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Characterisation of cysteine proteinases responsible for digestive proteolysis in guts of larval western corn rootworm (Diabrotica virgifera) by expression in the yeast Pichia pastoris.
Insect Biochem Mol Biol. 2004 Apr; 34(4):305-20.IB

Abstract

Cysteine proteinases are the major class of enzymes responsible for digestive proteolysis in western corn rootworm (Diabrotica virgifera), a serious pest of maize. A larval gut extract hydrolysed typical cathepsin substrates, such as Z-phe-arg-AMC and Z-arg-arg-AMC, and hydrolysis was inhibited by Z-phe-tyr-DMK, specific for cathepsin L. A cDNA library representing larval gut tissue mRNA contained cysteine proteinase-encoding clones at high frequency. Sequence analysis of 11 cysteine proteinase cDNAs showed that 9 encoded cathepsin L-like enzymes, and 2 encoded cathepsin B-like enzymes. Three enzymes (two cathepsin L-like, DvRS5 and DvRS30, and one cathepsin B-like, DvRS40) were expressed as recombinant proteins in culture supernatants of the yeast Pichia pastoris. The cathepsin L-like enzymes were active proteinases, whereas the cathepsin B-like enzyme was inactive until treated with bovine trypsin. The amino acid residue in the S2 binding pocket, the major determinant of substrate specificity in cathepsin cysteine proteinases, predicted that the two cathepsin L-like enzymes, DvRS5 and DvRS30, should differ in substrate specificity, with the latter resembling cathepsin B in hydrolysing substrates with a positively charged residue at P2. This prediction was confirmed; DvRS5 only hydrolysed Z-phe-arg-AMC and not Z-arg-arg-AMC, whereas DvRS30 hydrolysed both substrates. The enzymes showed similar proteolytic activity towards peptide substrates.

Authors+Show Affiliations

School of Biological and Biomedical Sciences, University of Durham, South Road, Durham DH1 3LE, UK.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

15041015

Citation

Bown, David P., et al. "Characterisation of Cysteine Proteinases Responsible for Digestive Proteolysis in Guts of Larval Western Corn Rootworm (Diabrotica Virgifera) By Expression in the Yeast Pichia Pastoris." Insect Biochemistry and Molecular Biology, vol. 34, no. 4, 2004, pp. 305-20.
Bown DP, Wilkinson HS, Jongsma MA, et al. Characterisation of cysteine proteinases responsible for digestive proteolysis in guts of larval western corn rootworm (Diabrotica virgifera) by expression in the yeast Pichia pastoris. Insect Biochem Mol Biol. 2004;34(4):305-20.
Bown, D. P., Wilkinson, H. S., Jongsma, M. A., & Gatehouse, J. A. (2004). Characterisation of cysteine proteinases responsible for digestive proteolysis in guts of larval western corn rootworm (Diabrotica virgifera) by expression in the yeast Pichia pastoris. Insect Biochemistry and Molecular Biology, 34(4), 305-20.
Bown DP, et al. Characterisation of Cysteine Proteinases Responsible for Digestive Proteolysis in Guts of Larval Western Corn Rootworm (Diabrotica Virgifera) By Expression in the Yeast Pichia Pastoris. Insect Biochem Mol Biol. 2004;34(4):305-20. PubMed PMID: 15041015.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Characterisation of cysteine proteinases responsible for digestive proteolysis in guts of larval western corn rootworm (Diabrotica virgifera) by expression in the yeast Pichia pastoris. AU - Bown,David P, AU - Wilkinson,Hillary S, AU - Jongsma,Maarten A, AU - Gatehouse,John A, PY - 2003/09/29/received PY - 2003/11/18/revised PY - 2003/11/19/accepted PY - 2004/3/26/pubmed PY - 2004/6/16/medline PY - 2004/3/26/entrez SP - 305 EP - 20 JF - Insect biochemistry and molecular biology JO - Insect Biochem Mol Biol VL - 34 IS - 4 N2 - Cysteine proteinases are the major class of enzymes responsible for digestive proteolysis in western corn rootworm (Diabrotica virgifera), a serious pest of maize. A larval gut extract hydrolysed typical cathepsin substrates, such as Z-phe-arg-AMC and Z-arg-arg-AMC, and hydrolysis was inhibited by Z-phe-tyr-DMK, specific for cathepsin L. A cDNA library representing larval gut tissue mRNA contained cysteine proteinase-encoding clones at high frequency. Sequence analysis of 11 cysteine proteinase cDNAs showed that 9 encoded cathepsin L-like enzymes, and 2 encoded cathepsin B-like enzymes. Three enzymes (two cathepsin L-like, DvRS5 and DvRS30, and one cathepsin B-like, DvRS40) were expressed as recombinant proteins in culture supernatants of the yeast Pichia pastoris. The cathepsin L-like enzymes were active proteinases, whereas the cathepsin B-like enzyme was inactive until treated with bovine trypsin. The amino acid residue in the S2 binding pocket, the major determinant of substrate specificity in cathepsin cysteine proteinases, predicted that the two cathepsin L-like enzymes, DvRS5 and DvRS30, should differ in substrate specificity, with the latter resembling cathepsin B in hydrolysing substrates with a positively charged residue at P2. This prediction was confirmed; DvRS5 only hydrolysed Z-phe-arg-AMC and not Z-arg-arg-AMC, whereas DvRS30 hydrolysed both substrates. The enzymes showed similar proteolytic activity towards peptide substrates. SN - 0965-1748 UR - https://www.unboundmedicine.com/medline/citation/15041015/Characterisation_of_cysteine_proteinases_responsible_for_digestive_proteolysis_in_guts_of_larval_western_corn_rootworm__Diabrotica_virgifera__by_expression_in_the_yeast_Pichia_pastoris_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0965174803002200 DB - PRIME DP - Unbound Medicine ER -