Molecular analysis of astacin-like metalloproteases of Ostertagia ostertagi.
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.
De Maere
V
V
Laboratory of Parasitology, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium. veerle.demaere@UGent.be
Vercauteren
I
I
Geldhof
P
P
Gevaert
K
K
Vercruysse
J
J
Claerebout
E
E
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000469:parasitology / Q000517:prevention & control; D005243:Feces / Q000469:parasitology; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D008666:Metalloendopeptidases; D045726:Metalloproteases / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D010028:Ostertagia / Q000201:enzymology* / Q000235:genetics; D010029:Ostertagiasis / Q000517:prevention & control / Q000662:veterinary; D010270:Parasite Egg Count; D010802:Phylogeny; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014612:Vaccines
D014612:Vaccines; D045726:Metalloproteases; D008666:Metalloendopeptidases; C071288:astacin