Response of the digestive system of Helicoverpa zea to ingestion of potato carboxypeptidase inhibitor and characterization of an uninhibited carboxypeptidase B.
Carboxypeptidase activity participates in the protein digestion process in the gut of lepidopteran insects, supplying free amino-acids to developing larvae. To study the role of different carboxypeptidases in lepidopteran protein digestion, the effect of potato carboxypeptidase inhibitor (PCI) on the digestive system of larvae of the pest insect Helicoverpa zea was investigated, and compared to that of Soybean Kunitz Trypsin Inhibitor. Analysis of carboxypeptidase activity in the guts showed that ingested PCI remained active in the gut, and completely inhibited the activity of carboxypeptidases A and O. Interestingly, carboxypeptidase B activity was not affected by PCI. All previously described enzymes from the same family, both from insect or mammalian origin, have been found to be very sensitive to PCI. Analysis of several lepidopteran species showed the presence of carboxypeptidase B activity resistant to PCI in most of them. The H. zea carboxypeptidase B enzyme (CPBHz) was purified from gut content by affinity chromatography. N-terminal sequence information was used to isolate its corresponding full-length cDNA, and recombinant expression of the zymogen of CPBHz in Pichia pastoris was achieved. The substrate specificity of recombinant CPBHz was tested using peptides. Unlike other CPB enzymes, the enzyme appeared to be highly selective for C-terminal lysine residues. Inhibition by PCI appeared to be pH-dependent.
Bayés
Alex
A
Departament de Bioquímica i Biologia Molecular, Facultat de Ciències and Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain.
de la Vega
Mónica Rodríguez
MR
Vendrell
Josep
J
Aviles
Francesc X
FX
Jongsma
Maarten A
MA
Beekwilder
Jules
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D043424:Carboxypeptidase B / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000187:drug effects; D004068:Digestive System Physiological Phenomena / Q000187:drug effects; D015870:Gene Expression; D019476:Insect Proteins; D007814:Larva / Q000187:drug effects; D045727:Metalloexopeptidases / Q000187:drug effects; D008969:Molecular Sequence Data; D009036:Moths / Q000187:drug effects* / Q000235:genetics; D010936:Plant Extracts / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins; D017422:Sequence Analysis, DNA; D011198:Solanum tuberosum / Q000737:chemistry; D013379:Substrate Specificity
D019476:Insect Proteins; D010936:Plant Extracts; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D045727:Metalloexopeptidases; D043424:Carboxypeptidase B