Mammalian metallopeptidase inhibition at the defense barrier of Ascaris parasite.
Roundworms of the genus Ascaris are common parasites of the human gastrointestinal tract. A battery of selective inhibitors protects them from host enzymes and the immune system. Here, a metallocarboxypeptidase (MCP) inhibitor, ACI, was identified in protein extracts from Ascaris by intensity-fading MALDI-TOF mass spectrometry. The 67-residue amino acid sequence of ACI showed no significant homology with any known protein. Heterologous overexpression and purification of ACI rendered a functional molecule with nanomolar equilibrium dissociation constants against MCPs, which denoted a preference for digestive and mast cell A/B-type MCPs. Western blotting and immunohistochemistry located ACI in the body wall, intestine, female reproductive tract, and fertilized eggs of Ascaris, in accordance with its target specificity. The crystal structure of the complex of ACI with human carboxypeptidase A1, one of its potential targets in vivo, revealed a protein with a fold consisting of two tandem homologous domains, each containing a beta-ribbon and two disulfide bonds. These domains are connected by an alpha-helical segment and a fifth disulfide bond. Binding and inhibition are exerted by the C-terminal tail, which enters the funnel-like active-site cavity of the enzyme and approaches the catalytic zinc ion. The findings reported provide a basis for the biological function of ACI, which may be essential for parasitic survival during infection.
Sanglas
Laura
L
Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Facultat de Ciències, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Barcelona, Spain.
Aviles
Francesc X
FX
Huber
Robert
R
Gomis-Rüth
F Xavier
FX
Arolas
Joan L
JL
D000595:Amino Acid Sequence; D000818:Animals; D001200:Ascaris / Q000737:chemistry* / Q000502:physiology; D043422:Carboxypeptidases A / Q000737:chemistry*; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry*; D005260:Female; D006801:Humans; D045726:Metalloproteases / Q000037:antagonists & inhibitors*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014018:Tissue Distribution
D004791:Enzyme Inhibitors; D045726:Metalloproteases; D043422:Carboxypeptidases A