Vol. 178, No. 3, 1991 August 15, 1991

BIOCHEMICAL

Detection

of ll-kDa Group II Phospholipase in Human Seminal Plasma

Kiyoshi TAKAYAMA,

11,

A2

Shuntaro HARA, Ichiro KUDO and Keizo INOUE

Faculty of Pharmaceutical Received July

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1505-l 511

Sciences, University

of Tokyo,

Tokyo

113, Japan

1991

Summary - About 90% of phospholipase AZ activity detected in human seminal plasma reacted with monoclonal antibodies raised against human synovial fluid phospholipase A2. The crude seminal plasma yielded a pure immuno-cross-reactive phospholipase A2 preparation in a single purification step using immuno-affinity chromatography. The amino acid sequence of the N-terminal 20 residues of this seminal enzyme was determined and found to be identical with that of human synovial phospholipase A2. Thus, it is suggested that human seminal plasma contains phospholipase AZ, belonging to the 14-kDa group II enzyme family, as the major isoenzyme. RI1991Academic Press, Inc.

Phospholipase

which hydrolyzes

A2.

glycerophospholipids, prostaglandins,

is thought

fatty acids esterified

to be a rate-limiting

at the C-2 position

enzyme

leukotrienes

and even a platelet-activating

extracellular

phospholipases

for biosynthesis

factor

in various

of of cells

and tissues (1). Mammalian based on their mainly

primary

in digestive

distributed

group

that the seminal

the seminal

contains

the

phospholipase prostanoids

plasma

highest A2

(17).

I enzymes

in various

are present

tissues, such as spleen (5), liver

A2

vesicles

activity

and prostate

animal

among

so far.

in

All

It has

also secrete phospholipase

examined

have purified

1505

stimulus.

Human

to be involved

and Kunze

cells (15) secret

species (16).

those

(6) and

vascular smooth muscle

in response to an appropriate

of several

is thought Wurl

(8-ll),

(13), astrocytes (14) and renal mesangial

group II phospholipase

been reported into

1bkDa

(7). Various kinds of cell, such as platelets

cells (12), chondrocytes 14-kDa

(2). The

into two groups

organs such as the pancreas (3,4), whereas the 14-kDa group II

enzymes are widely intestine

structures

have been classified

A2

the

seminal This

biosynthesis

phospholipase Copyright 0 1991 rights of reproduction

A2

A2

plasma particular

of

seminal

from

human

0006-291X/91 $1.50 by Academic Press, Inc. in any form reserved.

Vol.

178, No.3,

seminal

BIOCHEMICAL

1991

plasma (18), but no detailed

AND BIOPHYSICAL

information

RESEARCH COMMUNICATIONS

on the molecular

properties

of this

enzyme has been available. We have established phospholipase

A2

phospholipase

A2

four monoclonal

by immunizing

antibodies

a mouse

against human

with

purified

(19). In this study, we attempted

present in human seminal

14-kDa

human

to identify

group II

synovial

fluid

the phospholipase

A2

plasma using of these antibodies.

MATERIALS

AND METHODS

Biological Materials - Human semen was obtained from two healthy volunteers. The semen was diluted with 3 vol. of 10 mM Tris-HCl (pH 7.4) containing 0.15 M NaCl, and the seminal plasma separated from the spermatozoa by centrifugation for 15 min at 5000 x g at 4°C Phospholipase Az Assay - E. coli [14C]phosphatidylethanolamine was prepared as described previously (20). Phospholipase A2 activity was assayed by incubating the substrate (specific activity 3000 dpm/nmol) in 0.1 M Tris-HCl, pH 9.0, 4 mM CaClz with an appropriate amount of enzyme. Liberated ‘q-labeled fatty acid was extracted using a modification of Dole’s procedure (21). Immunoblotting - Immunoblot analysis was performed as described previously (22). Briefly, test samples were resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose paper, then treated sequentially with 1% bovine serum albumin in phosphate-buffered saline for blocking, antibodies, and horseradish peroxidase-conjugated anti-mouse IgG. Color development was performed using a horseradish peroxidase-color developing reagent as a substrate. Immunoaffinity Chromatography of Human Seminal Phospholipase AZ Human seminal plasma was loaded onto a monoclonal antibody-conjugated Sepharose column (10 mm x 3 cm) with an attached precolumn of Sepharose 4B. The column was washed extensively with a buffer of 10 mM Tris-HCl (pH 7.4) containing 1.0 M NaCl, and then eluted with 0.1 M glycine-HCl buffer (pH 2.3).

RESULTS We have reported fluid

the purification

(23) and the establishment

and HP-4)

against

seminal

plasma

protein

band with

with HP-l

(Fig.

molecular

was loaded onto an HP-l-conjugated healthy

volunteers

activity

was retained

A2

was carried

Sepharose column. seminal

on the HP-1-Separose, 1506

human

(HP-l,

synovial

HP-2,

HP-3

out on the human

reacted exclusively

mass of about

in human

from

antibodies

immunoblotting l), the antibody

an estimated

A2

of phospholipase

of four monoclonal

it (19). When

90% of phospholipase

AND DISCUSSION

with a single

14 kDa. Seminal

plasma

As shown in Table I, about plasma

from

two independent

whereas a large amount

of

Vol.

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No.

3, 1991

BIOCHEMICAL

AND

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RESEARCH

COMMUNICATIONS

Fig. 1. Immunoblotting analysis of human seminal plasma. Human seminal plasma (lane 1) and purified human synovial fluid phospholipase AZ (lane 2) were loaded onto a 15% acrylamide gel, ,transferred to nitrocellulose paper, and blotted with 5 p g/ml HP-l antibody.

protein

appeared

in the flow-through

major

phospholipase

A2

in human

human

synovial

family.

Good

release

of the immuno-cross-reactive

buffer

phospholipase recoveries

(about

A2,

fractions.

seminal

These findings

plasma

which belongs

90-958)

at pH 2.3 to break the antigen-antibody

to the 16kDa activity A2

group

by elution

interaction.

Exp. 1 crude non-binding fraction binding fraction Exp.2 crude non-binding fraction binding fraction

Protein (mg)

elution

Yield (%)

129 60 640

585 349 0.04

1.24 0.17 1.60 x 10’

100 8 80

214 20 253

278 139 0.015

0.98 0.14 1.69 x 104

100 8 93

1507

after

with glycine-HCl

A typical

Specific activity (nmol/min/mg)

with

II enzyme

were achieved

TABLE I. Immuno-crossreactivity of human seminal plasma phospholipase AZ activity with anti-human synovial fluid phospholipase AZ antibody Total activity (nmol/min)

that the

was immuno-cross-reactive

of enzymatic

phospholipase

indicated

pattern

Vol.

178,

No.

BIOCHEMICAL

3, 1991

AND

100

50

0

BIOPHYSICAL

Etutbll

RESEARCH

COMMUNICATIONS

150

vdume(ml)

Fig. 2. Immunoaffinity chromatography of human seminal plasma phospholipase AZ. Human seminal plasma was applied to HP-l-conjugated Sepharose which had been equilibrated with 10 mM ‘R-is-HCI (pH 7.4) containing 1.0 M NaCl. The column was washed extensively and then eluted with 0.1 M glycine-HCl (pH 2.3). The fraction volume was 1.4 ml. The elution of protein was ). The phospholipase AZ followed by monitoring of absorbance at 280 nm (activity in a 20- ,U 1 aliquot of each fraction was measured ( . . e. . . . . . ).

is illustrated

in Fig.

polyacrylamide

gel

approximately yielded

the

preparation

with

appreciable

plasma

phospholipase

The

a

yielded

molecular

HPLC,

phospholipase

essentially

band on SDSmass

this

preparation

A2 activity

a pure

of

(Fig. 3B).

phospholipase

A2

step.

N-terminal

AZ determined

to

protein

On reverse-phase

from reverse-phase

eluted

analysis.

corresponding 3A).

peak

seminal

gave a single

(Fig.

in a single purification

The enzyme Edman

daltons

protein

crude

enzyme

electrophoresis

14,000

a single

Thus,

2. The eluted

HPLC

20 amino

was then subjected

acid

residues

to automated

of the human

seminal

were:

NLVNFHRMIKLTTGKEAALSThis

sequence

is identical

belongs

to the group

human

haploid

previously protein.

This

seminal

plasma

the

idea

for

seminal was

family.

group

enzyme.

II

enzyme

further

phospholipase

of the human synovial requirement

that of the human

II enzyme

genome

(1 l),

with

A2

(24),

of a single

phospholipase

has

by

exhibited

enzymatic

the fact

high affinity

activity 1508

A2

be identical

supported

for Ca2+ ions. The optimum

enzyme

Since the presence

might

It showed

synovial

been

to the that

the

properties for heparin

was observed

gene per suggested

synovial purified

identical

which

fluid human

to those

and an absolute

at pH 9.0.

Vol.

178,

No.

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

-68kDa -4SkDa -25kDa -

17kDa

% 55

5

Fig.

3. Purity

of

human

seminal

plasma

phospholipase

AZ preparation.

A. Immunopurified human seminal plasma phospholipaseA2 was loaded onto a 15 % acrylamide gel, and stained with Coomassiebrilliant blue. B. Immunopurified human seminal plasma phospholipaseA2 was applied to a column of Tosoh TSK-gel ODS 120-T pre-equilibrated with buffer A (acetonitrile/O.l% trifluoroacetic acid (395, v/v) at a flow rate of 0.5 ml/min. The column was washed with buffer A, then eluted with a gradient of O-100% buffer B (acetonitrile/O.l% trifluoroacetic acid (55/45, v/v)). The elution of protein was followed by monitoring of absorbance at 220nm ().

At present, the physiological roles of 1CkDa group II phospholipase A2 are not yet fully

understood. Recently,

we found that extracellular

group II phospholipase

A2 augmented the production of prostaglandin E2 from activated leukocytes (25). A similar

phenomenon

may

occur

in

seminal fluid.

phospholipase A2 may play a role in exocytosis, reported a potential

Alternatively,

the group

II

since several investigators have

role of sperm phospholipase A2 in exocytosis of the sperm

acrosome, an essential process in the series of phenomena leading to fertilization (26, 27). It

should be noted that part (about 10%) of the phospholipase AZ activity

detected in human seminal plasma was not adsorbed to HP-1-Sepharose (Fig. 2, Table I).

The activity

recovered

from the flow-through

fractions

of the HP-l-

Sepharose was not adsorbed to both heparin-Sepharose and Sepharose conjugated with

HP-4,

previously

another

monoclonal

antibody

(data not

shown).

shown that the binding sites for antibodies HP-l,

human synovial

fluid

We

have

shown

HP-4 and heparin on

phospholipase A2 are located separately from one another.

Thus, the phospholipase A2 in the flow-through

fractions

from

HP-1-Sepharose

appeared to be totally different from the 16kDa group II enzyme. This activity 1509

was

Vol.

178,

No.

not reactive suggesting different

BIOCHEMICAL

3, 1991

with that

anti-human this

from either

not yet been further

enzyme

16kDa might

AND

BIOPHYSICAL

group

RESEARCH

I phospholipase

be a novel

extracellular

group I or group II enzyme.

The activity

COMMUNICATIONS

A2

antibody

(28),

phospholipase was labile

A2

and has

characterized.

ACKNOWLEDGMENTS: We thank Dr. K. Yamamoto Sciences, University of Tokyo) for kindly supplying the work was supported in part by Grants-in-Aid for 02557090, 02954171 and 03680163) from the Ministry Culture of Japan.

(Faculty of Pharmaceutical amino acid sequencer. This Scientific Research (Nos. of Education, Science and

REFERENCES 1. Van den Bosch, H. (1982) in Phospholipids (Hawthorne, J. N. and Ansell, G. B., eds), pp. 313-358, Elsevier, Amsterdam. 2. Forst, S., Weiss, J., Elsbach, P., Maraganore, J.M., Reardon, I., and Heinrikson, R.L. (1986) Biochemistry 25:8381-8385 3. Ohara, O., Tamaki, M., Nakamura, E., Tsuruta, Y., Fujii, Y., Shin, M., Teraoka, H., and Okamoto, M. (1986) J. B&hem. 99:733-739 4. Sakata, T., Nakamura, E., Tsuruta, Y., Tamaki, M., Teraoka, H., Tojo, H., Ono, T., and Okamoto, M. (1989) B&him. Biophys. Acta 1007:124-126 5. Ono, T., Tojo, H., Kuramitsu, S., Kagamiyama, H., and Okamoto, M. (1988) J. Biol. Chem. 263:5732-5738 6. Aarsman, A.J., De Jong, J.G.N., Arnoldussen, E., Neys, F.W., Van Wassennaar, P.D., and Van den Bosch, H. (1989) J. Biol. Chem. 264:10008-10014 7. Verger, R., Farrato, F., Mansbach, C., and Pieroni, G. (1982) Biochemistry 21:68836889 8. Hayakawa, M., Horigome, K., Kudo, I., Tomita, M., Nojima, S., and Inoue, K. (1987) J. Biochem. 101:1311-1314 9. Hayakawa, M., Kudo, I., Tomita, M., Nojima, S., and Inoue, K. (1988) J. B&hem. 104:767-772 10. Mizushima, H., Kudo, I., Horigome, K., Murakami, M., Hayakawa, M., Kim, D.K., Kondo, E., Tomita, M., and Inoue, K. (1989) J. Biochem. 105:520-525 11. Kramer, R.M., Hession, C., Johansen, B., Hayes, G., McGray, P., Chow, E.P., Tizard, R., and Pepinsky, R.B. (1989) J. Biol. Chem. 264:5768-5775 12. Nakano, T., Ohara, O., Teraoka, H., and Arita, H. (1990) FEBS Lett. 261:171-174 13. Lyons-Giordano, B., Davis, G. L., Galbraith, W., Pratta, M., and Arner, E.C. (1989) Biochem. Biophys. Res. Commun. 164:488-495 14. Oka, S., and Arita, H. (1991) J. Biol. Chem. 266:9956-9960 15. Schalkwijk, C., Pfeilschifter, J., Marki, F., and Van den Bosch, H. (1991) B&hem. Biophys. Res. Commun. 174:268-275 16. Kunze, H., Nahas, N., and Wurl, M. (1974) B&him. Biophys. Acta 348:35-44 17. Kunze, H., and Bohn, E. (1978) Adv. Prostagl. Thrombox. Res. 3, 159-165 18. Wurl, M., and Kunze, H. (1985) B&him. Biophys. Acta 834:411-418 1510

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19. Takayama, K., Kudo, I., Hara, S., Murakami, M., Matsuta, K., Miyamoto, T., and Inoue, K. (1990) Biochem. Biophys. Res. Commun. 167:1309-1315 20, Doi, O., and Nojima, S. (1971) Biochim. Biophys. Acta 248:234-244 21. Natori, Y., Karasawa, K., Arai, H., Tamori-Natori, Y ., and Nojima, S. (1983) 3. Biochem. 93:631-637 22. Murakami, M ., Kobayashi, T., Umeda, M., Kudo, I., and Inoue, K. (1988) J. Biochem. 104:884-888 23. Hara, S., Kudo, I., Chang, H. W., Matsuta, K., Miyamoto, T., and Inoue, K. (1989) J. Biochem. 105:395-399 24. Hara, S., Kudo, I., Matsuta, K., Miyamoto, T., and Inoue, K. (1988) J. Biochem. 104:326-328 25. Hara, S., Kudo, I., and Inoue, K. (1991) 3. Biochem. 1lO:in press 26. Ono, K., Yanagimachi, R., and Huang, T.T.F. (1982) Develop. Growth Diff. 24:305310 27. Llanos, M.N., Lui, C.W., and Me&l, S. (1982) J. Exp. Zool. 221:107-117 28. Misaki, A., Ogawa, M., Kono, M., and Okamoto, M. (1988) Eur. Pat. Appl. 881019:8

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Detection of 14-kDa group II phospholipase A2 in human seminal plasma.

About 90% of phospholipase A2 activity detected in human seminal plasma reacted with monoclonal antibodies raised against human synovial fluid phospho...
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