title: Characterization of a digestive carboxypeptidase from the insect pest corn earworm (Helicoverpa armigera) with novel specificity towards C-terminal glutamate residues.
abstract: Carboxypeptidases were purified from guts of larvae 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-terminal 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 glutamate-specific metallocarboxypeptidase from clan MC to be identified and characterized. The genome of Drosophila melanogaster contains genes encoding enzymes with similar sequences and predicted specificity, and a cDNA encoding a similar enzyme has been isolated from gut tissue in tsetse fly. We suggest that digestive carboxypeptidases with sequence similarity to the classical mammalian enzymes, but with specificity towards C-terminal glutamate, are widely distributed in insects.
authors.affiliation: School of Biological and Biomedical Sciences, University of Durham, UK.
authors.forename: David P
authors.initials: DP
authors.lastname: Bown
authors.forename: John A
authors.initials: JA
authors.lastname: Gatehouse
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002268:Carboxypeptidases / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D041981:Gastrointestinal Tract / Q000201:enzymology*; D015870:Gene Expression; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism*; D007814:Larva / Q000201:enzymology; D007915:Lepidoptera / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D010843:Pichia / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014357:Trypsin / Q000494:pharmacology; D001615:beta-Endorphin / Q000378:metabolism
chemical_list: D010455:Peptides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D018698:Glutamic Acid; D001615:beta-Endorphin; D002268:Carboxypeptidases; D014357:Trypsin