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Response of digestive cysteine proteinases from the Colorado potato beetle (Leptinotarsa decemlineata) and the black vine weevil (Otiorynchus sulcatus) to a recombinant form of human stefin A.
Arch Insect Biochem Physiol. 1996; 31(4):451-64.AI

Abstract

The effects of the cystatins, human stefin A (HSA) and oryzacystatin I (OCI) on digestive cysteine proteinases of the Colorado potato beetle (CPB), Leptinotarsa decemlineata, and the black vine weevil (BVW), Otiorynchus sulcatus, were assessed using complementary inhibition assays, cystatin-affinity chromatography, and recombinant forms of the two inhibitors. For both insects, either HSA and OCI used in excess (10 or 20 microM) caused partial and stable inhibition of total proteolytic (azocaseinase) activity, but unlike for OCI the HSA-mediated inhibitions were significantly increased when the inhibitor was used in large excess (100 microM). As demonstrated by complementary inhibition assays, this two-step inhibition of the insect proteases by HSA was due to the differential inactivation of two distinct cysteine proteinase populations in either insect extracts, the rapidly (strongly) inhibited population corresponding to the OCI-sensitive fraction. After removing the cystatin-sensitive proteinases from CPB and BVW midgut extracts using OCI- (or HSA-) affinity chromatography, the effects of the insect "non-target" proteases on the structural integrity of the two cystatins were assessed. While OCI remained essentially stable, HSA was subjected to hydrolysis without the accumulation of detectable stable intermediates, suggesting the presence of multiple exposed cleavage sites sensitive to the action of the insect proteases on this cystatin. This apparent susceptibility of HSA to proteolytic cleavage may partially explain its low efficiency to inactivate the insect OCI-insensitive cysteine proteinases when not used in large excess. It could also have major implications when planning the use of cystatin-expressing transgenic plants for the control of coleopteran pests.

Authors+Show Affiliations

Michaud D
Département de phytologie, Université Laval, Québec, Canada.
Nguyen-Quoc B
No affiliation info available
Vrain TC
No affiliation info available
Fong D
No affiliation info available
Yelle S
No affiliation info available

MeSH

AnimalsBase SequenceColeopteraCystatin ACystatinsCysteine EndopeptidasesCysteine Proteinase InhibitorsDNA PrimersDigestive SystemHumansMolecular Sequence DataRecombinant Fusion Proteins

Pub Type(s)

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

Language

eng

PubMed ID

8920105

Citation

Michaud, D, et al. "Response of Digestive Cysteine Proteinases From the Colorado Potato Beetle (Leptinotarsa Decemlineata) and the Black Vine Weevil (Otiorynchus Sulcatus) to a Recombinant Form of Human Stefin A." Archives of Insect Biochemistry and Physiology, vol. 31, no. 4, 1996, pp. 451-64.
Michaud D, Nguyen-Quoc B, Vrain TC, et al. Response of digestive cysteine proteinases from the Colorado potato beetle (Leptinotarsa decemlineata) and the black vine weevil (Otiorynchus sulcatus) to a recombinant form of human stefin A. Arch Insect Biochem Physiol. 1996;31(4):451-64.
Michaud, D., Nguyen-Quoc, B., Vrain, T. C., Fong, D., & Yelle, S. (1996). Response of digestive cysteine proteinases from the Colorado potato beetle (Leptinotarsa decemlineata) and the black vine weevil (Otiorynchus sulcatus) to a recombinant form of human stefin A. Archives of Insect Biochemistry and Physiology, 31(4), 451-64.
Michaud D, et al. Response of Digestive Cysteine Proteinases From the Colorado Potato Beetle (Leptinotarsa Decemlineata) and the Black Vine Weevil (Otiorynchus Sulcatus) to a Recombinant Form of Human Stefin A. Arch Insect Biochem Physiol. 1996;31(4):451-64. PubMed PMID: 8920105.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Response of digestive cysteine proteinases from the Colorado potato beetle (Leptinotarsa decemlineata) and the black vine weevil (Otiorynchus sulcatus) to a recombinant form of human stefin A. AU - Michaud,D, AU - Nguyen-Quoc,B, AU - Vrain,T C, AU - Fong,D, AU - Yelle,S, PY - 1996/1/1/pubmed PY - 2000/6/20/medline PY - 1996/1/1/entrez SP - 451 EP - 64 JF - Archives of insect biochemistry and physiology JO - Arch Insect Biochem Physiol VL - 31 IS - 4 N2 - The effects of the cystatins, human stefin A (HSA) and oryzacystatin I (OCI) on digestive cysteine proteinases of the Colorado potato beetle (CPB), Leptinotarsa decemlineata, and the black vine weevil (BVW), Otiorynchus sulcatus, were assessed using complementary inhibition assays, cystatin-affinity chromatography, and recombinant forms of the two inhibitors. For both insects, either HSA and OCI used in excess (10 or 20 microM) caused partial and stable inhibition of total proteolytic (azocaseinase) activity, but unlike for OCI the HSA-mediated inhibitions were significantly increased when the inhibitor was used in large excess (100 microM). As demonstrated by complementary inhibition assays, this two-step inhibition of the insect proteases by HSA was due to the differential inactivation of two distinct cysteine proteinase populations in either insect extracts, the rapidly (strongly) inhibited population corresponding to the OCI-sensitive fraction. After removing the cystatin-sensitive proteinases from CPB and BVW midgut extracts using OCI- (or HSA-) affinity chromatography, the effects of the insect "non-target" proteases on the structural integrity of the two cystatins were assessed. While OCI remained essentially stable, HSA was subjected to hydrolysis without the accumulation of detectable stable intermediates, suggesting the presence of multiple exposed cleavage sites sensitive to the action of the insect proteases on this cystatin. This apparent susceptibility of HSA to proteolytic cleavage may partially explain its low efficiency to inactivate the insect OCI-insensitive cysteine proteinases when not used in large excess. It could also have major implications when planning the use of cystatin-expressing transgenic plants for the control of coleopteran pests. SN - 0739-4462 UR - https://www.unboundmedicine.com/medline/citation/8920105/Response_of_digestive_cysteine_proteinases_from_the_Colorado_potato_beetle__Leptinotarsa_decemlineata__and_the_black_vine_weevil__Otiorynchus_sulcatus__to_a_recombinant_form_of_human_stefin_A_ L2 - https://doi.org/10.1002/(SICI)1520-6327(1996)31:4<451::AID-ARCH7>3.0.CO;2-Y DB - PRIME DP - Unbound Medicine ER -