Secretion of a novel, developmentally regulated chitinase (family 19 glycosyl hydrolase) into the perivitelline fluid of the parasitic nematode, Ascaris suum

The early development of the parasitic nematode, Ascaris suum , occurs within a chitinous eggshell and an abundant chitinase (As-p50) has been identified in the perivitelline fluid (PVF) surrounding the infective larva prior to hatching. A cDNA encoding As-p50 was cloned, sequenced and the protein expressed in Escherichia coli. As-p50 is a member of glycosyl hydrolase family 19, previously identified only in plants, making the characterization of As-p50 the first family 19 glycosyl hydrolase from any animal species. Structural modeling indicates that two glutamate residues in As-p50 are spatially aligned with the two conserved Glu residues in the Hordeum vulgare structure. As expected, the chitinase activity of recombinant As-p50 or isolated PVF was insensitive to allosamidin.As-p50 expression was developmentally regulated. As-p50 mRNA appeared between days 5 and 8 of development prior to the formation of the first-stage larva (L1). The As-p50 protein and chitinase activity appeared later between days 8 and 15 and remained at constant levels until hatching. GFP-promoter constructs of C08B6.4, the most closely related Caenorhabditis elegans As-p50 homologue, were expressed in hypodermal cells of three-fold stage larvae and L1s with a timing similar to that of As-p50 and the fusion protein was secreted into the space between the hypodermis and the cuticle. Taken together, these results suggest that As-p50 is involved in the formation of the L1 cuticle and/or the initial molt however, As-p50 may be multifunctional and also responsible for the digestion of the eggshell during hatching.