Full‐fledged proteomic analysis of bioactive wheat amylase inhibitors by a 3‐D analytical technique: Identification of new heterodimeric aggregation states

Abstract

Full-fledged proteomic analysis of bioactive wheat amylase inhibitors by a 3-D analytical technique: Identification of new heterodimeric aggregation statesWheat proteinaceous a-amylase inhibitors (a-AIs) are increasingly investigated for their agronomical role as natural defence molecules of plants against the attack of insects and pests, but also for their effects on human health. The wheat genomes code for several bioactive a-AIs that share sequence homology, but differ in their specificity against a-amylases from different species and for their aggregation states. Wheat a-AIs are traditionally classified as belonging to the three classes of tetrameric, homodimeric and monomeric forms, each class being constituted by a number of polypeptides that display different electrophoretic mobilities. Here we describe a proteomic approach for the identification of bioactive a-AIs from wheat and, in particular, a 3-D technique that allows to best identify and characterize the dimeric fraction. The technique takes advantage of the thermal resistance of a-AIs (resistant to T . 707C) and consists in the separation of protein mixtures by 2-D polyacrylamide/starch electrophoresis under nondissociating PAGE (ND-PAGE, first dimension) and dissociating (urea-PAGE or U-PAGE second dimension) conditions, followed by in-gel spontaneous reaggregation of protein complexes and identification of the a-amylase inhibitory activity (antizymogram, third dimension) using enzymes from human salivary glands and from the larvae of Tenebrio molitor coleopter (yellow mealworm). Dimeric a-AIs from Triticum aestivum (bread wheat) were observed to exist as heterodimers. The formation of heterodimeric complexes was also confirmed by in vitro reaggregation assays carried out on RP-HPLC purified wheat dimeric a-AIs, and their bioactivity assayed by antizymogram analysis. The present 3-D analytical technique can be exploited for fast, full-fledged identification and characterization of wheat a-AIs.

Expression of α-amylase inhibitors in diploid Triticum species

“…To evaluate the presence of aAI that are not recognized by PABs but that still exert an inhibition activity towards amylases, a reverse zymographic technique was applied (Zoccatelli et al, 2007). The accessions were challenged with human salivary amylase and with a pest amylase and showed that T.b.…”

“…The activity of the potential aAIs present in AA accessions were analyzed by means of a recent technique called anti-zymogram, or reverse zymogram (Zoccatelli et al, 2007). U-PAGE has been selected as separation technique since although the quaternary structure of aAIs is denatured, the tertiary structure is reversibly affected by urea, leading to the restoration of the inhibition activ- ity.…”

“…Two a-amylases were employed: human salivary a-amylase (HSA) from Sigma and T. molitor a-amylase (TMA), prepared as described by Zoccatelli et al (2007) from pest larvae.…”

“…Electrophoresis in the presence of 8 M urea (U-PAGE) was performed as described previously (Zoccatelli et al, 2007) by Mini Protean III apparatus. The running gel (7% polyacrylamide) contained 0.25% gelatinized starch from potato (Sigma, Milano, Italy).…”

Minor Wheat Protein Fractions Analysis by Using Capillary Zone Electrophoresis

“…The efficiency against the amylases from a particular source is correlated with the inhibitor aggregation state and structural features of protein sequences [4]. Based on the degree of polypeptides aggregation, monomeric, homodimeric and heterotetrameric forms have been identified in wheat flour [9]. Due to the related structures of these proteins, efficient separation techniques are crucial to elucidate their nature.…”