Secretion of a Novel, Developmentally Regulated Fatty Acid-binding Protein into the Perivitelline Fluid of the Parasitic Nematode, Ascaris suum

Abstract

Early development of the parasitic nematode, Ascaris suum, occurs inside a highly resistant eggshell, and the developing larva is bathed in perivitelline fluid. Two-dimensional gel analysis of perivitelline fluid from infective larvae reveals seven major proteins; a cDNA encoding one of these, As-p18, has been cloned, sequenced, and protein expressed in Escherichia coli. The predicted amino acid sequence of As-p18 exhibits similarities to the intracellular lipid-binding protein (iLBP) family including retinoid- and fatty acid-binding proteins (FABP). As-p18 is unusual in that it possesses a hydrophobic leader that is not present in the mature protein, the developmental regulation of its expression, and in terms of its predicted structure. Recombinant As-p18 is a functional FABP with a high affinity for both a fluorescent fatty acid analog (11(((5-(dimethylamino)-1-naphthalenyl)sulfonyl)amino) undecanoic acid) and oleic acid, but not retinol. Circular dichroism of rAs-p18 reveals a high beta-sheet content (62%), which is consistent with secondary structure for the protein predicted from sequence algorithms, and the structure of iLBPs. Unusual features are apparent in a structural model of As-p18 generated from existing crystal structures of iLBPs. As-p18 is not found in unembryonated eggs, begins to be synthesized at about day 3 of development, reaches a maximal concentration with the formation of the first-stage larva and remains abundant in the perivitelline fluid of the second-stage larva. Since As-p18 is not present in the post-infective third-stage larva or adult worm tissues, it appears to be exclusive to the egg. Surprisingly, however, Northern blot analysis yields mRNA for As-p18 not only in the early larval stages, but also the unembryonated egg, third-stage larvae, and ovaries of adult worms, even though the protein is not detectable from any of those sources. As-p18 may play a role in sequestering potentially toxic fatty acids and their peroxidation products, or it may be involved in the maintenance of the impermeable lipid layer of the eggshell.