Partial characterization of a major gut thiol proteinase from larvae of <i>Callosobruchus maculatus</i> F.

Kitch, L. W.; Murdock, Larry L.

doi:10.1002/arch.940030607

Cited by 90 publications

(51 citation statements)

References 34 publications

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“…Proteolysis of the rGSII-Y134D or BSA was reduced substantially by trans-epoxysuccinyl-L-leucylamido (4-guanidino)-butane (E-64), an irreversible inhibitor of cysteine proteases (data not shown). This result is in accordance with earlier evidence indicating that cysteine proteases are the major protein digestive enzymes used by cowpea bruchid (34,38). The rGSII protein did not prevent proteolysis of BSA by C. maculatus gut extract (data not shown), indicating that biochemical stability and insecticidal activity of rGSII is not due to inherent protease inhibitor activity.…”

Section: Resultssupporting

confidence: 93%

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Carbohydrate binding and resistance to proteolysis control insecticidal activity of Griffonia simplicifolia lectin II

Zhu‐Salzman

Shade²,

Koiwa³

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

119

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Griffonia simplicifolia leaf lectin II (GSII), a plant defense protein against certain insects, consists of an N-acetylglucosamine (GlcNAc)-binding large subunit with a small subunit having sequence homology to class III chitinases. Much of the insecticidal activity of GSII is attributable to the large lectin subunit, because bacterially expressed recombinant large subunit (rGSII) inhibited growth and development of the cowpea bruchid, Callosobruchus maculatus (F). Site-specific mutations were introduced into rGSII to generate proteins with altered GlcNAc binding, and the different rGSII proteins were evaluated for insecticidal activity when added to the diet of the cowpea bruchid. At pH 5.5, close to the physiological pH of the cowpea bruchid midgut lumen, rGSII recombinant proteins were categorized as having high (rGSII, rGSII-Y134F, and rGSII-N196D mutant proteins), low (rGSII-N136D), or no (rGSII-D88N, rGSII-Y134G, rGSII-Y134D, and rGSII-N136Q) GlcNAc-binding activity. Insecticidal activity of the recombinant proteins correlated with their GlcNAc-binding activity. Furthermore, insecticidal activity correlated with the resistance to proteolytic degradation by cowpea bruchid midgut extracts and with GlcNAc-specific binding to the insect digestive tract. Together, these results establish that insecticidal activity of GSII is functionally linked to carbohydrate binding, presumably to the midgut epithelium or the peritrophic matrix, and to biochemical stability of the protein to digestive proteolysis.

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Section: Resultssupporting

confidence: 93%

“…The physiological pH of C. maculatus larval midguts has been determined to be about 6.1 by using a pH microelectrode (34). However, the midgut contains a thiol-dependent protease with pH optimum at 5.0 (34).…”

Section: Resultsmentioning

confidence: 99%

Carbohydrate binding and resistance to proteolysis control insecticidal activity of Griffonia simplicifolia lectin II

Zhu‐Salzman

Shade²,

Koiwa³

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

119

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“…Interestingly, arcelin has been shown to possess activity against the bruchid Zabrotes subfasciatus but not against another bean bruchid Acanthoscelides obtectus (19). Further, the pH optimum of inhibition of porcine a-amylase by P. vulgaris aAI is 5.7 (15), which is close to the pH of the cowpea weevil midgut contents (13). These data suggest that the bean aAI has evolved as one of the major determinants in protecting common bean from attack by bruchid beetles such as the cowpea weevil (10,16).…”

Section: And Discussionmentioning

confidence: 97%

α-Amylase Inhibitor, Not Phytohemagglutinin, Explains Resistance of Common Bean Seeds to Cowpea Weevil

Huesing

Shade²,

Chrispeels³

et al. 1991

Plant Physiol.

109

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There are claims that phytohemagglutinin (PHA), the lectin of common bean, Phaseolus vulgaris, is toxic when fed to the cowpea weevil, Callosobruchus maculatus, and that PHA serves as the chemical defense against this seed-feeding bruchid beetle (DH Janzen, HB Juster, IE Liener [1976] (1,4,8,9), one role was proposed by Janzen et al. (12), who showed that 1% (w/w) Phaseolus vulgaris lectin (PHA) from black bean caused mortality when incorporated into the diet of the cowpea weevil, Callosobruchus maculatus F. (Coleoptera: Bruchidae). They concluded that "a major part ofthe adaptive significance of phytohemagglutinins (PHA) in black bean and other legume seeds is to protect them from attack by insect seed predators" (12). Subsequently, the PHA-mediated cowpea/ common bean interaction has become a prime example of protein-mediated plant-insect interactions.Gatehouse et al. (6) (also P. vulgaris) was toxic to the cowpea weevil. Curiously, they noted that a highly purified preparation (>95%) of PHA was less toxic than a less pure commercial preparation obtained from Sigma Chemical Co., London (6). PHA is a tetrameric protein composed of two different subunit types: one erythrocyte-binding (E-type) and one lymphocyte-binding (L-type) (9). Gatehouse et al. (6) concluded that the Etype subunit was the major antimetabolite. However, subsequent studies by Boulter (2) indicated that purified E-type or L-type isolectins were both ineffective when tested alone against the cowpea weevil. To account for the biological activity of the mixture described in the earlier paper (6), Boulter (2) concluded that there must be a synergistic effect between the two different lectin subunits.These provocative results and conclusions led us to determine ifplant lectins represent a general category ofinsecticidal proteins (18). We screened 17 different commercially available plant lectins in our artificial seed bioassay system (21) using the cowpea weevil (18). Included in the study were a PHA lectin mixture (PHA isolectins composed of both the E and L subunits), as well as purified PHA-E and PHA-L. Our results showed that none ofthe three PHA lectin preparations were effective against the cowpea weevil when fed at 1 % (w/ w) in the diet (18), a level claimed effective in the published studies (6,12). Since we had shown that other lectins show great promise as biotechnology based plant chemical defenses (18,22) and because PHA is being advanced as a potential candidate for this role by some investigators, we decided to investigate the putative toxic qualities of PHA further. MATERIALS AND METHODS Artificial SeedsTo test the effects ofvarious potential toxins on seed feeding insects, we used an artificial seed system previously developed in our laboratory (21). Briefly, the system involved milling decorticated cowpea seeds (cultivar California Blackeye No. 5) into flour, adding aqueous solutions of test compounds to the flour to make a paste, injecting the paste into Teflon molds, freezing the paste on dry ice and then liquid N2, ...

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“…This cleavage removes a small C-terminal peptide and makes the inhibitor ineffective against pancreatic a-amylase. Cys proteases rather than Ser proteases are abundant in larval midguts of bruchids and may be primarily responsible for protein digestion (Kitch and Murdock, 1986;Campos et al, 1989). However, prevention of proteolysis by protease inhibitors shows that the midgut protease responsible for the cleavage of aAI-1 is a Ser protease.…”

Section: How Do Bruchids Tolerate High Levels Of Aai In Beans?mentioning

confidence: 99%

Protective Mechanism of the Mexican Bean Weevil against High Levels of α--Amylase Inhibitor in the Common Bean

1996

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~The seeds of starchy grain legumes are an important staple in many countries, but as with other crops, insect pests can cause considerable crop losses. The postharvest damage done by larvae of bruchids (Co1eoptera:Bruchidae) can be quite extensive. Crops of the common bean (Pkaseolus vulgaris), the scarlet runner bean (Phaseoltis coccineus), and the tepary bean (Phaseolus acutifolius) can be severely damaged by the bean weevil (Acanthoscelides obtectus) and the Mexican bean weevil (Zabrotes subfasciatus) . This damage occurs ín spite of the presence in the seeds of a family of plant defense proteins that comprises PHA and aAI (see Chrispeels and Raikhel, 1991). The three Phaseolus species are not equally sensitive to the bruchids. For example, tepary bean is more resistant than common bean to the bean weevil (Shade et al., 1987).

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Partial characterization of a major gut thiol proteinase from larvae of Callosobruchus maculatus F.

Cited by 90 publications

References 34 publications

Carbohydrate binding and resistance to proteolysis control insecticidal activity of Griffonia simplicifolia lectin II

Carbohydrate binding and resistance to proteolysis control insecticidal activity of Griffonia simplicifolia lectin II

α-Amylase Inhibitor, Not Phytohemagglutinin, Explains Resistance of Common Bean Seeds to Cowpea Weevil

Protective Mechanism of the Mexican Bean Weevil against High Levels of α--Amylase Inhibitor in the Common Bean

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