M. V. Coutinho

The effect of arcelin-1 on the structure of the midgut of bruchid larvae and immunolocalization of the arcelin protein

Some wild accessions of the common bean (Phaseolus vulgaris) contain a family of proteins called arcelins, that are toxic to the larvae of certain bruchid species. Among the six allelic variants of arcelin tested so far, arcelin-5 and arcelin-1 confer the highest level of resistance against the Mexican bean weevil, Zabrotes subfasciatus. The same proteins are not toxic to the bean weevil, Acanthoscelides obtectus, which is also a serious pest of cultivated beans. Arcelins belong to the bean lectin family that includes phytohemaggutinins and alpha-amylase inhibitors. Although homologous to lectins, arcelins are themselves only very weak lectins, and their binding properties have not been clearly established. The toxic properties of arcelins may be related to their recognition of and interaction with the glycoproteins and other constituents of the membranes along the digestive tract of insects. Since arcelin-1 was shown to have growth inhibitory effects for the larvae of Z. subfasciatus but not of A. obtectus, we examined the effect of an arcelin-1 containing diet on the structure of the cells that line the intestinal tract of the larvae of these two bruchid species, and used antibodies against arcelin to examine the distribution of arcelin within the cells and tissues. Here we show that dietary arcelin-1 caused an alteration of the gut structure and the penetration of arcelin into the haemolymph in Z. subfasciatus but not in A. obtectus. These results lead us to suggest that arcelins exert their toxic effect by severely damaging the epithelial cells.

Mutantes do inibidor-2 de alfa-amilase do feijão-comum para investigação da especificidade de ligação a alfa-amilases

Despite the presence of a family of defense proteins, Phaseolus vulgaris can be attacked by bruchid insects resulting in serious damage to stored grains. The two distinct active forms of a-amylase inhibitors, a-AI1 and a-AI2, in P. vulgaris show different specificity toward a-amylases. Zabrotes subfasciatus a-amylase is inhibited by a-AI2 but not by a-AI1. In contrast, porcine a-amylase is inhibited by a-AI1 but not by a-AI2. The objective of this work was to understand the molecular basis of the specificity of two inhibitors in P. vulgaris (a-AI1 and a-AI2) in relation to a-amylases. Mutants of a-AI2 were made and expressed in tobacco plants. The results showed that all the a-AI2 mutant inhibitors lost their activity against the insect a-amylases but none exhibited activity toward the mammalian a-amylase. The replacement of His33 of a-AI2 with the a-AI1-like sequence Ser-Tyr-Asn abolished inhibition of Z. subfasciatus a-amylase. From structural modeling, the conclusion is that the size and complexity of the amylase-inhibitor interface explain why mutation of the N-terminal loop and resultant abolition of Z. subfasciatus a-amylase inhibition are not accompanied by gain of inhibitory activity against porcine a-amylase.

Isolation characterization of the major albumin from Colocasia esculenta Corms

Abstract The major albumin from Colocasia esculenta corms was isolated using differential solubility and gel-filtration chromatography. It corresponds to 11% of the total soluble protein that accumulates in the corms. The native protein has an Mr of 50 000 and appears to be composed of homogenous subunits with Mr of approximately 8300 in denaturing gels. Isoelectric focussing and two-dimensional electrophoresis demonstrate the presence of three isoforms. The major one, accounting for more than 90% of the protein, has an pI of 6.1. The two minor ones have pI values of 5.3 and 5.7. The isolated protein has a relatively high content of essential amino acids such as phenylalamine and leucine but is poor in sulphur-containing amino acids. Western blotting analysis has shown that the expression of the protein is enhanced in the corm and that the residual expression observed in lamina and petiole corresponds to a larger protein, although lamina expresses both species. No expression has been observed in roots. The protein also seems to accumulate in Caladium spp. corms, where it appears to be larger, but not in the storage Xanthosoma sagitifolium, Xanthosoma atrovirens, Ipomoea batatas, Daucus carota, Solanum tuberosum and Manihot esculenta.

Meloidogyne incognita: Molecular cloning and characterization of a cDNA encoding a cathepsin D-like aspartic proteinase

Herein we describe the cloning and characterization of a cDNA encoding an aspartic proteinase from the root-knot nematode Meloidogyne incognita. Using PCR techniques, a 1471-bp cDNA fragment encoding a cathepsin D-like (Mi-asp1) transcript was isolated from second-stage larvae mRNA. Its predicted amino acid sequence comprises a pro-region of 71 amino acid residues and a mature protease of 378 amino acid residues with a predicted molecular mass of 41.502kDa. Protein sequence comparisons of Mi-asp1 with GenBank (DQ360827) sequences showed 59-71% identity with nematode-specific cathepsin D-like aspartic proteinases. Southern blot analysis, RT-PCR amplification and EST mining suggest the existence of a developmentally expressed gene family encoding aspartic proteinases in M. incognita. Mi-asp1 may represent a potential target for molecular intervention for the purposes of plant-parasitic nematode control.