Molecular cloning and characterisation of a developmentally regulated putative metallopeptidase present in a host protective extract of Haemonchus contortus.
Antisera from lambs immunised with the Haemonchus contortus integral membrane protein complex, Haemonchus galactose-containing glycoprotein (H-gal-GP), the lambs being refractory to subsequent challenge, were used to identify several clones from an adult H. contortus lambda gt11 cDNA library. Using gene-specific oligonucleotide primers in conjunction with primers directed to a conserved nematode Spliced Leader (SL) sequence and to the polyA+ tail of mRNA, the remaining 5' and 3' sequences of one of these clones, metallopeptidase-1 (MEP1), were amplified. The 2.4 kb full-length coding sequences was subsequently amplified in a single reaction. Sequence analysis identified MEP1 as encoding a putative zinc metallopeptidase, which shared limited homology with the mammalian type II integral membrane protein neutral endopeptidase (NEP). Southern blotting indicated that MEP1 belonged to a multigene family. MEP1 was expressed in bacteria as a glutathione-S-transferase (GST) fusion protein, and a specific antiserum raised in sheep. This antiserum recognised several polypeptide components of H-gal-GP. Immunolocalisation studies showed that MEP1 encoded a protein located on the luminal surface of the nematode gut. Both MEP1 mRNA and protein are developmentally regulated with expression being limited to the blood-feeding stages of H. contortus.
Redmond
D L
DL
Moredun Research Institute, Edinburgh, UK.
Knox
D P
DP
Newlands
G
G
Smith
W D
WD
D000595:Amino Acid Sequence; D000818:Animals; D000947:Antigens, Helminth / Q000235:genetics*; D002451:Cell Compartmentation; D018076:DNA, Complementary / Q000235:genetics; D010450:Endopeptidases / Q000276:immunology; D018507:Gene Expression Regulation, Developmental; D015723:Gene Library; D006188:Haemonchiasis / Q000517:prevention & control; D006189:Haemonchus / Q000201:enzymology / Q000235:genetics*; D015801:Helminth Proteins / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000276:immunology; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D015260:Neprilysin / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D013045:Species Specificity
D000947:Antigens, Helminth; D018076:DNA, Complementary; D015801:Helminth Proteins; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D010450:Endopeptidases; D008666:Metalloendopeptidases; C106178:metallopeptidase-1; D015260:Neprilysin; C119206:galactose-containing glycoprotein, Haemonchus contortus