title: Molecular cloning and demonstration of an aminopeptidase activity in a filarial nematode glycoprotein.
abstract: 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.
authors.affiliation: Department of Immunology, University of Strathclyde, The Todd Centre, Glasgow, UK. w.harnett@strath.ac.uk
authors.forename: W
authors.initials: W
authors.lastname: Harnett
authors.forename: K M
authors.initials: KM
authors.lastname: Houston
authors.forename: R
authors.initials: R
authors.lastname: Tate
authors.forename: T
authors.initials: T
authors.lastname: Garate
authors.forename: H
authors.initials: H
authors.lastname: Apfel
authors.forename: R
authors.initials: R
authors.lastname: Adam
authors.forename: S M
authors.initials: SM
authors.lastname: Haslam
authors.forename: M
authors.initials: M
authors.lastname: Panico
authors.forename: T
authors.initials: T
authors.lastname: Paxton
authors.forename: A
authors.initials: A
authors.lastname: Dell
authors.forename: H
authors.initials: H
authors.lastname: Morris
authors.forename: H
authors.initials: H
authors.lastname: Brzeski
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D000909:Antibodies, Helminth; D001483:Base Sequence; D004153:Dipetalonema / Q000201:enzymology* / Q000235:genetics; D005260:Female; D015723:Gene Library; D006023:Glycoproteins / Q000235:genetics / Q000302:isolation & purification*; D015801:Helminth Proteins / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D018510:RNA, Helminth / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
chemical_list: D000909:Antibodies, Helminth; D006023:Glycoproteins; D015801:Helminth Proteins; D018510:RNA, Helminth; D012333:RNA, Messenger; D000626:Aminopeptidases