title: Molecular characterization of a family of metalloendopeptidases from the intestinal brush border of Haemonchus contortus.
abstract: Substantial protection against the economically important parasitic nematode Haemonchus contortus has been achieved by immunizing sheep with a glycoprotein fraction isolated from the intestinal membranes of the worm (H-gal-GP). Previous studies showed that one of the major components of H-gal-GP is a family of at least 4 zinc metalloendopeptidases, designated MEPs 1-4. This paper describes aspects of the molecular architecture of this protease family, including the proteomic analysis of the MEP fraction of the H-gal-GP complex. These enzymes belong to the M13 zinc metalloendopeptidase family (EC 3.4.24.11), also known as neutral endopeptidases or neprilysins. The sequences of MEPs 1 and 3 suggested a typical Type II integral membrane protein structure, whilst MEPs 2 and 4 had putative cleavable signal peptides, typical of secreted proteins. Proteomic analysis of H-gal-GP indicated that the extracellular domain of all 4 MEPs had been cleaved close to the transmembrane region/signal peptide with additional cleavage sites mid-way along the polypeptide. MEP3 was present as a homo-dimer in H-gal-GP, whereas MEP1 or MEP2 formed hetero-dimers with MEP4. It was found that expression of MEP3 was confined to developing 4th-stage larvae and to adult worms, the stages of Haemonchus which feed on blood. MEP-like activity was detected in the H-gal-GP complex over a broad pH range (5-9). Since all 4 MEPs must share a similar microenvironment in the complex, this suggests that each might have a different substrate specificity.
authors.affiliation: Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 OPZ, Scotland. george.newlands@moredun.ac.uk
authors.forename: G F J
authors.initials: GF
authors.lastname: Newlands
authors.forename: P J
authors.initials: PJ
authors.lastname: Skuce
authors.forename: A J
authors.initials: AJ
authors.lastname: Nisbet
authors.forename: D L
authors.initials: DL
authors.lastname: Redmond
authors.forename: S K
authors.initials: SK
authors.lastname: Smith
authors.forename: D
authors.initials: D
authors.lastname: Pettit
authors.forename: W D
authors.initials: WD
authors.lastname: Smith
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000909:Antibodies, Helminth / Q000276:immunology; D016282:Aspartic Acid Endopeptidases / Q000302:isolation & purification; D003001:Cloning, Molecular / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000096:biosynthesis / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D006189:Haemonchus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000276:immunology; D015801:Helminth Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007814:Larva / Q000201:enzymology / Q000254:growth & development; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D016415:Sequence Alignment
chemical_list: D000909:Antibodies, Helminth; D015801:Helminth Proteins; D008562:Membrane Glycoproteins; D011480:Protease Inhibitors; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C119206:galactose-containing glycoprotein, Haemonchus contortus