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.
178,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
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
Vol.
178,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
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
1511