Acanthocheilonema viteae: Characterization of a molt-associated excretory/secretory 18-kDa protein

Postinvasive third-stage larvae (pL3) of Acanthocheilonema viteae were labe led with [S-35]-methionine in vivo, and proteins released into the culture supernatant before and during the third molt were analyzed. The molting sup ernatant (MSN) contained abundant proteins of 14. 18, 29, and 36 kDa. The 1 4- and 29-kDa proteins were exclusively found in the MSN, while the 18- and 36-kDa proteins were also produced by nonmolting pL3, albeit in much lower quantities. The cDNA for the most abundant protein in the MSN. an 18-kDa p rotein (Av18), was isolated by polymerase chain reaction (PCR) with reverse transcribed (RT) RNA of pL3, using information of the protein sequence. Th e Av18 full-length cDNA of 583 base pairs contained the 5' spliced leader s equence of nematodes, an open reading frame of 427 base pairs, and a poly(A ) tail in typical distance to a polyadenylation signal. The deduced amino a cid sequence encodes for a protein with a calculated size of 15.8 kDa. The N-terminus starts with a hydrophobic signal sequence and a predicted cleava ge site after amino acid 20. The Av18 protein showed homologies to the dedu ced amino acid sequence of the larval transcripts Bm-alt-l and alt-2 of Bru gia malayi and to the Dirofilaria immitis proteins Di20/22 as well as to th e Onchocerca volvulus proteins Ov-alt-1 and Ov-alt-2. Av18 is present in al l parasite stages within the mammalian host, as determined by immunoblot wi th sera against the Escherichia coli-expressed protein and RT-PCR experimen ts. However, it was released into culture medium only by L3 and adult femal e worms. In female worms Av18 was localized in the cuticular region as demo nstrated by immunofluorescent antibody tests using cryosections. (C) 1999 A cademic Press.