1 Department of Molecular Physiology and Biological Physics, University of Virginia, Health Sciences Center, Charlottesville, VA 22906-0011, USA and 2 Department of Health Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan
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Abstract |
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Keywords: dimerization/mammalian brain/platelet-activating factor acetylhydrolase (Ib)
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Introduction |
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All of the above PLA2s hydrolyze phospholipids with long acyl chains in the sn2 position. In contrast, platelet-activating factor acetylhydrolases (PAF-AHs) are distinct PLA2s with high substrate specificity towards 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor, PAF), where an acetyl group occupies the sn2 position. The physiological function of PAF-AHs is to inactivate PAF, a potent phospholipid messenger molecule with both paracrine and autocrine functions, through a hydrolytic deacetylation of the sn-2 postion, with concomitant formation of the biologically inert lyso-PAF molecule. There are several isoforms, intra- and extracellular (for recent reviews, see Stafforini et al., 1997; Derewenda and Ho, 1999). The plasma (Tjoelker et al., 1995) isoform and the so-called isoform II (Hattori et al., 1996
) are members of the ubiquitous
/ß hydrolase superfamily (Heikinheimo et al., 1999
), as inferred from the crystal structure of a bacterial homologue (Wei et al., 1998
). Consequently, these enzymes are related to neither secretory nor cytosolic PLA2s, but show more similarity to neutral lipases (Derewenda, 1994
). The third well-characterized mammalian isoform of PAF-AHs is the brain intracellular isoform Ib (Hattori et al., 1993
). It is an oligomeric complex, which consists of three types of subunits: two homologous
-subunits,
1 and
(63% amino acid identity), which harbor all of the catalytic activity and which are unrelated to any other PLA2s (Hattori et al., 1994a
, 1995
), and the ß-subunit, a member of the WD40 family of proteins and the product of the causal gene for MillerDieker lissencephaly (Reiner et al., 1993
; Hattori et al., 1994b
). The two catalytic subunits associate with high affinity to form a dimer. Interestingly, the composition of this dimer varies during the early development of the organism because of different expression patterns of the
1 and
2 proteins: the
1 is expressed almost exclusively during the fetal stage, whereas
2 is expressed in adult life (Manya et al., 1998). All three dimers, i.e. the two possible homodimers and the heterodimeric species, are catalytically active, although active-site labeling in the heterodimer suggests that only the
1-subunit is active in that form. The head-group specificity differs depending on the composition: 1-O-alkyl-2-acetyl-sn-glycero-3-phosphoric acid (AAGPA) is hydrolyzed preferentially by both the
1-homodimer and the heterodimer, whereas PAF and 1-O-alkyl-2-acetyl-sn-glycero-3-phosphatidylethanolamine (AAGPE) are the preferred substrates for the
2-homodimer (Manya et al., 1999
). This pattern of substrate preference is consistent with the notion of the
1-subunit being the catalytically active one in a functionally asymmetric heterodimer.
In an effort to understand better the structurefunction relationships in the intracellular mammalian brain PAF-AH(Ib), we have recently determined the crystal structure of the 1-homodimer (Ho et al., 1997
). We reported that the tertiary fold of this protein is unique among PLA2s, with unexpected similarities to the low molecular weight GTPases. The active site contains a classical trypsin-like triad of SerHisAsp. More recently, we showed that three residues are largely responsible for the observed strict specificity towards an acetyl moiety, i.e. Leu48, Leu194 and Thr103 (Ho et al., 1999
). The dimer-forming interface is extensive (1150 Å2 per monomer), and involves 18 key residues, which form a contiguous region around the active site. When the two monomers form a dimer, a donut-shaped structure is created with a deep gorge providing access to two active sites, which are brought to relatively close proximity (Ho et al., 1997
).
Dimerization is an unusual feature in PLA2s. Although some secretory phospholipases A2 are known to form dimers, the functional implications are not clear. Of all the PLA2s characterized to date, only the E.coli OMPLA requires dimerization for activity, because in a monomer neither the oxyanion holerequired to stabilize the transition statesnor the acyl-binding pocket are formed within a single site. Dimerization places these features of one monomer in proximity of the SerHisAsn triad in the other, and so two half-sites in either molecule contribute to the formation of a catalytically competent dimer (Snijder et al., 1999). In contrast, the
-subunits of PAF-AH(Ib) harbor the complete catalytic machinery within a contiguous site in a single monomer, and the functional consequences of dimerization are not intuitively obvious. In this paper, we describe a study designed to probe this issue. Originally our intention was to disrupt the dimer using site-directed mutagenesis, but this approach resulted in the accumulation of insoluble protein in inclusion bodies, suggesting that the monomers are highly unstable and that dimerization is essential for the stability of the protein. We found, however, that in the presence of high Ca2+ concentration both the
1- and
2-homodimers dissociate into monomers, which are significantly less stableas assessed by differential scanning calorimetryand catalytically inert. Structural analysis of the dimer interface suggested that some residues in one monomer, notably Arg22, Leu26 and Arg29, come into close contact with the loop containing the active site Asp192 and His195 in the other. Our site-directed mutagenesis data show that the interactions of Arg22 and Arg29 across the interface are critical for full expression of activity. This may have important physiological implications. Although differential expression of the
-subunits is not fully understood, the present study shows that their dimerization is clearly essential for both stability and activity. The inherent instability of monomers and the absence of any evidence of interdimer
-subunit exchange suggest that the composition of the dimers is effectively regulated exclusively at the transcriptional level.
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Materials and methods |
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The cDNAs for both bovine -subunits were subcloned into the gluthathione-S-transferase (GST) expression plasmid, pGEX4T1 (Pharmacia), as EcoRI/SalI fragments to produce pGEX4T1-
1 and pGEX4T1-
2. These plasmids were individually transformed into the E.coli strain XL1blue (Stratagene). Expression was carried out in liquid cultures in 12 l of Luria broth. Growth was initiated at 37°C until an optical density (600 nm) of 0.40.6 was reached, and expression was induced by the addition of 0.51.0 mM IPTG; growth continued for a further 16 h at 25°C. The cells were harvested by centrifugation (10 min, 5000 g), resuspended in lysis buffer (50 mM TrisHCl, pH 8.5, 300 mM NaCl, 5 mM EDTA) and lysed by sonication. The soluble fraction was retained after centrifugation (30 min, 15 000 g). GST-fusion proteins were isolated by glutathione Sepharose affinity chromatography. Glutathione Sepharose affinity columns were pre-equilibrated with 20 column volumes of buffer I (50 mM TrisHCl, pH 8.0, 300 mM NaCl) at room temperature. Soluble protein fractions were allowed to bind to the column for 48 h at room temperature with gentle agitation. Columns then drained and washed with 2 l of buffer I overnight at 4°C. Fusion protein was eluted using 20 ml of buffer II (50 mM TrisHCl, pH 8.0, 500 mM NaCl, 10 mM reduced glutathione). The
1- or
2-homodimers were cleaved from their GST tags by thrombin (Sigma) digestion, dialyzed against lysis buffer to remove excess glutathione and GST was removed by passing the resultant GST/
-subunit mixture again through a glutathione Sepharose column. Pure
1- or
2-homodimers were obtained after a final purification step involving size-exclusion chromatography on an S-75 column (Pharmacia, Uppsala, Sweden).
Mutagenesis
cDNA of wild-type bovine 1-subunit was subcloned into the GST expression vector pGEX4T1 (Pharmacia) as an EcoRI/SalI fragment. The resulting plasmid, pGEX4T1-
1, was used as the template for site-directed mutagenesis. Mutations were done using the mega-primer polymerase chain reaction (PCR) method (Saiki et al., 1988
, Higuchi and Ochman, 1989
). The mega-primer PCR method requires two rounds of amplification, the first with the mutagenic primer in combination with a 5' primer for the pGEX4T1 vector and pGEX4T1-
1 as template DNA. The PCR product produced was then used as the mega-primer in the second reaction in combination with a 3' pGEX4T1 primer. pGEX4T1-
1 is used again as template DNA in the second reaction. The second reaction produced the full length, mutated PAF-Ah
1 gene, flanked by EcoRI and SalI restriction sites, which was then subcloned into pGEX4T1. Positive clones were isolated and verified by DNA sequencing. Mutants were expressed as a GST-fusion protein in E.coli strain BL21(DE3). Cultures were grown and
1-mutant proteins were isolated and purified as described above.
Differential scanning calorimetry (DSC)
Protein solutions were dialyzed into 20 mM sodium phosphate buffer with 120 mM NaCl and 0.5 mM DTT. For experiments containing calcium, the buffer Tris was substituted for sodium phosphate. The protein concentration varied from ~3.5 to 12.0 mg/ml. The protein concentration was determined from a stock solution of protein using quantitative amino acid analysis. Samples and reference solutions were degassed prior to loading into the calorimeter. All of the calorimetric experiments were performed on an MC-2 differential scanning calorimeter (MicroCal, Northampton, MA). The scan rate was 60°C/h unless noted otherwise and the sample volume was 1.23 ml. The calorimeter was interfaced with an IBM PC and the data were analyzed using Origin 4.1 (MicroCal).
Size-exclusion chomatography
Size-exclusion chromatography was performed on a BioCad Sprint system (PerSeptive Biosystems) using a Superdex 75 column (Pharmacia) capable of separating proteins in the range of 500060 000 kDa. A standard buffer (50 mM TrisHCl, pH 8.0, 200 mM NaCl) was used throughout the purification runs or for the determination of molecular weights. The column was standardized using aldolase, bovine serum albumin, chymotrypsin and ribonuclease. The flow rate was constant at 0.65 ml/min and the amount of protein sample applied to the column varied from 0.01 mg to 0.10 mg. The samples and buffers used to equilibrate the column were identical with those prepared for DSC analysis.
Activity assays
The activity of the 1- and
2-homodimer or monomer species was determined in 50 mM TrisHCl at pH 7.4 with 5 mM EDTA and 20 nmol [3H]acetyl-PAF (DuPontNew England Nuclear) in a total volume of 125 µl. The reaction was allowed to proceed for 30 min at 37°C prior to quenching with 1.25 ml of chloroformmethanol (4:1) and an additional 125 µl of doubly distilled water. An aliquot of 300 µl was taken from the upper phase for radioactivity measurements with a ß-counter (Pharmacia Biotech). For samples preincubated with Ca2+, 100 mM Ca2+ was added to the
1- or
2-subunit protein solution and incubated on ice for various times prior to addition of [3H]acetyl-PAF.
X-ray diffraction studies
The Arg22Lys mutant protein was expressed and purified as described above, and crystallized according to the procedure established for wild-type enzyme (Ho et al., 1997, 1999
). Data were collected at the X9B synchrotron beamline (NSLS) at the Brookhaven National Laboratory, and processed using DENZO and SCALEPACK (Otwinowski and Minor, 1997
). Subsequent calculations were carried out using the CCP4 suite of programs (CCP4, 1994), CNS (Brunger et al., 1998
) and the program O (Jones et al., 1991
).
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Results and discussion |
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We then used differential scanning calorimetry to probe the stability of the wild-type 1- and
2-homodimers, searching for conditions that might induce their dissociation and/or reversible unfolding. The DSC scans for the
1- and
2-homodimers as a function of pH are shown in Figure 1
. The thermally induced unfolding of the
1- or
2-subunit from pH 6.5 to 9.5 is irreversible under all conditions examined and the protein precipitated out of solution following denaturation. Below pH 6.0, both
1- and
2-homodimers denatured spontaneously and therefore all experiments were conducted above this value. The irreversibility of the thermal denaturation of the
1- and
2-subunits of platelet-activating factor acetylhydrolase precluded a formal and detailed analysis of the thermodynamics of unfolding. We therefore limited our calorimetric analysis to the transition temperature and enthalpy of thermal denaturation. For the
1-homodimer, we found that both the enthalpy and the temperature of unfolding depend, as expected, on pH (Figure 1A
). From a maximum of 52.3°C at pH 6.5, the temperature of unfolding decreases to 47.5°C as the pH increases to 9.5. The overall enthalpy of the unfolding transition of the
1-subunit homodimer also decreases as a function of increasing pH, with a maximum value of 164 kcal/mol at pH 6.5 and a minimum value of ~130 kcal/mol at pH 9.5. Similarly, the transition temperature and enthalpy of the thermally induced unfolding of the
2-subunit homodimers, shown in Figure 1B
, is also pH dependent. The trend is similar to that shown for the
1-subunit with both the temperature and enthalpy decreasing as a function of pH. The transition temperature and enthalpy, 51.7°C and 156 kcal/mol, respectively, decrease markedly to 46.2°C and 113 kcal/mol as the pH is increased from 6.5 to 9.5. These parameters did not change in any significant way when the protein concentration was decreased 5-fold, when the scan rate was decreased by a factor of 23 or when the ionic strength of the solution was varied (Table I
). Similar results were obtained with the
2-homodimers at various scan rates and protein concentrations (results not shown). Overall, the DSC data indicate that the
1- and
2-homodimers undergo thermal denaturation and we do not see a stable monomeric species. This was confirmed by size-exclusion chromatography as described later.
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The catalytic assays are indicative of the activity of the 1- and
2-subunits being highly dependent on the oligomeric state of the enzyme. As the proportion of the enzyme in monomeric state increases at the expense of the dimer with increasing levels of calcium, the relative catalytic activity decreases accordingly (Figure 5
). In contrast, when the pH is varied from 6.5 to 9.5 the relative activity level of both enzymes remains constant (results not shown). These results are consistent with the notion that amino acids from both monomers contribute to the active site, in such a way that a monomer is catalytically incompetent. Furthermore, the isolated monomer is highly unstable. We therefore set out to identify which specific amino acids from one monomer might contribute to the active site in the other.
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Recent literature suggests that the brain isoform of PAF-AH is distantly related to a diverse family of microbial hydrolases, which use complex saccharides and lipids as substrates (Upton and Buckley, 1995; Dalrymple et al., 1997
). While relationships inferred from limited amino acid sequence similarities are sometimes misleading, structural information is more convincing. Indeed, the crystal structure of the rhamnogalacturonan acetylesterase from Aspergillus aculeatus (Mølgaard et al., 2000
) confirms the notion of evolutionary link between these enzymes. It is not known, however, if dimerization plays any role in the microbial proteins. Among eukaryotes, only Drosophila has been so far shown to harbor a gene coding for a homologue of the
-subunit of the brain PAF-AH (Sheffield et al., 2000
). However, the protein is catalytically inert, at least against PAF-related substrates and probably also against other esters, given that two of the three residues in the active site triad are changed. In contrast to the mammalian protein, the Drosophila homologue is largely monomeric, as judged by gel filtration experiments. Hence, the evolutionary origins and roots of substrate specificity, in addition to the phylogeny of gene duplication, continue to be an enigma. As more eukaryotic genome data become available, it will be interesting to see if they contain more clues to the physiological roles of the brain PAF-AH. Our work demonstrates, however, that the dimerization of the mammalian protein is essential for full catalytic activity and stability. In addition, the instability of the
-monomeric species suggests that the composition of the catalytic
-dimers is regulated at the transcriptional level and not by dimer rearrangement at the protein level, unless accessory proteins of some kind are involved.
The coordinates of the R29K mutant reported in this paper have been deposited with the Protein Data Bank under accession code 1ES9.
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Notes |
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Acknowledgments |
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References |
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Received June 1, 2000; revised August 9, 2000; accepted September 15, 2000.