present in the corresponding region of the human gene. The AP-2 binding sites are known to exist on the 5'-flanking regions of genes whose transcription is induced by CAMP The

(21). carry

two

5'-flanking Nakano

et

vascular (22). It

is

induction

that

genes

sequences

for

mammalian group II phospholipase to an AP-2 binding site at homologous

A2 the

region seems to be consistent with the recent report by who have shown the expression of the gene in rat al., smooth muscle cells treated with CAMP-elevating agents has recently

such as expression It

fact

been reported

that

some inflammatory

interleukin-1 and tumor necrosis of mammalian group II phospholipase

of

great of

phospholipase

interest

the

to

expression

A2 by these

elucidate of

the

inflammatory

the gene

for

signals,

factor, elevated the A2 genes (22,23,24). precise

mechanism

mammalian

group

of II

signals.

It should be noted that one difference between the gene and the cDNA was found in a protein-coding region. The nucleotide residue of

the

second

was T on the

letter gene,

of

the

instead

as the 114th amino acid polymorphism of the 114th

codon

coding

of C on the

for

the

114th

amino

cDNA. The gene codes

acid

leucine

whereas the cDNA codes proline. The amino acid in the phospholipase A2 was

previously observed on both amino acid analysis of purified rat platelet phospholipase A2 (25) and nucleotide sequence analysis of cDNA for rat spleen membrane-bound phospholipase A2 (26). The present findings suggest that the observed polymorphism is due to a genetic polymorphism. Furthermore, two additional differences between region; insertion. present.

the gene and the one is a one base The reasons

ACKNOWLEDGMENTS:This (No. 63480490) for Education, Science, Mitsubishi Foundation.

for

cDNA were found in the 3' non-coding exchange and the other is a six bases these two differences are not known at

work

was supported in part by Grant-in-Aid Scientific research from the Ministry of and Culture of Japan, and also by The

1064

Vol.

168, No. 3, 1990

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES

1. Verger, R., Ferrato, F., Mansbach, C-M., and Pieroni, G. (1982) Biochemistry 21:6883-6889 2. Hayakawa, M., Horigome, K., Kudo, I., Tomita, M., Nojima, S., and Inoue, K. (1987) J. Biochem. 101:1311-1314 3. Ono, T., Tojo, H., Kuramitsu, S., Kagamiyama, H., and Okamoto, M. (1988) J. Biol. Chem. 263:5732-5738 4. Aarsman, A.J., De Jong, J.G.N., Arnolddussen, E., Neyes, F.W., van Wassenaar, P.D., and van den Bosch, H. (1989) J. Biol. Chem. 264:10008-10014 5. Mizushima, H., Kudo, I., Horigome, K., Murakami, M., Hayakawa, M Kim, D-K., Kondo, E., Tomita, M., and Inoue, K. (1989) J. Bibchem. 105:520-525 6. Kramer, R.M., Hession, C., Johansen, B., Hayes, G., McGray, P., Chow, E.P., Tizard, R., and Pepinsky, B. (1989) J. Biol. Chem. 264:5768-5775 7. Heinrikson, R.L., Trueger, E.T., and Kein, P.S. (1977) J. Biol. Chem. 252:4913-4921 8. Forst, S., Weiss, J., Elsbach, P., Maraganore, J.M., Reardon, I ., and Heinrikson, R.L. (1986) Biochemistry 25:8381-8385 9. Chang, H.W., Kudo, I., Tomita, M., and Inoue, K. (1987) J. Biochem. 102:147-154 10. Hara, S., Kudo, I., Matsuta, K., Miyamoto, T., and Inoue, K. (1988) J. Biochem. 104:326-328 11. Horigome, K., Hayakawa, M., Inoue, K., and Nojima, S. (1987) J. Biochem. 101:52-61 12. Komada, M., Kudo, I., Mizushima, H., Kitamura, N., and Inoue, K. (1989) J. Biochem. 106:545-547 13. Blin, N. and Stafford, D.W. (1976) Nucleic Acids Res. 3:23032308 14. Southern, E.M. (1975) J. Mol. Biol. 98:503-517 15. Feinberg, A.P. and Vogelstein, B. (1983) Anal. Biochem. 132:6-13 16. Yanish-Perron, C., Vieira, J., and Messing, J. (1985) Gene 33:103-119 17. Sanger, F. (1981) Science 214:1205-1210 18. Seilhamer, J-J., Pruzanski, W., Vadas, P., Plant. S., Miller, J.A., Kloss, J., and Johnson, L.K. (1989) J. Biol. Chem. 264:5335-5338 19. Corden, J., Wasylyk, B., Buchwalder, A., Sassone-Corsi, P., Kedinger, C., and Chambon, P. (1980) Science 209:1406-1414 20. Jones, N.C., Rigby, P.W.J., and Ziff, E.B. (1988) Genes Dev. 2:267-281

21. 22. 23. 24. 25. 26.

Roesler, W.J., Vandenbark, G.R., and Hanson, R.W. (1988) J. Biol. Chem. 263:9063-9066 Nakano, T., Ohara, O., Teraoka, H., and Arita, H. (1990) FEBS 261:171-174 Lett. Lyons-Giordano, B., Davis, G.L., Galbraith, W., Pratta, M-A., and Arner, E.C. (1989) Biochem. Biophys. Res. Commun. 164:488495 Kerr, J.S., Stevens. T.M., Davis, G.L., McLaughlin, J.A., and (1989) Biochem. Biophys. Res. Commun. 165:1079Harris, R.R. 1084 Hayakawa, M., Kudo, I., Tomita, M., Nojima, S., and Inoue, K. (1988) J. Biochem. 104:767-772 Ishizaki, S., Ohara, O., Nakamura, E., Tamaki, M., Ono, T., Kanda, A. Yoshida, N., Teraoka, H., Tojo, H., and Okamoto, M. (1989) Biochem. Biophys. Res. Commun. 162:1030-1036

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