title: Molecular cloning and characterization of Ac-MTP-2, an astacin-like metalloprotease released by adult Ancylostoma caninum.
abstract: Ac-MTP-2 is an astacin-like metalloprotease secreted by adult Ancylostoma caninum hookworms. Ac-mtp-2 cDNA was cloned by immunoscreening a cDNA library with antisera prepared against adult A. caninum excretory/secretory (ES) products. The full-length Ac-mtp-2 contains 850 bp cDNA encoding a 233 amino acid open reading frame (ORF) with 32% amino acid identity to Ce-NSP-4, a pharyngeal cell-derived secreted metalloprotease of the nematode Caenorhabditis elegans. The predicted ORF contained a conserved Met-turn sequence (SXMHY), but only a partial zinc-binding signature sequence (GXXXEHXRXER instead of HEXXHXXGXXHEXXRXDR) found in other astacins. However, by both gelatin gel electrophoresis and azocasein digestion, the recombinant Ac-MTP-2 exhibited proteolytic activity that was inhibited by the zinc chelator 1,10-phenanthroline and Ac-TMP, a putative tissue inhibitor of metalloprotease that was previously shown to be a highly abundant component of adult A. caninum ES products. By RT-PCR, Western blot Ac-MTP-2 was found only expressed in adult hookworms and secreted in the adult ES products. Immunolocalization with antisera shows that Ac-MTP-2 is located to the esophageal glands (confirming its role as a secretory protein), as well as to the parasite uterus. It is hypothesized that Ac-MTP-2 functions in the extracorporeal digestion of the intestinal mucosal plug lodged in the buccal capsule of the adult parasite.
authors.affiliation: Department of Microbiology, Immunology and Tropical Medicine, The George Washington University and Sabin Vaccine Institute, Washington, DC 20037, USA.
authors.forename: Jianjun
authors.initials: J
authors.lastname: Feng
authors.forename: Bin
authors.initials: B
authors.lastname: Zhan
authors.forename: Yueyuan
authors.initials: Y
authors.lastname: Liu
authors.forename: Sen
authors.initials: S
authors.lastname: Liu
authors.forename: Angela
authors.initials: A
authors.lastname: Williamson
authors.forename: Gaddam
authors.initials: G
authors.lastname: Goud
authors.forename: Alex
authors.initials: A
authors.lastname: Loukas
authors.forename: Peter
authors.initials: P
authors.lastname: Hotez
mesh_terms: D000595:Amino Acid Sequence; D000722:Ancylostoma / Q000201:enzymology* / Q000378:metabolism; D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015801:Helminth Proteins / Q000235:genetics* / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics; D045726:Metalloproteases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment
chemical_list: D018076:DNA, Complementary; D015801:Helminth Proteins; D011994:Recombinant Proteins; D045726:Metalloproteases; D008666:Metalloendopeptidases; C071288:astacin