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, 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.
Harnett
W
W
Department of Immunology, University of Strathclyde, The Todd Centre, Glasgow, UK. w.harnett@strath.ac.uk
Houston
K M
KM
Tate
R
R
Garate
T
T
Apfel
H
H
Adam
R
R
Haslam
S M
SM
Panico
M
M
Paxton
T
T
Dell
A
A
Morris
H
H
Brzeski
H
H
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
D000909:Antibodies, Helminth; D006023:Glycoproteins; D015801:Helminth Proteins; D018510:RNA, Helminth; D012333:RNA, Messenger; D000626:Aminopeptidases