Brain Platelet-activating Factor Acetylhydrolase Research Articles

Platelet-Activating Factor Acetylhydrolase and Brain Development.

The heterotrimeric brain platelet-activating factor acetylhydrolase (PAFAH1B1) contains two catalytic subunits and a regulatory subunit. This complex plays important, surprising roles in brain development and in spermatogenesis. The regulatory subunit, PAFAH1B1 (LIS1 protein), is critically regulated and when deficient leads to the devastating human neurological disorder Lissencephaly, or smooth brain. The role of the protein in brain development is not the catalysis of platelet-activating factor, rather the entire brain platelet-activating factor acetylhydrolase complex serves a signaling role, coordinating important pathways in brain development. The role of this complex in spermatogenesis was not foreseen, but appears to function to regulate a critical level of the PAFAH1B1 protein, such that too much of this protein or too little of this protein can lead to a disruption of spermatogenesis. Brain platelet-activating factor is thus a signaling complex, important for brain development and for spermatogenesis.

Serum Activity of Platelet-Activating Factor Acetylhydrolase Is a Potential Clinical Marker for Leptospirosis Pulmonary Hemorrhage

Pulmonary hemorrhage has been recognized as a major, often lethal, manifestation of severe leptospirosis albeit the pathogenesis remains unclear. The Leptospira interrogans virulent serogroup Icterohaemorrhagiae serovar Lai encodes a protein (LA2144), which exhibited the platelet-activating factor acetylhydrolase (PAF-AH) activity in vitro similar to that of human serum with respect to its substrate affinity and specificity and thus designated L-PAF-AH. On the other hand, the primary amino acid sequence of L-PAF-AH is homologous to the α1-subunit of the bovine brain PAF-AH isoform I. The L-PAF-AH was proven to be an intracellular protein, which was encoded unanimously and expressed similarly in either pathogenic or saprophytic leptospires. Mongolian gerbil is an appropriate experimental model to study the PAF-AH level in serum with its basal activity level comparable to that of human while elevated directly associated with the course of pulmonary hemorrhage during severe leptospirosis. Mortality occurred around the peak of pulmonary hemorrhage, along with the transition of the PAF-AH activity level in serum, from the increasing phase to the final decreasing phase. Limited clinical data indicated that the serum activity of PAF-AH was likely to be elevated in the patients infected by L. interrogans serogroup Icterohaemorrhagiae, but not in those infected by other less severe serogroups. Although L-PAF-AH might be released into the micro-environment via cell lysis, its PAF-AH activity apparently contributed little to this elevation. Therefore, the change of PAF-AH in serum not only may be influential for pulmonary hemorrhage, but also seems suitable for disease monitoring to ensure prompt clinical treatment, which is critical for reducing the mortality of severe leptospirosis.

The Pafah1b Complex Interacts with the Reelin Receptor VLDLR

Reelin is an extracellular protein that directs the organization of cortical structures of the brain through the activation of two receptors, the very low-density lipoprotein receptor (VLDLR) and the apolipoprotein E receptor 2 (ApoER2), and the phosphorylation of Disabled-1 (Dab1). Lis1, the product of the Pafah1b1 gene, is a component of the brain platelet-activating factor acetylhydrolase 1b (Pafah1b) complex, and binds to phosphorylated Dab1 in response to Reelin. Here we investigated the involvement of the whole Pafah1b complex in Reelin signaling and cortical layer formation and found that catalytic subunits of the Pafah1b complex, Pafah1b2 and Pafah1b3, specifically bind to the NPxYL sequence of VLDLR, but not to ApoER2. Compound Pafah1b1+/−;Apoer2−/− mutant mice exhibit a reeler-like phenotype in the forebrain consisting of the inversion of cortical layers and hippocampal disorganization, whereas double Pafah1b1+/−;Vldlr−/− mutants do not. These results suggest that a cross-talk between the Pafah1b complex and Reelin occurs downstream of the VLDLR receptor.

CMP-N-Acetylneuraminic Acid Synthetase from Escherichia coli K1 Is a Bifunctional Enzyme: IDENTIFICATION OF MINIMAL CATALYTIC DOMAIN FOR SYNTHETASE ACTIVITY AND NOVEL FUNCTIONAL DOMAIN FOR PLATELET-ACTIVATING FACTOR ACETYLHYDROLASE ACTIVITY

Escherichia coli CMP-NeuAc synthetase (EC 2.7.7.43) catalyzes the synthesis of CMP-NeuAc from CTP and NeuAc, which is essential for the formation of capsule polysialylate for strain K1. Alignment of the amino acid sequence of E. coli CMP-NeuAc synthetase with those from other bacterial species revealed that the conserved motifs were located in its N termini, whereas the C terminus appeared to be redundant. Based on this information, a series of deletions from the 3'-end of the CMPNeuAc synthetase coding region was constructed and expressed in E. coli. As a result, the catalytic domain required for CMP-NeuAc synthetase was found to be in the N-terminal half consisting of amino acids 1-229. Using the strategy of tertiary structure prediction based on the homologous search of the secondary structure, the C-terminal half was recognized as an alpha1-subunit of bovine brain platelet-activating factor acetylhydrolase isoform I. The biochemical analyses showed that the C-terminal half consisting of amino acids 228-418 exhibited platelet-activating factor acetylhydrolase activity. The enzyme properties and substrate specificity were similar to that of bovine brain alpha1-subunit. Although its physiological function is still unclear, it has been proposed that the alpha1-subunit-like domain of E. coli may be involved in the traversal of the blood-brain barrier.

The functional implications of the dimerization of the catalytic subunits of the mammalian brain platelet-activating factor acetylhydrolase (Ib).

The mammalian brain contains significant amounts of the cytosolic isoform Ib of the platelet-activating factor acetylhydrolase (PAF-AH), a unique type of PLA2. This oligomeric protein complex contains three types of subunits: two homologous (63% identity) 26 kDa catalytic subunits (alpha(1) and alpha(2)) which harbor all the PAF-AH activity, and the 45 kDa beta-subunit (LIS1), a product of the causal gene for Miller-Dieker lissencephaly. During fetal development, the preferentially expressed alpha(1)-subunit forms a homodimer, which binds to a homodimer of LIS1, whereas in adult organisms alpha(1)/alpha(2) and alpha(2)/alpha(2) dimers, also bound to dimeric LIS1, are the prevailing species. The consequences of this "switching" are not understood, but appear to be of physiological significance. The alpha(1)- and alpha(2)-subunits readily associate with very high affinity to form homodimers. The nature of the interface has been elucidated by the 1.7 A resolution crystal structure of the alpha(1)/alpha(1) homodimer (Ho et al., 1997). Here, we examined the functional consequences of the dimerization in both types of alpha-subunits. We obtained monomeric protein in the presence of high concentrations (>50 mM) of Ca2+ ions, and we show that it is catalytically inactive and less stable than the wild type. We further show that Arg29 and Arg22 in one monomer contribute to the catalytic competence of the active site across the dimer interface, and complement the catalytic triad of Ser47, Asp192 and His195, in the second monomer. These results indicate that the brain PAF-acetylhydrolase is a unique PLA2 in which dimerization is essential for both stability and catalytic activity.

Homologs of the alpha- and beta-subunits of mammalian brain platelet-activating factor acetylhydrolase Ib in the Drosophila melanogaster genome.

The mammalian intracellular brain platelet-activating factor acetylhydrolase, implicated in the development of cerebral cortex, is a member of the phospholipase A2 superfamily. It is made up of a homodimer of the 45 kDa LIS1 protein (a product of the causative gene for type I lissencephaly) and a pair of homologous 26-kDa alpha-subunits which account for all the catalytic activity. LIS1 is hypothesized to regulate nuclear movement in migrating neurons through interactions with the cytoskeleton, while the alpha-subunits, whose structure is known, contain a trypsin-like triad within the framework of a unique tertiary fold. The physiological significance of the association of the two types of subunits is not known. In an effort to better understand the function of the complex we turned to genomic data mining in search of related proteins in lower eukaryotes. We found that the Drosophila melanogaster genome contains homologs of both alpha- and beta-subunits, and we cloned both genes. The alpha-subunit homolog has been overexpressed, purified and crystallized. It lacks two of the three active-site residues and, consequently, is catalytically inactive against PAF-AH (Ib) substrates. Our study shows that the beta-subunit homolog is highly conserved from Drosophila to mammals and is able to interact with the mammalian alpha-subunits but is unable to interact with the Drosophila alpha-subunit. Proteins 2000;39:1-8.

Lissencephaly associated mutations suggest a requirement for the PAFAH1B heterotrimeric complex in brain development

Human brain malformations, such as Miller–Dieker syndrome (MDS) or isolated lissencephaly sequence (ILS) may result from abnormal neuronal migration during brain development. MDS and ILS patients have a hemizygous deletion or mutation in the LIS1 gene ( PAFAH1B1), therefore, the LIS1 encoded protein (Lis1) may play a role in neuronal migration. Lis1 is a subunit of a brain platelet-activating factor acetylhydrolase (PAFAH1B) where it forms a heterotrimeric complex with two hydrolase subunits, referred to as 29 kDa (PAFAH1B3) and 30 kDa (PAFAH1B2). In order to determine whether this heterotrimer is required for the developmental functions of PAFAH1B, we examined the binding properties of 29 and 30 kDa subunits to mutant Lis1 proteins. The results defined the critical regions of Lis1 for PAFAH1B complex formation and demonstrated that all human LIS1 mutations examined resulted in abolished or reduced capacity of Lis1 to interact with the 29 and 30 kDa subunits, suggesting that the PAFAH1B complex participates in the process of neuronal migration.

Molecular cloning of cDNAs encoding α1, α2, and β subunits of rat brain platelet-activating factor acetylhydrolase

Brain intracellular platelet-activating factor acetylhydrolase (PAF-AH(Ib)) is a tertiary G-protein-complex-like heterotrimeric enzyme which is composed of α1, α2, and β subunits and is implicated in stages of brain development such as the formation of the brain cortex. We have isolated and sequenced cDNA clones encoding these three subunits of rat brain PAF-AH(Ib). The amino acid sequences of brain PAF-AH has shown an extremely high homology among mammalian species. The tissue distribution of the three subunits was examined by Northern blot analysis. Although the mRNAs were detected in various organs, the ratio of the level of mRNA expression for the three subunits differed among rat tissues, raising the possibility that isoform(s) other than the heterotrimeric isoform exist in certain tissues.

PAF analogues capable of inhibiting PAF acetylhydrolase activity suppress migration of isolated rat cerebellar granule cells

Intracellular platelet-activating factor (PAF) acetylhydrolase in the bovine brain is a heterotrimeric enzyme composed of α1, α2 and β subunits. The trimeric enzyme may be involved in neural cell migration, since the human homolog of the non-catalytic β subunit is a product of the LIS-1 gene which is a causative gene for Miller–Dieker syndrome. Miller–Dieker syndrome is a form of lissencephaly that is characterized by widespread agyria of the brain and defects of neuronal cell migration. In the present study, we attempted to determine whether the catalytic activity of either the α1 or α2 subunit is required for the regulation of granule cell migration. Granule cells prepared from rat cerebellum at postnatal day 0 express all three subunit proteins ( α1, α2 and β) as determined by western blotting. Granule cell migration, which was observed in vitro on a layer coated with laminin, was effectively blocked by PAF analogs which showed PAF receptor-antagonistic activity (CV-6209 and CV-3988) and PAF receptor-agonistic activity (carbamoyl PAF). These PAF analogs also inhibited the activity of bovine brain PAF acetylhydrolase. Cell migration was restored when the inhibitors were removed by washing the treated cells with buffer, indicating that the inhibitory effect of PAF analogs is reversible. Structurally-unrelated PAF antagonists (SM-12502, TCV-309 and YM-264), none of which showed any appreciable inhibitory activity against PAF acetylhydrolase, did not block granule cell migration under the same conditions. It is suggested that the catalytic activity of PAF acetylhydrolase may play a crucial role in neural cell migration.

Predominant localization of the LIS family of gene products to Cajal-Retzius cells and ventricular neuroepithelium in the developing human cortex.

Mutations that perturb neuronal migration provide important biological clues that can lead to an understanding of the role of specific cells and molecules in the formation of the cortex. The human neuronal migration disorder, Miller-Dieker Lissencephaly, results from a hemideletion of LIS-1, which encodes a subunit of a brain platelet-activating factor acetylhydrolase. The cellular localization of the LIS-1 gene product in human fetal brain and its normal role in neuronal migration have yet to be determined. LIS-1 belongs to a family of genes that have identical coding sequences (LIS-1 [chromosome 17] and LIS-2 [chromosome 2]). In the brain, LIS-1 is the more abundant gene as determined by Northern blot analysis. Using antibodies raised against 2 epitopes of the LIS-1/LIS-2 protein sequence, we have localized the LIS family of gene products in the developing human brain to the Cajal-Retzius cells, some subplate neurons, thalamic neurons, the ventricular neuroepithelium, and at later gestational ages, to the ependyma. Therefore, LIS-1 bears some resemblance to reelin, the gene product involved in the cortical mouse mutant reeler, in that Cajal-Retzius cells demonstrate immunolocalization. However, unlike reelin, LIS proteins are expressed not only in the Cajal Retzius cells, but also in the ventricular neuroepithelium, suggesting a potential role for this structure in neuronal migration.

Cloning and Expression of a cDNA Encoding the β-Subunit (30-kDa Subunit) of Bovine Brain Platelet-activating Factor Acetylhydrolase

Bovine brain platelet-activating factor (PAF) acetylhydrolase isoform Ib is a heterotrimeric enzyme. Its gamma-subunit (which, formerly, we called the 29-kDa subunit) acts as a catalytic subunit, whereas the alpha-subunit (45 kDa) is the bovine homolog of the product of human LIS-1, the causative gene of Miller-Dieker lissencephaly, indicating that this intracellular PAF acetylhydrolase plays a key role in brain development. In the current study, we cloned the cDNA for the beta-subunit (30 kDa) of bovine brain PAF acetylhydrolase Ib. The predicted 229-amino acid sequence was homologous (63.2% identity) to that of the gamma-subunit, especially (86% identity) in the catalytic and PAF receptor homologous domains. The recombinant beta-protein produced in Escherichia coli showed significant PAF acetylhydrolase activity. A mutant protein, in which Ser48, which corresponds to the active serine residue of the gamma-subunit, was replaced with cysteine showed no enzymatic activity, suggesting Ser48 is the active serine residue. Although the beta- and gamma-subunits form a heterocomplex in the native enzyme, both recombinant beta- and gamma-proteins exist as a homodimer. The purified recombinant beta-protein was labeled readily with [1,3-H]diisopropyl fluorophosphate, whereas the beta-subunit in the native complex was only labeled with higher concentrations of [1,3-3H]diisopropyl fluorophosphate to a lesser extent than the gamma-subunit. Combined with our previous data, the present study demonstrated that bovine brain PAF acetylhydrolase Ib is a unique enzyme possessing two catalytic subunits and another, possibly regulatory, subunit.

The catalytic subunit of bovine brain platelet-activating factor acetylhydrolase is a novel type of serine esterase.

Platelet-activating factor (PAF) acetylhydrolase is the key enzyme in PAF inactivation. We recently purified one isoform of the enzyme from a bovine brain soluble fraction and revealed it as a heterotrimeric enzyme consisting of 29-, 30-, and 45-kDa subunits. Among them, the 29-kDa subunit possesses an active serine residue since diisopropyl fluorophosphate (DFP), an inhibitor of the enzyme, labeled only this subunit (Hattori, M., Arai, H., and Inoue, K. (1993) J. Biol. Chem. 268, 18748-18753). In the current study, we cloned the cDNA for the 29-kDa catalytic subunit. The predicted sequence of 232 amino acids is unique and is not homologous with those of any other proteins reported so far. When transfected into either Escherichia coli or COS7 cells, the cDNA produced PAF acetylhydrolase activity in both types of cells, indicating that this subunit alone is enough for catalysis. The recombinant 29-kDa protein was also inhibited and labeled by DFP. Furthermore, we isolated and sequenced the [3H]DFP-labeled peptide fragment, revealing that Ser47 is the active serine residue. The sequence surrounding it is different from the consensus sequence of the serine esterase family. Interestingly, the sequence of about 30 amino acids located 6 residues downstream from the active serine site exhibits significant homology to the first transmembrane region of the PAF receptor. These data demonstrate that the catalytic subunit of brain PAF acetylhydrolase is a novel type of serine esterase.

Erratum: Miller-Dicker lissencephaly gene encodes a subunit of brain platelet-activating factor acetylhydrolase

Nature 370, 216-218 (1994) THE word 'acetylhydrolase' was accidentally omitted from the end of the title of this paper. The correct title should read "Miller-Dieker lissencephaly gene encodes a subunit of brain platelet-activating factor acetylhydrolase".

Purification and characterization of bovine brain platelet-activating factor acetylhydrolase.

Platelet-activating factor (PAF) acetylhydrolase, which removes the acetyl moiety at the sn-2 position, has been found in plasma and tissue cytosol. PAF acetylhydrolase in bovine brain cytosol was chromatographically separated into three distinct fractions, all of which exhibited pH optima in the neutral to mild alkaline region and were unaffected by EDTA. We have purified the major fraction of the enzyme to near homogeneity. The purified enzyme had a molecular mass of about 100 kDa, as estimated by gel filtration chromatography, and gave three distinct bands of 45, 30, and 29 kDa, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These polypeptides exclusively co-migrated with the activity throughout the purification steps. These data suggest that this set of polypeptides corresponds to the subunits of bovine brain PAF acetylhydrolase. Diisopropyl fluorophosphate completely inhibited the activity at 0.1 mM. [3H]Diisopropyl fluorophosphate labeled only the 29-kDa polypeptide, suggesting that this polypeptide possesses an active serine residue(s). The purified enzyme displayed similar activity against PAF and oxidatively modified phosphatidylcholine, but did not hydrolyze phosphatidylcholine or phosphatidylethanolamine with two long chain acyl groups. Thus, the intracellular PAF acetylhydrolase is likely to be a new member of the calcium-independent phospholipases A2 in mammalian tissues.