Calcium modulates protease resistance and carbohydrate binding of a plant defense legume lectin, Griffonia simplicifolia lectin II (GSII)
2002
Zhu-Salzman, K. | Hammen, P.K. | Salzman, R.A. | Koiwa, H. | Bressan, R.A. | Murdock, L.L. | Hasegawa, P.M.
Site-directed mutagenesis previously identified the residues responsible for the biological activity of the plant defense legume lectin, Griffonia simplicifolia lectin II (GSII) [Proc. Natl. Acad. Sci. USA 95, (1998) 15123-15128]. However, these results were inconclusive as to whether these residues function as direct defense determinants through carbohydrate binding, or whether substantial changes of the protein structure had occurred in mutated proteins, with this structural disruption actually causing the loss of biochemical and biological functions. Evidence shown here supports the former explanation: circular dichroism and fluorescence spectra showed that mutations at carbohydrate-binding residues of GSII do not render it disfunctional because of substantial secondary or tertiary structure modifications; and trypsin treatment confirmed that rGSII structural integrity is retained in these mutants. Reduced biochemical stability was observed through papain digestion and urea denaturation in mutant versions that had lost carbohydrate-binding ability, and this was correlated with lower Ca(2+) content. Accordingly, the re-addition of Ca(2+) to demetalized proteins could recover resistance to papain in the carbohydrate-binding mutant, but not in the non-binding mutant. Thus, both carbohydrate binding (presumably to targets in the insect gut) and biochemical stability to proteolytic degradation in situ indeed contribute to anti-insect activity, and these activities are Ca(2+)-dependent.
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