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Molecular analysis of astacin-like metalloproteases of Ostertagia ostertagi

Published online by Cambridge University Press:  13 December 2004

V. DE MAERE
Affiliation:
Laboratory of Parasitology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan, 133, B-9820 Merelbeke, Belgium
I. VERCAUTEREN
Affiliation:
Laboratory of Parasitology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan, 133, B-9820 Merelbeke, Belgium
P. GELDHOF
Affiliation:
Laboratory of Parasitology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan, 133, B-9820 Merelbeke, Belgium Current address: Moredun Research Institute, Edinburgh EH26 OPZ, Scotland, UK.
K. GEVAERT
Affiliation:
Department of Medical Protein Research, Flanders Interuniversity Institute for Biotechnology and Ghent University, Albert Baertsoenkaai, 3, B-9000 Ghent, Belgium
J. VERCRUYSSE
Affiliation:
Laboratory of Parasitology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan, 133, B-9820 Merelbeke, Belgium
E. CLAEREBOUT
Affiliation:
Laboratory of Parasitology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan, 133, B-9820 Merelbeke, Belgium

Abstract

In this study, we describe the molecular analysis of zinc-metalloproteases from the abomasal nematode Ostertagia ostertagi which were exclusively recognized by local antibodies of immune cattle. Full-length or partial coding sequences of 4 different zinc-metalloprotease cDNAs of Ostertagia (met-1, -2, -3 and -4) were amplified using gene-specific primers using the 3′- and 5′-Rapid Amplification of cDNA Ends (RACE) technique. Sequence analysis identified the cDNAs as encoding zinc-metalloproteases, which showed between 62% and 70% homology to a metalloprotease 1 precursor of Ancylostoma caninum. The full-length cDNA of met-1 consists of an open reading frame (ORF) of 586 amino acids which contains 5 potential N-glycosylation sites and a predicted zinc-binding domain (HEBXHXBGFXHEXXRXDRD). The complete coding sequence of met-3 contains an ORF of 508 aa and the same conserved zinc-binding domain. These domains are signature sequences of the astacin family of the superfamily of metzincin metalloproteases. The presence of a threonine amino acid after the third histidine in MET-1 and MET-3, however, may place them in a new family or subfamily. Real-time PCR analysis of L3, exsheathed L3, L4 and adult cDNA identified transcription of the 4 metalloproteases in different life-stages. The protein MET-1 was expressed in insect cells using the baculovirus expression system but the immunization of calves with this molecule did not lead to protection against challenge infection.

Type
Research Article
Copyright
© 2004 Cambridge University Press

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