Molecular characterisation of the Ancylostoma-secreted protein family from the adult stage of Ancylostoma caninum

The Ancylostoma-secreted proteins are a family of nematode-specific cysteine-rich secreted proteins belonging to the pathogenesis-related protein superfamily. Previously we reported that third stage infective larvae of Ancylostoma caninum produce two different Ancylostoma-secreted proteins, a single and double-domain Ancylostoma-secreted protein, designated as Ancylostoma- secreted protein-1 and Ancylostoma-secreted protein-2. respectively. Here we report that adult A. caninum hookworms produce and release four additional Ancylostoma-secreted proteins (Ancylostoma-secreted protein-3-6). Using antiserum against adult excretory/secretory products, Ancylostoma-secreted protein cDNAs were isolated from cDNA expression libraries. Immunolocalisation experiments using specific antisera indicated that the single-domain Ac-Ancylostoma-secreted protein-3 is located in the adult pharyngeal and oesophageal glands. Ac-Ancylostoma-secreted protein-4, Ancylostoma-secreted protein-5 and Ancylostoma-secreted protein-6 are composed of two pathogenesis-related protein domains linked in tandem as a heterodimorphic repeat. Ac-Ancylostoma-secreted protein-4 is localised to the cuticular surface of the adult hookworm. whereas Ac-Ancylostoma-secreted protein-5 was found in the intestinal brush border membrane, and Ancylostoma-secreted protein-6 in the cephalic and excretory glands. All of the adult Ancylostoma-secreted proteins were identified in excretory/secretory products of adult hookworms by Western blotting and are presumably released by the parasite. None of the adult Ancylostoma-secreted proteins were detected by immunoblotting in L3 extracts, although mRNAs of Ac-Ancylostoma-secreted protein-3 and Ac-Ancylostoma-secreted protein-4 were resent in the larval stage. The functions of the adult Ancylostoma-secreted proteins are unknown, although the P 2 secretion of multiple family members by the adult suggests an important role in the establishment or maintenance of the parasitic relationship. (C) 2003 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.