From the Department of Health Chemistry, Graduate
School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan, the § Laboratory of
Molecular Neuroanatomy, Division of Brain Science, Graduate School of
Medicine, Hokkaido University, Sapporo 060, Japan, and the
¶ Department of Cell Biology, Tokyo Metropolitan Institute of
Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo 173, Japan
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ABSTRACT |
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In a previous study, we demonstrated that
Platelet-activating Factor (PAF) acetylhydrolase purified from bovine
brain cortical cytosol consists of two mutually homologous catalytic
subunits (1 and
2) and one putative regulatory
subunit. The
latter is a product of the LIS1 gene, which is defective in
the Miller-Dieker syndrome, a form of lissencephaly. In this study, we
examined the expression patterns of these three subunits in the
developing rat brain. All three subunits were expressed in embryonic
brain, whereas only
2 and
subunit were detected in the adult
brain by Western blotting. Biochemical analyses revealed that the
1/
2 heterodimer and
2/
2 homodimer are major catalytic units
of embryonic and adult brain PAF acetylhydrolases, respectively. The
1 transcript and protein were detected predominantly in embryonic
and postnatal neural tissues, such as the brain and spinal cord.
Furthermore, we found using primary cultured cells isolated from
neonatal rat brain that
1 protein were expressed only in neurons but
not in glial cells and fibroblasts. In contrast,
2 and
transcripts and proteins were detected both in neural and non-neural
tissues, and their expression level was almost constant from fetal
stages through adulthood. These results indicate that
1 expression
is restricted to actively migrating neurons in rats and that switching of catalytic subunits from the
1/
2 heterodimer to the
2/
2 homodimer occurred in these cells during brain development, suggesting that PAF acetylhydrolase plays a role(s) in neuronal migration.
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INTRODUCTION |
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Platelet-activating factor (PAF)1 is a potent pro-inflammatory phospholipid produced by leukocytes, platelets, endothelial cells, and some neural cells. Mammalian brains contain significant amounts of PAF, which may act as a synapse messenger and transcription inducer of the early-response genes c-fos and c-jun (1). PAF has also been implicated as a messenger in long term potentiation, a cellular model of memory formation (2).
PAF is inactivated by a specific enzyme, PAF acetylhydrolase, which
removes the acetyl moiety at the sn-2 position of the glycerol backbone (3). Mammalian PAF acetylhydrolase is classified into
two types (4), plasma (extracellular) and tissue (intracellular). The
former is a 43-kDa monomeric enzyme that effectively abolishes the
inflammatory effects of PAF on leukocytes and the vasculature, indicating it is involved in maintaining plasma PAF at certain levels
(5). Recently, we succeeded in purifying and cloning intracellular PAF
acetylhydrolases. Tissue cytosol contains at least two types of
intracellular PAF acetylhydrolase, isoforms Ib and II (6). Isoform II
(PAF acetylhydrolase(II)) is a 40-kDa monomer, and its amino acid
sequence exhibits 41% identity with that of plasma PAF acetylhydrolase
(5, 7, 8), whereas isoform Ib is a heterotrimeric enzyme composed of
1,
2, and
subunits (6).
So far, cDNAs for the three subunits of PAF
acetylhydrolase(Ib) have been cloned from the cow (9-11), human (12,
13), mouse (14), and rat (15). Molecular cloning has revealed several characteristics of brain PAF acetylhydrolase (9-12, 15). (i) The amino
acid sequences of the three subunits showed extremely high homologies
among the above mammalian species. For example, the amino acid
sequences of the subunit from the mouse, rat, and cow were
identical and only one amino acid substitution was observed in the
human
subunit. Similarly, only one amino acid substitution was
observed in the human
2 subunit in comparison with those from the
other three species. The sequence identities of the
1 subunits are
lower than those of the
2 and
subunits, but are still over 95%
among these four species. (ii) The
1 (29 kDa) and
2 (30 kDa)
subunits show about 60% amino acid homology with each other, and both
1 and
2 have a catalytic center (9, 10). (iii) When these
catalytic subunits were expressed individually in Escherichia
coli, they formed catalytically competent homodimers (10),
although the
1 and
2 subunits of the PAF acetylhydrolase(Ib) purified from bovine brain formed a heterodimer. (iv) The
subunit, which does not possess enzymatic activity, has a unique domain structure, called WD40, which may interact with other proteins. Indeed,
the
subunit was shown to interact with spectrin through a
pleckstrin homology domain in vitro (16). (v) The
subunit gene is identical to the human LIS1 gene, the
causative gene of the Miller-Dieker syndrome (11).
We have also succeeded in crystallizing the recombinant 1/
1
homodimer at 1.7-Å resolution (17). Interestingly, the catalytic subunits of PAF acetylhydrolase (Ib) are very similar to those found in
p21ras and other GTPases, such as the
subunit of trimeric
G-protein. This and the fact that the PAF acetylhydrolase (Ib)
subunit shows limited but significant sequence homology with the G
protein
subunit indicate that PAF acetylhydrolase(Ib) is
essentially a trimeric G-protein-like (
1/
2)
molecule.2
Miller-Dieker syndrome is manifested by widespread agyria of the brain
and is thought to be due to a defect in the neuronal migration process
during brain development. Although the physiological function of PAF
acetylhydrolase is not fully understood, it has been speculated to play
an important role in neuronal cell migration in the developing brain.
Previous studies demonstrated that mRNAs of all three subunits were
expressed predominantly in premigrating or migrating neurons in the
fetal brains and developing cerebella of mice (14, 18), suggesting a
link between neuronal migration and brain PAF acetylhydrolase. It is
also interesting to note that NudF, a nuclear migration gene
in Aspergillus nidulans, shows 42% sequence identity to the
human LIS1 gene, suggesting that the LIS1 gene
product (the PAF acetylhydrolase subunit) has a function similar to
that of NudF and that nuclear migration plays a role in
neuronal cell migration.
As part of our continuing studies on the physiological role of PAF
acetylhydrolase, we have examined the developmental expression patterns
of the three PAF acetylhydrolase subunits in rats. In this study we
demonstrated that the expression of one catalytic subunit (1) is
regulated developmentally and that changes in
1 expression resulted
in the switching of brain PAF acetylhydrolase catalytic subunits
during brain development.
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EXPERIMENTAL PROCEDURES |
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Antibodies--
Polyclonal antibodies against the 1 and
2
subunits were prepared as follows. Rabbits (New Zealand White) were
immunized with 200 µg of purified recombinant bovine protein (10)
with Freund's complete adjuvant, followed by four boosters at 2-week intervals with 100 µg of protein, sera were prepared and used as
antisera against the
1 (antisera 453) and
2 (antisera 444) subunits. These antisera were affinity-purified using rat
1- and rat
2-coupled Sepharose 4B columns. Anti-
monoclonal antibody (clone
338.40) was a kindly gift from Dr. O. Reiner (28).
Immunoblotting Analysis-- Tissues and samples of brain at various developmental stages from Wistar rats were homogenized with four times their volumes (w/v) of SET buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 250 mM sucrose), as described (6), ultracentrifuged at 100,000 × g at 4 °C, and the supernatants were used as the cytosol fractions, the protein concentrations of which were determined by the BCA assay (Pierce). An aliquot (50 µg) of each cytosol fraction was separated by SDS-PAGE, and the proteins were transferred to nitrocellulose filters using the Bio-Rad protein transfer system. The filters were blocked with phosphate-buffered saline containing 5% (w/v) skim milk and 0.05% (v/v) Tween 20, incubated with the required antibody in phosphate-buffered saline containing 5% skim milk and 0.05% Tween 20, and then treated with anti-mouse or anti-rabbit IgG conjugated with horseradish peroxidase. Proteins bound to the antibodies were visualized using an enhanced chemiluminescence kit (ECL, Amersham Pharmacia Biotech).
Measurement of PAF Acetylhydrolase Activity-- Homogenates of various tissues and embryos were prepared as described above, and their PAF acetylhydrolase activities were measured, as described previously (6), using [3H]acetyl-PAF as the substrate.
Column Chromatography-- DEAE column chromatography was performed as described elsewhere (6). Hydroxyapatite (ECONOPACK CHT-II, Bio-Rad) and Mono Q FPLC column (Amersham Pharmacia Biotech) were linked to an FPLC system (Amersham Pharmacia Biotech). For hydroxyapatite column chromatography, the active fraction from the DEAE column was rechromatographed, and the proteins were eluted with a gradient of 0-200 mM potassium phosphate. For Mono Q FPLC column chromatography, the active fraction from the hydroxyapatite column was rechromatographed, and the proteins were eluted with a gradient of 0-500 mM NaCl.
In Situ Hybridization--
For isotopic detection of rat PAF
acetylhydrolase 1,
2, and
mRNAs, two or three
nonoverlapping antisense 45-mer oligonucleotide probes complementary to
nucleotide residues 304-348, 644-688, and 781-825 of
1 cDNA,
112-156 and 730-774 of
2 cDNA, and 268-313 and 1577-1621 of
cDNA were synthesized (15). Each probe was labeled with
35S-dATP using terminal deoxyribonucleotidyl transferase
(Life Technologies, Inc.) to produce a specific activity of 0.5 × 109 dpm/µg DNA.
Cross-linking-- Cross-linking was performed as described previously (10). Briefly, each sample was dialyzed against 100 mM sodium phosphate, cross-linked by adding 10 mM BS3 (Pierce) and then subjected to SDS-PAGE and Western blotting.
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RESULTS |
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Expression of each PAF Acetylhydrolase Subunit in Adult and
Embryonic Rat Brains--
Expression of each PAF acetylhydrolase
subunit in fetal (21-day-old embryo, E21) and adult (9 weeks old) rat
brains was examined by Western blotting with affinity-purified
anti-1 and -
2 polyclonal antibodies and an anti-
monoclonal
antibody. As shown in Fig. 1A,
all three subunits were detected in fetal brains. As the intensities of
the
1 and
2 subunits subjected to Western blotting were very similar (data not shown), it would appear that almost equal amounts of
1 and
2 proteins were expressed in the fetal rat brain. In contrast, only
2 and
subunits were detected in adult rat brains. In fact, the
1 subunit was not detected in any of the adult rat tissue tested (Fig. 1B). These data indicate that the PAF
acetylhydrolase complex exists in a form other than the classical
1/
2/
heterotrimer in adult rat tissues, including the
brain.
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Catalytic Subunit Switching during Brain Development--
As both
the 1 and
2 recombinant proteins expressed individually in
E. coli formed their respective homodimers (10), it is
likely that the
2 protein present in adult rat brain and other tissues also exists as a homodimer. To investigate this, we analyzed the subunit composition of PAF acetylhydrolase in adult (9 weeks) and
embryonic (E21) rat brains. PAF acetylhydrolase from fetal and adult
brain cytosols were partially purified by sequential DEAE ion-exchange
followed by hydroxyapatite column chromatography. Two peaks showing PAF
acetylhydrolase activity were obtained from the adult rat brain after
hydroxyapatite column chromatography (Fig.
2B), and each fraction was
subjected to PAF acetylhydrolase assay and Western blot analysis. As
shown in Fig. 2D, the first peak showing PAF acetylhydrolase
activity, which was eluted by about 80 mM phosphate,
contained only
2 polypeptides, whereas the second, which was eluted
by 150 mM phosphate, contained
2 and
polypeptides.
To examine complex formation in each fraction, a cross-linking
experiment using the cross-linking reagent BS3 was
performed. Mixing the first peak fraction with 10 mM
BS3 resulted in conversion of the 30-kDa
2 polypeptide
to a ~60-kDa band, detected using SDS-PAGE (Fig.
3, lane 6), suggesting that the
2 polypeptide present in the first peak fraction had formed a
homodimer. Cross-linking of the second peak fraction unexpectedly yielded a faint but significant ~200-kDa band in addition to the ~60-kDa band (Fig. 3, lane 8). We could not ascertain
whether the ~200-kDa band contained the
polypeptide, because the
monoclonal antibody against
polypeptide loses its reactivity after
chemical modification with BS3.
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Expression Patterns of the Three Subunits during Brain
Development--
To elucidate the developmental changes in the
expression of the PAF acetylhydrolase subunit mRNAs, parasagittal
sections of rat whole embryos and postnatal brains were examined by
in situ hybridization with subunit-specific antisense
oligonucleotide probes (Fig. 5). During
the embryonic stages, high levels of all three subunit mRNAs were
expressed in the brain, spinal cord, sensory ganglia (dorsal root and
trigeminal ganglia), and thymus (Fig. 5, A-C,
I-K, Q-S). The signals in the brain were
distributed throughout the ventricular and marginal zones. Expression
of 1 subunit mRNA was observed mainly in neural tissues, whereas
other non-neural embryonic tissues expressed low to moderate levels of
2 and
subunit mRNAs. The hepatic levels of
subunit
mRNA were particularly high in comparison with those of other
non-neural tissues.
2 subunit mRNA was expressed ubiquitously in
rat embryos (Fig. 5, I-K). At birth (P0), expression of all
three subunit mRNAs in the brain was marked (Fig. 5, D,
L, T). Thereafter, the levels of
1 subunit
mRNA declined gradually and reached the background level by the
adult stage (Fig. 5, E-H). High
2 and
subunit mRNA expression levels were ubiquitous in the gray matter of the brain until the adult stage (Fig. 5, M-P,
U-X).
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1 Subunit Expression in Isolated Neural Cells--
To identify
the cell type(s) that expressed each subunit, we isolated neural cells
from neonatal rat cerebellum, then performed Western blotting analysis
on these cells in the primary cultures. As shown in Fig.
7, the
1 subunit was expressed
exclusively in granule cells, whereas the
2 and
subunits were
expressed in granule cells, astroglial cells, and oligodendrocytes.
Granule cells are known to migrate actively from the external to the
internal granular layer in the cerebella of postnatal rats.
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DISCUSSION |
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This study is, the first to demonstrate the existance of the
2/
2 homodimer, as well as the
1/
2 heterodimer, in
vivo. In rats, switching of the catalytic complex from the
1/
2 heterodimer to the
2/
2 homodimer occurs in the brain
during development. The
1 subunit (or
1/
2 heterodimer) appears
to be expressed specifically in neurons of fetal and neonatal brains.
Furthermore, high
1 subunit expression levels were observed between
E16 and E21 and between E19 and P7 in the cerebrum and cerebellum,
respectively. Neuronal migration is well known to be particularly
active during these periods in rats, suggesting a link between brain
PAF acetylhydrolase and neuronal migration.
Significant levels of the 1 subunit were not expressed in any of the
adult rat tissues, including the brain, tested. This was also the case
in the mouse (data not shown). In our previous study, however, we
demonstrated that PAF acetylhydrolase purified from the brains of
young-adult cows (1 to 2 years old) comprised
1,
2, and
subunits, and this complex was also detected in the bovine kidney (6).
Moreover, Northern blot analysis of various human tissues revealed that
significant levels of the
1 subunit were expressed in the kidney,
thymus, and colon of the adult human, although the highest
1 subunit
expression level was observed in the fetal human brain (12). Thus, the
1 subunit expression pattern seems to differ among mammalian
species. Cell migration remains prominent in the adult organism under
normal physiological, as well as pathological, conditions. During the inflammatory response, for example, leukocytes migrate into areas subjected to insult, and migration of fibroblasts and vascular endothelial cells is essential for wound healing. It is, therefore, possible that cells expressing the
1 subunit possess the ability to
migrate in adult tissues. Alternatively, PAF acetylhydrolase possessing
1,
2, and
subunits may play roles in processes other than
cell migration. It is essential to determine the cell type(s)
expressing the
1 subunit in adult bovine and human tissues to
elucidate the physiological function(s) of intracellular PAF acetylhydrolase. In contrast to the
1 subunit, expression of the
2 and
subunits was found to be fairly universal in all species
tested (13, 15). Therefore, the
2/
2 and
2/
2/
complexes
may have more generalized functions than cell migration.
What is the functional difference between the 1/
2 heterodimer and
2/
2 homodimer? The
1 and
2 catalytic subunits show approximately 60% amino acid homology and then can form their respective homodimers and the heterodimer. According to the crystal structure of the
1/
1 homodimer (17), most of the amino acids that
are not conserved in
2 subunits are located on the surface of the
complex and are not utilized for dimer formation or catalysis. This
suggests that the surface natures of the
1/
2 heterodimer and
2/
2 homodimer complexes are distinctly different. In fact, the
Km and Vmax values of each
catalytic complex are roughly the same (9, 10). According to our
preliminary experiments, all three catalytic complexes (
1/
1,
1/
2, and
2/
2) interact with the
subunit, but their
affinities for it differ. Moreover, the catalytic activity of each
complex is affected in a different manner upon binding to the
subunit. For example, the catalytic activity of the
1/
1 and
1/
2 complexes is suppressed, whereas that of the
2/
2
complex is stimulated, upon binding to the
subunit.3 Therefore,
regulation of catalytic activity toward PAF by the
subunit may be
crucial to cells during migration. It is also possible that, in
addition to the
subunit, a different complex interacts with a
different protein, although we have no data to support this idea.
An unexpected finding was that cross-linking of the fractions
containing the 1,
2, and
subunits and
2 and
subunits yielded products of approximately 200 kDa, demonstrated by SDS-PAGE. Gel filtration column chromatography showed that the complex purified from bovine brain had a molecular mass of about 100 kDa, and SDS-PAGE revealed it was composed of three subunits (10). Therefore, we
concluded that purified PAF acetylhydrolase is a heterotrimer complex.
However, it is possible that PAF acetylhydrolase present in
vivo is associated with other protein(s) in a reversible manner and that this protein(s) dissociated from the complex during the purification procedures. Indeed, the
subunit can be dissociated from the complex by heparin column chromatography (11) and can be
re-associated with a catalytic complex.3
Morris and his group (19-21) isolated a set of mutants defective in
nuclear migration and distribution in the multinuclear filamentous
fungus A. nidulans and cloned several genes (nud
genes) required for nuclear migration using these mutants.
Interestingly, nudF encodes a protein with 42% sequence
identity to the human LIS1 gene product (i.e. the
PAF acetylhydrolase subunit) (21). Cytological observations
suggested that nuclear translocation is an essential feature of
neuronal migration both in the cerebral cortex (22) and the cerebellum
(23). If nuclear migration is essential for neuronal migration, it is
reasonable to conclude that a defect in nuclear translocation is the
cause of the neuronal migration defect observed in Miller-Dieker
lissencephaly. An other gene, nudC, encodes a 22-kDa protein
of unknown function, but it shows 68% identity to the C-terminal half
of C15 protein, which was originally identified as a
prolactin-inducible gene in activated T cells (24). Complementation
experiments showed that the full-length mammalian (rat) C15 protein,
which has a molecular mass of 45 kDa, is capable of rescuing the
nuclear movement defect of nudC mutants (25), indicating
that rat C15 protein and fungal nudC protein not only have
similar structures, but also serve similar functions. Studies on
A. nidulans mutants have shown that the nudC
protein regulates the nudF protein post-transcriptionally, suggesting that nudF and nudC proteins interact
within cells. In addition to nudF and nudC, two
other genes have been identified, nudA and nudG,
which encode a cytoplasmic dynein heavy and light chain, respectively
(19, 20), indicating that cytoplasmic dynein is involved in nuclear
migration. Microtubules have been shown to be required for nuclear
migration in a wide variety of organisms. Cytoplasmic dynein, a
microtubule-dependent, minus end-directed motor (26),
apparently provides the motive force for nuclear migration. Genetic
studies have located nudF (and also nudC)
upstream of dynein (27). Therefore, the nudF gene product,
which may be a homolog of the LIS1 gene product, and the PAF
acetylhydrolase
subunit may interact with multiple proteins involved in nuclear migration. In fact, Sapir et al. (28)
recently demonstrated that the
subunit interacts directly with
tubulin and regulates microtubular dynamics. A complex of >200 kDa may contain such components in addition to the PAF acetylhydrolase subunits. Identification of a protein(s) that interacts with (the subunits of) PAF acetylhydrolase will be essential to elucidate the
physiological function of the enzyme.
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FOOTNOTES |
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* This work was supported in part by research grants from the Ministry of Education, Science, Sports and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
81-3-3812-2111 (ext. 4723); Fax: 81-3-3818-3173; E-mail:
harai{at}mol.f.u-tokyo.ac.jp.
1 The abbreviations used are: PAF, platelet-activating factor; PAGE, polyacrylamide gel electrophoresis; FPLC, fast protein liquid chromatography.
2
Originally, we named the three subunits of PAF
acetylhydrolase, 1 (29 kDa),
2 (30 kDa), and
(45 kDa),
,
, and
subunits, respectively. In view of their structural
similarities to trimeric G-proteins, we have changed their nomenclature
to the form in this paper.
3 H. Manya, J. Aoki, H. Arai, and K. Inoue, manuscript in preparation.
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REFERENCES |
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