Molecular cloning and demonstration of an aminopeptidase activity in a filarial nematode glycoprotein

ES-62 is an abundant phosphorylcholine-containing secreted glycoprotein of the filarial nematode Acanthocheilonema viteae. Using an antiserum directed against the parasite molecule, 3 cDNAs of size, similar to 1.5-1.6 kbp wer e isolated from an A. viteae expression library. Sequence analysis in combi nation with N-terminal amino acid sequencing of purified ES-62 revealed tha t each clone contained a full-length cDNA for ES-62 corresponding to 474 am ino acid residues but differed in their 5' and 3' untranslated regions. Cha racterisation of the 5' end of ES-62 mRNA using 5' rapid amplification of c DNA ends showed that it coded for a signal sequence. Several tryptic peptid es were independently sequenced using quadruple-time-of-flight mass spectro metry and used to confirm the cDNA sequence. The mature protein was found t o contain three potential N-linked glycosylation sites. Comparison of the d erived amino acid sequence of ES-62 with the SwissProt database identified a sequence (between amino acid residues approximately 250 and 350 of mature ES-62) with significant similarity to several bacterial/fungal aminopeptid ases. Incubation of ES-62 with leucine-7-amino-4-methylcoumarin as substrat e confirmed that ES-62 possessed aminopeptidase activity. (C) 1999 Elsevier Science B.V. All rights reserved.