PAF-acetylhydrolase (PAF-AH) is an enzyme that inactivates PAF (Platelet-activating factor) by hydrolyzing the acetyl group attached at the sn-2 position of the glycerol backbone. Type II PAF-AH (PAF-AH (II)) is a 40-kDa monomeric enzyme and myristoylated at the N-terminus. PAF is not detected in C. elegans, however, orthologous genes of PAF-AH (II) are conserved in many species, including C. elegans. The physiological function and substrate(s) of this enzyme have not been fully understood so far. In this study, we cloned two PAF-AH (II) genes in C. elegans (
paf-1,
paf-2) and disrupted these genes to elucidate the biological function. Amino acid sequences of PAF-1 and PAF-2 exhibited 70% identity with each other and 35% identity with mammalian PAF-AH (II)s. Both PAF-1 and PAF-2 conserved a Gly-X-Ser-X-Gly catalytic center and the N-myristoylation sequence, and exhibited similar substrate specificity as mammalian PAF-AH (II)s in vitro. Next we generated deletion mutants of these two enzymes and examined their phenotypes.
paf-2(-/-) was lethal during embryogenesis, whereas
paf-1(-/-) grew normally. All
paf-2(-/-) embryos failed to elongate and the development was arrested at the lima bean stage. Analysis using SEM (scanning electron microscope) and immuno-staining revealed that the organization of the hypodermis in the
paf-2(-/-) mutant embryo is severely obstructed. To further analyze the function of
paf-2 during elongation process and following postembryonic development, we generated transgenic animals in which heat-shock induces
paf-2 RNAi, producing both senses of
paf-2 RNAs in vivo. Heat-shocked hsp::
paf-2 RNAi animals showed negligible PAF acetylhydrolase activity, indicating that PAF-2 is a major isoform for enzyme activity in C. elegans. These worms exhibited variable abnormal morphology (Vab) at the late embryonal and early larval stages including ectopic protrusions due to epithelial organization defects. These observations demonstrate that PAF-AH (II) is required for hypodermal formation and suggest that particular lipids, the substrates or products of PAF-2, are critically involved in epithelial morphogenesis.