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

Mol Biochem Parasitol. 1999 Oct 25;104(1):11-23. doi: 10.1016/s0166-6851(99)00113-9.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Aminopeptidases / genetics
  • Aminopeptidases / isolation & purification*
  • Animals
  • Antibodies, Helminth
  • Base Sequence
  • Dipetalonema / enzymology*
  • Dipetalonema / genetics
  • Female
  • Gene Library
  • Glycoproteins / genetics
  • Glycoproteins / isolation & purification*
  • Helminth Proteins / genetics
  • Helminth Proteins / isolation & purification*
  • Molecular Sequence Data
  • RNA, Helminth / genetics
  • RNA, Messenger / genetics
  • Sequence Analysis, DNA
  • Sequence Homology, Amino Acid

Substances

  • Antibodies, Helminth
  • Glycoproteins
  • Helminth Proteins
  • RNA, Helminth
  • RNA, Messenger
  • Aminopeptidases

Associated data

  • GENBANK/AF077194