Exploiting molecular diversity in legume Bowman-Birk inhibitors to investigate their potential role as colorectal chemopreventive agents

???????Recent studies suggest that Bowman-Birk inhibitors (BBI) from legumes, such as soybean, pea, lentil and chickpea, could exert preventive and/or suppressive effects on carcinogenic and inflammatory disorders within the gastrointestinal tract.  Physiologically relevant amounts of BBI can reach the large intestine in active form due to the resistance of these proteins to extreme conditions, including acidic pH, action of digestive enzymes, and the proteolytic and metabolic activity of intestinal microbiota.  We have investigated the effect of a pea albumin extract enriched in BBI in the dextran sodium sulphate (DSS) induced colitis model in mice; the BBI-rich albumin preparation ameliorated DSS-induced damage in rodents significantly.  In addition, we demonstrated a significant concentration- and time-dependent decrease in the proliferation of human colorectal cancer cells, following treatment with BBI natural variants from pea, lentil and soybean.  In order to investigate the relationship between protease inhibitory properties, protein structure and possible health beneficial effects of BBI within the gastrointestinal tract, we have engineered a set of BBI mutants using a major pea BBI isoinhibitor, TI1, having both trypsin and chymotrypsin inhibitory activity, as a template.  An inactive TI1 variant as well as modified TI1 variants, having trypsin or chymotrypsin inhibitory activity only, were generated.  Although all the BBI variants were internalised by cultured HT29 human colorectal cancer cells, only those with enzyme inhibitory activity decreased HT29 cell growth in a dose-dependent manner.  Studies are in progress to identify the HT29 trypsin- and chymotrypsin-like proteases that may present as potential therapeutic targets of BBI proteins.