Biochemical Characterization of Various Catalytic Complexes of the Brain Platelet-activating Factor Acetylhydrolase

Abstract

Brain intracellular platelet-activating factor acetylhydrolase (PAF-AH) isoform I is a member of a family of complex enzymes composed of mutually homologous ␣ 1 and ␣ 2 subunits, both of which account for catalytic activity, and the ␤ subunit. We previously demonstrated that the expression of one catalytic subunit, ␣ 1 , is developmentally regulated, resulting in a switching of the catalytic complex from ␣ 1 /␣ 2 to ␣ 2 /␣ 2 during brain development (Manya, H., Aoki, J., Watanabe, M., Adachi, T., Asou, H., Inoue, Y., Arai, H., and Inoue, K. (1998) J. Biol. Chem. 273, 18567-18572). In this study, we explored the biochemical differences in three possible catalytic dimers, ␣ 1 /␣ 1 , ␣ 1 /␣ 2 , and ␣ 2 /␣ 2 . The ␣ 2 /␣ 2 homodimer exhibited different substrate specificity from the ␣ 1 /␣ 1 homodimer and the ␣ 1 /␣ 2 heterodimer, both of which showed similar substrate specificity. The ␣ 2 /␣ 2 homodimer hydrolyzed PAF and 1-O-alkyl-2-acetyl-snglycero-3-phosphorylethanolamine (AAGPE) most efficiently among 1-O-alkyl-2-acetyl-phospholipids. In contrast, both ␣ 1 /␣ 1 and ␣ 1 /␣ 2 hydrolyzed 1-O-alkyl-2-acetyl-sn-glycero-3-phosphoric acid more efficiently than PAF. AAGPE was the poorest substrate for these enzymes. The ␤ subunit bound to all three catalytic dimers but modulated the enzyme activity in a catalytic dimer composition-dependent manner. The ␤ subunit strongly accelerated the enzyme activity of the ␣ 2 /␣ 2 homodimer but rather suppressed the activity of the ␣ 1 /␣ 1 homodimer and had little effect on that of the ␣ 1 /␣ 2 heterodimer. The (His 149 to Arg) mutant ␤, which has been recently identified in isolated lissencephaly sequence patients, lost the ability to either associate with the catalytic complexes or modulate their enzyme activity. The enzyme activity of PAF-AH isoform I may be regulated in multiple ways by switching the composition of the catalytic subunit and by manipulating the ␤ subunit.Brain platelet-activating factor acetylhydrolase (PAF-AH 1 isoform I) was first identified in bovine brain as an oligomeric enzyme consisting of two catalytic ␣ 1 and ␣ 2 subunits and a noncatalytic ␤ subunit (1). The oligomeric brain PAF-AH contains a dimer of two highly homologous catalytic subunits, ␣ 1 and ␣ 2 , that share 63% amino acid sequence identity but are not related to any other known proteins (2, 3). The ␣ 1 and ␣ 2 subunits form a heterodimer in the PAF-AH purified from the bovine brain cortex (1). They can also form a catalytically active homodimer when expressed individually in Escherichia coli (3). Recently, the crystal structure of the ␣ 1 /␣ 1 homodimer was determined (4). Surprisingly, the ␣ 1 subunit has a tertiary fold reminiscent of small GTPases such as p21 ras and harbors a chymotrypsin-like Ser-Asp-His triad in its active site (4). Although the catalytic subunits of PAF acetylhydrolase were first identified as a ␣ 1 /␣ 2 heterodimer from young adult bovine brain (1), recent studies have revealed that the ␣ 2 /␣ 2 homodimers are present in vivo as well (5). We have demonstrated in a pr...

Platelet-Activating Factor Acetylhydrolases: Broad Substrate Specificity and Lipoprotein Binding Does Not Modulate the Catalytic Properties of the Plasma Enzyme

“…It has been proposed that PAF in the brain plays an important role as a messenger in excitatory neurotransmitter release, neuronal plasticity, memory formation, and long-term potentiation (reviewed in ref 44). It was also postulated that 1-Oalkyl-2-acetyl-phospholipids other than PAF such as alk-1-enyl-acetyl-glycerophosphoethanolamine (2-acetylplasmalogen) may be physiological substrates for PAF-AH(Ib) (45). A crystallographic study of PAF-AH(Ib) R1/ R1 homodimers reveals that the active site structure can account for the exclusion of phospholipids with sn-2 acyl chains longer than acetyl (46).…”

LIS1—no more no less

“…This may result in a local increase of the normal protein that can enhance specifically the enzymatic activity of PAF-AH ␣2 dimers. 72 The net effect of higher enzymatic activity will be reduced PAF concentration that may in turn affect the cytoskeleton. 73 The mutant protein may also alter the interactions with dynein, mNudE or NudeL in Lis1/sLis1 cells.…”

Novel isoforms of intracellular platelet activating factor acetylhydrolase (PAFAH1b2) in human testis; encoded by alternatively spliced mRNAs

“…The Alpha-2 homodimer and the Alpha-1/Alpha-2 heterodimer both have higher rates of PAF hydrolysis than the Alpha-1 homo-dimer (12). The regulatory effect of the beta subunit on pafah-Ib activity is also dependent on subunit composition.…”

“…This narrow substrate specificity is more consistent with regulatory enzymes in signaling pathways (11) than a generalized scavenger of reactive lipids. In addition to PAF, other structurally related lipids such as 1-OAlkyl-2-acetyl-sn-glycero-3-phosphoric acid (AAGPA-an intermediate in PAF synthesis) and 1-O-Alkyl-2-acetyl-sn-glycero-3-phosphatidylethanolamine (AAGPE) may act as second messengers whose signals are terminated by intracellular paf-ah-Ib catalyzed hydrolysis (12).…”

“…The alpha subunits form a catalytic dimer, the activity of which is modulated by, but not dependant upon, association with the beta subunit. Alpha subunit composition of the dimer varies depending on tissue type and developmental stage (12,13). Alpha-2 expression is ubiquitous, whereas Alpha-1 has a more tissue specific and developmentally regulated pattern of expression (13,9).…”