Switching of Platelet-activating Factor Acetylhydrolase Catalytic Subunits in Developing Rat Brain*

Hiroshi ManyaDagger , Junken AokiDagger , Masahiko Watanabe§, Tomoya AdachiDagger , Hiroaki Asou, Yoshirou Inoue§, Hiroyuki AraiDagger parallel , and Keizo InoueDagger

From the Dagger  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

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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 (alpha 1 and alpha 2) and one putative regulatory beta  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 alpha 2 and beta  subunit were detected in the adult brain by Western blotting. Biochemical analyses revealed that the alpha 1/alpha 2 heterodimer and alpha 2/alpha 2 homodimer are major catalytic units of embryonic and adult brain PAF acetylhydrolases, respectively. The alpha 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 alpha 1 protein were expressed only in neurons but not in glial cells and fibroblasts. In contrast, alpha 2 and beta  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 alpha 1 expression is restricted to actively migrating neurons in rats and that switching of catalytic subunits from the alpha 1/alpha 2 heterodimer to the alpha 2/alpha 2 homodimer occurred in these cells during brain development, suggesting that PAF acetylhydrolase plays a role(s) in neuronal migration.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha 1, alpha 2, and beta  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 beta  subunit from the mouse, rat, and cow were identical and only one amino acid substitution was observed in the human beta  subunit. Similarly, only one amino acid substitution was observed in the human alpha 2 subunit in comparison with those from the other three species. The sequence identities of the alpha 1 subunits are lower than those of the alpha 2 and beta  subunits, but are still over 95% among these four species. (ii) The alpha 1 (29 kDa) and alpha 2 (30 kDa) subunits show about 60% amino acid homology with each other, and both alpha 1 and alpha 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 alpha 1 and alpha 2 subunits of the PAF acetylhydrolase(Ib) purified from bovine brain formed a heterodimer. (iv) The beta  subunit, which does not possess enzymatic activity, has a unique domain structure, called WD40, which may interact with other proteins. Indeed, the beta  subunit was shown to interact with spectrin through a pleckstrin homology domain in vitro (16). (v) The beta  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 alpha 1/alpha 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 alpha  subunit of trimeric G-protein. This and the fact that the PAF acetylhydrolase (Ib) beta  subunit shows limited but significant sequence homology with the G protein beta  subunit indicate that PAF acetylhydrolase(Ib) is essentially a trimeric G-protein-like (alpha 1/alpha 2)beta 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 beta  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 (alpha 1) is regulated developmentally and that changes in alpha 1 expression resulted in the switching of brain PAF acetylhydrolase catalytic subunits during brain development.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Antibodies-- Polyclonal antibodies against the alpha 1 and alpha 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 alpha 1 (antisera 453) and alpha 2 (antisera 444) subunits. These antisera were affinity-purified using rat alpha 1- and rat alpha 2-coupled Sepharose 4B columns. Anti-beta 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 alpha 1, alpha 2, and beta  mRNAs, two or three nonoverlapping antisense 45-mer oligonucleotide probes complementary to nucleotide residues 304-348, 644-688, and 781-825 of alpha 1 cDNA, 112-156 and 730-774 of alpha 2 cDNA, and 268-313 and 1577-1621 of beta  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.

The rats were anesthetized with pentobarbital, and whole embryos were removed on embryonic days (E) 13, 15, and 18 and brains on postnatal days (P) 1, 7, 14, and 21 and from adults (4 months old). The tissue specimens were frozen immediately in powdered dry ice, fresh frozen sections 20 mm thick were prepared using a cryostat and treated at room temperature as follows: fixation with 4% paraformaldehyde for 10 min, acetylation with 0.25% (w/v) acetic anhydride in 0.1 M triethanolamine HCl, pH 8.0, for 10 min, and prehybridization for 1 h in a buffer comprising 50% (v/v) formamide, 50 mM Tris-HCl, pH 7.5, 0.02% (v/v) Ficoll, 0.02% (v/v) polyvinylpyrrolidone, 0.02% (w/v) bovine serum albumin, 600 mM NaCl, 0.25% (w/v) SDS, 200 µg/ml tRNA, 1 mM EDTA, and 10% (w/v) dextran sulfate. Then, hybridization was performed at 42 °C for 10 h in the prehybridization buffer supplemented with 10,000 cpm/ml S-labeled oligonucleotide probes and 100 mM dithiothreitol. The slides were washed twice at 55 °C for 40 min with 0.1 × SSC containing 0.1% (w/v) Sarkosyl and autoradiographed using either Hyperfilm-beta max (Amersham Pharmacia Biotech) for 20 days or nuclear track emulsion (Kodak NTB-2, Eastman Kodak Co.) for 2 months.

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.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

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-alpha 1 and -alpha 2 polyclonal antibodies and an anti-beta monoclonal antibody. As shown in Fig. 1A, all three subunits were detected in fetal brains. As the intensities of the alpha 1 and alpha 2 subunits subjected to Western blotting were very similar (data not shown), it would appear that almost equal amounts of alpha 1 and alpha 2 proteins were expressed in the fetal rat brain. In contrast, only alpha 2 and beta  subunits were detected in adult rat brains. In fact, the alpha 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 alpha 1/alpha 2/beta heterotrimer in adult rat tissues, including the brain.


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Fig. 1.   Western blot analysis of rat brain PAF acetylhydrolase. A, analysis of alpha 1, alpha 2, and beta  subunits in embryonic (lane 1) and adult (lane 2) brains: 50 µg each of cytosol fraction was subjected to SDS-PAGE and Western blotting with subunit-specific antibodies. B, tissue distribution of alpha 1, alpha 2, and beta  proteins in adult rat tissues. Lane 1, cerebrum; lane 2, cerebellum; lane 3, heart; lane 4, lung; lane 5, stomach; lane 6, pancreas. Aliquots (50 µg) of the cytosol fractions were subjected to SDS-PAGE and Western blotting with subunit-specific antibodies.

Catalytic Subunit Switching during Brain Development-- As both the alpha 1 and alpha 2 recombinant proteins expressed individually in E. coli formed their respective homodimers (10), it is likely that the alpha 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 alpha 2 polypeptides, whereas the second, which was eluted by 150 mM phosphate, contained alpha 2 and beta  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 alpha 2 polypeptide to a ~60-kDa band, detected using SDS-PAGE (Fig. 3, lane 6), suggesting that the alpha 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 beta  polypeptide, because the monoclonal antibody against beta  polypeptide loses its reactivity after chemical modification with BS3.


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Fig. 2.   Hydroxyapatite column chromatographic elution profiles of PAF acetylhydrolase activity (A and B) and each subunit protein (C and D) in embryonic (E21, A and C) and adult (9 weeks, B and D) rat brains. The active fraction yielded by DEAE column chromatography was subjected to hydroxyapatite column chromatography, and the PAF acetylhydrolase activity was eluted with a gradient of potassium phosphate (dashed line), as described under "Experimental Procedures." The elution profiles of both PAF acetylhydrolase activity (A and B) and protein (C and D) are shown.


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Fig. 3.   Cross-linking of alpha 2 protein in embryonic and adult rat brain PAF acetylhydrolase. The alpha 2 proteins in the first (lanes 1 and 2) and the second (lanes 3 and 4) peaks showing PAF acetylhydrolase activity after hydroxyapatite column chromatography (Fig. 2) were cross-linked using BS3 and detected by Western blotting, as described under "Experimental Procedures." The results before (lanes 1-4) and after (lanes 4-7) cross-linking are shown.

PAF acetylhydrolase activity in the fetal brain was also separated into two peaks by hydroxyapatite column chromatography (Fig. 2A). The first peak contained alpha 1 and alpha 2 polypeptides and second contained alpha 1, alpha 2, and beta  polypeptides. Incubation of the first peak fraction with BS3 also yielded a ~60-kDa band, which cross-reacted with both the anti-alpha 1 and anti-alpha 2 antibodies, indicating that it represented a alpha 1/alpha 2 heterodimer complex. This fraction may have contained a mixture of alpha 1/alpha 1 and alpha 2/alpha 2 homodimers, but this possibility can be ruled out by the observation that the activity eluted in a single peak at a position different from that of the alpha 2/alpha 2 homodimer by mono Q FPLC chromatography (Fig. 4). Incubation of the second peak fraction with BS3 also produced a ~200-kDa band, in addition to the 60-kDa band (Fig. 3, lane 7).


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Fig. 4.   Mono Q FPLC column chromatographic elution profiles of embryonic and adult brain PAF acetylhydrolase. The first peak showing PAF acetylhydrolase activity yielded by hydroxyapatite column chromatography (fractions 9-11 in Fig. 2A and 8 in Fig. 2B) was subjected to Mono Q FPLC column chromatography, and the PAF acetylhydrolase activity was eluted with a gradient of NaCl (dashed line). Western blotting of the peak fraction is shown in the inset.

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 alpha 1 subunit mRNA was observed mainly in neural tissues, whereas other non-neural embryonic tissues expressed low to moderate levels of alpha 2 and beta  subunit mRNAs. The hepatic levels of beta  subunit mRNA were particularly high in comparison with those of other non-neural tissues. alpha 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 alpha 1 subunit mRNA declined gradually and reached the background level by the adult stage (Fig. 5, E-H). High alpha 2 and beta  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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Fig. 5.   Developmental changes in the expression of PAF acetylhydrolase alpha 1 (A-H), alpha 2 (I-P), and beta  subunit mRNAs (Q-X) in the rat. A, I, Q, E13; B, J, R, E15; C, K, S, E18; D, L, T, P1; E, M, U, P7; F, N, V, P14; G, O, W, P21; H, P, X, adult. Adjacent parasagittal sections were prepared and processed for in situ hybridization in the same experiment. Photographs were printed directly from x-ray film autoradiograms at the same magnification. The arrowhead in S indicates the thymus, while arrows in R and S indicate the trigeminal ganglion and dorsal root ganglia, respectively. Li, liver; 1, olfactory bulb; 2, cerebral cortex; 3, hippocampus; 4, diencephalon; 5, midbrain; 6, pons; 7, medulla oblongata; 8, cerebellum. Rostral and dorsal are to the right and top, respectively. Scale bar, 0.5 mm.

Western blot analysis on the expression of PAF acetylhydrolase subunit expression during brain development was also carried out. The embryonic brain expressed high levels of all three polypeptides (Fig. 6A). High alpha 1 subunit expression levels in the cerebrum and cerebellum were maintained until P4 and P7, respectively, and then declined gradually to adult levels (P63) (Fig. 6, B and C). In contrast, the alpha 2 and beta  subunit expression did not change dramatically, although alpha 2 subunit expression increased slightly as that of alpha 1 started to decline.


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Fig. 6.   Western blot analysis of rat brain PAF acetylhydrolase. A, rat embryonic brain. Cytosol fractions were prepared from brains at various embryonic stages and a 50-µg aliquot of each protein was subjected to Western blot analysis, as described under "Experimental Procedures." B and C, postnatal rat brain. Cytosol fractions were prepared from brains at various postnatal stages, and a 50-µg aliquot of each protein was subjected to Western blot analysis, as described under "Experimental Procedures." B, cerebrum, C, cerebellum.

alpha 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 alpha 1 subunit was expressed exclusively in granule cells, whereas the alpha 2 and beta  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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Fig. 7.   alpha 1 subunit expression predominates in neurons. Expression of the alpha 1, alpha 2, and beta  subunits in primary cultured oligodendrocytes (lane 1), astroglial cells (lane 2), and granule cells (lane 3) was examined by Western blotting. Bovine brain PAF acetylhydrolase(Ib), composed of alpha 1, alpha 2, and beta  subunits, was loaded onto lane 4 as a positive control.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
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References

This study is, the first to demonstrate the existance of the alpha 2/alpha 2 homodimer, as well as the alpha 1/alpha 2 heterodimer, in vivo. In rats, switching of the catalytic complex from the alpha 1/alpha 2 heterodimer to the alpha 2/alpha 2 homodimer occurs in the brain during development. The alpha 1 subunit (or alpha 1/alpha 2 heterodimer) appears to be expressed specifically in neurons of fetal and neonatal brains. Furthermore, high alpha 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 alpha 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 alpha 1, alpha 2, and beta  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 alpha 1 subunit were expressed in the kidney, thymus, and colon of the adult human, although the highest alpha 1 subunit expression level was observed in the fetal human brain (12). Thus, the alpha 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 alpha 1 subunit possess the ability to migrate in adult tissues. Alternatively, PAF acetylhydrolase possessing alpha 1, alpha 2, and beta  subunits may play roles in processes other than cell migration. It is essential to determine the cell type(s) expressing the alpha 1 subunit in adult bovine and human tissues to elucidate the physiological function(s) of intracellular PAF acetylhydrolase. In contrast to the alpha 1 subunit, expression of the alpha 2 and beta  subunits was found to be fairly universal in all species tested (13, 15). Therefore, the alpha 2/alpha 2 and alpha 2/alpha 2/beta complexes may have more generalized functions than cell migration.

What is the functional difference between the alpha 1/alpha 2 heterodimer and alpha 2/alpha 2 homodimer? The alpha 1 and alpha 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 alpha 1/alpha 1 homodimer (17), most of the amino acids that are not conserved in alpha 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 alpha 1/alpha 2 heterodimer and alpha 2/alpha 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 (alpha 1/alpha 1, alpha 1/alpha 2, and alpha 2/alpha 2) interact with the beta  subunit, but their affinities for it differ. Moreover, the catalytic activity of each complex is affected in a different manner upon binding to the beta  subunit. For example, the catalytic activity of the alpha 1/alpha 1 and alpha 1/alpha 2 complexes is suppressed, whereas that of the alpha 2/alpha 2 complex is stimulated, upon binding to the beta subunit.3 Therefore, regulation of catalytic activity toward PAF by the beta  subunit may be crucial to cells during migration. It is also possible that, in addition to the beta  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 alpha 1, alpha 2, and beta  subunits and alpha 2 and beta  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 beta  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 beta  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 beta  subunit may interact with multiple proteins involved in nuclear migration. In fact, Sapir et al. (28) recently demonstrated that the beta  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.

    FOOTNOTES

* 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.

parallel 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, alpha 1 (29 kDa), alpha 2 (30 kDa), and beta  (45 kDa), gamma , beta , and alpha  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.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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