cDNA Cloning and Expression of Intracellular Platelet-activating Factor (PAF) Acetylhydrolase II

Abstract

Platelet-activating factor (PAF) acetylhydrolase, which inactivates PAF by removing the acetyl group at the sn-2 position, is widely distributed in plasma and tissues. We previously demonstrated that tissue cytosol contains at least two types of PAF acetylhydrolase, isoforms Ib and II, and that isoform Ib is a heterotrimer comprising 45-, 30-, and 29-kDa subunits, whereas isoform II is a 40-kDa monomer.In this study, we isolated cDNA clones of bovine and human PAF acetylhydrolase isoform II. From the longest open reading frame of the cloned cDNAs, both bovine and human PAF acetylhydrolases II are predicted to contain 392 amino acid residues and to exhibit 88% identity with each other at the amino acid level. Both enzymes contain a Gly-X-Ser-X-Gly motif that is characteristic of lipases and serine esterases. Expression of isoform II cDNA in COS7 cells resulted in a marked increase in PAF acetylhydrolase activity. An immunoblot study using an established monoclonal antibody against the bovine enzyme revealed that the recombinant protein exists in the membranous fraction as well as the soluble fraction. Isoform II is expressed most abundantly in the liver and kidney in cattle, but low levels were also observed in other tissues. The amino acid sequence deduced from the cDNA of isoform II had no homology with any subunit of isoform Ib. Interestingly, however, the amino acid sequence of isoform II showed 41% identity with that of plasma PAF acetylhydrolase. Combined with previous data demonstrating that isoform II shows similar substrate specificity to plasma PAF acetylhydrolase, these results indicate that tissue type isoform II and the plasma enzyme may share a common physiologic function.

Anti-apoptotic Actions of the Platelet-activating Factor Acetylhydrolase I α2 Catalytic Subunit

“…0.1 mM DFP inhibits PAF-AH I ␣ 1 /␣ 1 or ␣ 1 /␣ 2 dimer activity, whereas 1 mM DFP inhibits PAF-AH I ␣ 1 /␣ 1 , PAF-AH I ␣ 1 /␣ 2 , and PAF-AH II activity. PAF-AH I ␣ 2 /␣ 2 dimers are DFP-resistant (21,23,35). 68 -70% of the total cytosolic PAF-AH activity was blocked by 0.1 mM DFP indicative of PAFAH I ␣ 1 /␣ 1 or ␣ 1 /␣ 2 activity (Table II).…”

“…First, we treated cells with 0.1 mM DFP to inhibit PAF-AH I ␣ 1 /␣ 1 and ␣ 1 /␣ 2 catalytic activity without changing the relative ratio of ␣ 1 to ␣ 2 protein expression (21,23,35). In the unlikely event that the anti-apoptotic effects of BN 52021 and FR 49175 are mediated by a loss of ␣ 1 catalytic activity, then 0.1 mM DFP should be cytoprotective.…”

“…PAF-AH II is a single 40-kDa polypeptide (23). This isoenzyme recognizes both PAF and acyl analogs of PAF with moderate length sn-2 chains as well as short chain diacylglycerols, triacylglycerols, and acetylated alkanols (24).…”

The Streptomyces coelicolor A3(2) lipAR operon encodes an extracellular lipase and a new type of transcriptional regulator The GenBank accession numbers for the sequences described in this paper are AF009336 and U03114.

“…Several Streptomyces species harbour sequences homologous to this family in their genomes (Cruz et al, 1994). The enzymes are related to a family of mammalian platelet activating factor acetylhydrolases (Hattori et al, 1996). The S. exfoliatus M11 lipase is a typical α\β hydrolase, lacking a structure similar to those in fungal lipases that are capable of functioning as a ' lid ' (Wei et al, 1998).…”

Type II platelet-activating factor-acetylhydrolase is essential for epithelial morphogenesis in Caenorhabditis elegans

“…The amino acid sequence of PAF-AH (II) does not show any significant similarity to any subunit of intracellular type I PAF-AH but shows Ϸ42% identity with that of the plasma-type PAF-AH (15). PAF-AH (II), as well as plasma PAF-AH, hydrolyze not only acetyl groups but also short-length acyl chains such as propionyl and butyloyl groups attached to phosphoglyceride (16).…”