tailieunhanh - Báo cáo khoa học: Characterization of a digestive carboxypeptidase from the insect pest corn earworm (Helicoverpa armigera) with novel specificity towards C-terminal glutamate residues

Carboxypeptidases were purified fromguts of larvae of corn earworm (Helicoverpa armigera), a lepidopteran crop pest, by affinity chromatography on immobilized potato carb-oxypeptidase inhibitor, and characterized by N-terminal sequencing. A larval gut cDNA library was screened using probes based on these protein sequences. cDNA HaCA42 encoded a carboxypeptidase with sequence similarity to enzymesofclan MC [Barrett,. D.& Woessner, J. F. (1998)Handbook of Proteolytic Enzymes. Academic Press, London.], but with a novel predicted spe-cificity towards C-terminal acidic residues | Eur. J. Biochem. 271 2000-2011 2004 FEBS 2004 doi Characterization of a digestive carboxypeptidase from the insect pest corn earworm Helicoverpa armigera with novel specificity towards C-terminal glutamate residues David P. Bown and John A. Gatehouse School of Biological and Biomedical Sciences University of Durham UK Carboxypeptidases were purified from guts oflarvae of corn earworm Helicoverpa armigera a lepidopteran crop pest by affinity chromatography on immobilized potato carboxypeptidase inhibitor and characterized by N-terminal sequencing. A larval gut cDNA library was screened using probes based on these protein sequences. cDNA HaCA42 encoded a carboxypeptidase with sequence similarity to enzymes of clan MC Barrett A. J. Rawlings N. D. Woessner J. F. 1998 Handbook of Proteolytic Enzymes. Academic Press London. but with a novel predicted specificity towards C-terminal acidic residues. This carboxypeptidase was expressed as a recombinant proprotein in the yeast Pichia pastoris. The expressed protein could be activated by treatment with bovine trypsin degradation of bound pro-region rather than cleavage of pro-region from mature protein was the rate-limiting step in activation. Activated HaCA42 carboxypeptidase hydrolysed a synthetic substrate for glutamate carboxypeptidases FAEE C-ter-minal Glu but did not hydrolyse substrates for carboxypeptidase A or B FAPP or FAAK C-terminal Phe or Lys or methotrexate cleaved by clan MH glutamate carboxypeptidases. The enzyme was highly specific for C-terminal glutamate in peptide substrates with slow hydrolysis of C-terminal aspartate also observed. Glutamate carboxypeptidase activity was present in larval gut extract from H. armigera. The HaCA42 protein is the first glutamatespecific metallocarboxypeptidase from clan MC to be identified and characterized. The genome of Drosophila mel-anogaster contains genes encoding enzymes with similar sequences and predicted specificity and a cDNA .