Molecular cloning and characterization of Ac-MTP-2, an astacin-like metalloprotease released by adult Ancylostoma caninum.
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.
Feng
Jianjun
J
Department of Microbiology, Immunology and Tropical Medicine, The George Washington University and Sabin Vaccine Institute, Washington, DC 20037, USA.
Zhan
Bin
B
Liu
Yueyuan
Y
Liu
Sen
S
Williamson
Angela
A
Goud
Gaddam
G
Loukas
Alex
A
Hotez
Peter
P
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
D018076:DNA, Complementary; D015801:Helminth Proteins; D011994:Recombinant Proteins; D045726:Metalloproteases; D008666:Metalloendopeptidases; C071288:astacin