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cDNA, synthesised from total RNA from Acanthocheilonema viteae , was amplified by PCR with a primer derived from the spliced leader 1 sequence of nematodes and oligo-dT. Due to the great number of side products observed in the reaction, a biotinylated oligo-dT primer was used for cDNA-synthesis and the first cycles of PCR. After binding of the PCR products to streptavidin/paramagnetic particles, the (+) strands of the cDNAs were recovered and reamplified. Analysis of the PCR products obtained revealed the presence of full-length cDNAs of at least 1.7 kbp in size in amplified total cDNA from microfilariae, postinfective L3, and adult worms. The total cDNA, from only 20 ex vivo recovered postinfective L3, was efficiently amplified.

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, approximately 1.5-1.6 kbp were isolated from an A. viteae expression library. Sequence analysis in combination with N-terminal amino acid sequencing of purified ES-62 revealed that each clone contained a full-length cDNA for ES-62 corresponding to 474 amino acid residues but differed in their 5' and 3' untranslated regions. Characterisation of the 5' end of ES-62 mRNA using 5' rapid amplification of cDNA ends showed that it coded for a signal sequence. Several tryptic peptides were independently sequenced using quadruple-time-of-flight mass spectrometry and used to confirm the cDNA sequence. The mature protein was found to contain three potential N-linked glycosylation sites. Comparison of the derived 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 aminopeptidases. Incubation of ES-62 with leucine-7-amino-4-methylcoumarin as substrate confirmed that ES-62 possessed aminopeptidase activity.

Abbreviations: hsp, heat shock protein(s); ER, endoplasmatic reticulum; GST, glutathione Stransferase; PCR, polymerase chain reaction; HPLC, high-performance liquid chromatography; bp, base pairs; IPTG, isopropyl-P-o-thiogalactopyranoside. Note: The nucleotide sequences reported here were deposited in the GenBanWEMBL data bank under accession numbers M63604 (EM-Grp78) and M63605 (EG-Grp78), respectively. ’ Presenr address: Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC, USA. * Corresponding author, Tel.: +49 51 I 5324352; Fax: +49 511 5324366. However, stimulators of grp78 expression include tunicamycin via inhibition of glycosylation, glucose starvation and calcium ionophores [3,4]. Important features of Grp78 that are absent in other hsp70 proteins include a hydrophobic signal sequence facilitating the translocation of Grp78 into the ER and a carboxy terminal ER retention signal (KDEL) maintaining the protein in the lumen of the ER [5]. Grp78 is believed to function as a molecular chaperone’ in the ER, promoting the correct folding and/or assembly of polypeptides in an ATP-dependent manner [6]. Members of the hsp70 family have been shown to be highly immunogenic in several parasitic diseases [7]. This includes the Plasmodium falciparum Grp78 [8] and the recently described Grp78 of Trypanosoma cruzi [9]. Furthermore hsp70-like polypeptides induce high levels of antibodies in infections caused by P. falciparum, Schistosoma mansoni, S. japonicum [7,10] and Brugia malayi [ll]. By screening of a cDNA library of the larval

Living Acanthocheilonema viteae microfilariae obtained from peripheral blood of parasitised Meriones unguiculatus were surface-labelled with 125I. Four major surface exposed proteins of approximately 14.50, 14.55, 17.5, 19 kDa and one less abundant protein of 40 kDa were identified. Under non-reducing conditions the low-molecular-weight (LMW) proteins were isolated as multimers suggesting the presence of intermolecular disulphide linkages. In gels containing Triton X-100 the labelled epicuticular proteins behaved lipophilically. By cultivation of surface-labelled and metabolically labelled microfilaria in vitro, a continuous shedding of two LMW proteins was demonstrated. These proteins were produced in large amounts and released into the culture supernatant as monomeric and pentameric molecules. Concomitant with this release, one of the proteins appeared to lose its lipophilic character, giving rise to a hydrophilic 14.50-kDa entity. Although most of the extracted surface proteins reacted with sera from patent jirds, these sera failed to recognise the surface of living microfilariae. However, microfilariae pretreated with glutaraldehyde or attenuated with Na-azide could be labelled with surface specific antibodies.