The Bowman-Birk inhibitors (BBIs) represent the most widespread class of serine proteinase inhibitors, and are widely found in legume seeds as well as in other legume organs or other plant families. BBIs are generally double-headed and their inhibitory domains are associated primarily with inhibition of the digestive enzymes, trypsin and chymotrypsin. The BBI trypsin inhibitor site has the ability to inhibit animal digestive enzymes and has been associated with the negative effect on bioavailability of dietary proteins and protein digestibility. The role of the trypsin proteinase inhibitors in the plant seems to be related to plant defence from attacks by insects, pathogens and other predators. On the other hand, many reports highlight the involvement of BBI chymotrypsin inhibitor site to prevent or suppress carcinogen-induced transformation in vitro and carcinogenesis in animal model systems. As a result, soybean extracts enriched in BBI have attained investigational new drug status with the US Food and Drug Administration and is being studied in the prevention of cancer. Two BBI gene classes have been reported in lentil, one coding for a trypsin/trypsin inhibitor, the other encoding a trypsin/chymotrypsin inhibitor, the sequence of the latter one being incomplete at the 3' end. In the present study, we report on the isolation of a complete cDNA sequence coding for lentil trypsin/chymotrypsin BBI. Two forms of the inhibitor were identified: a mature form, corresponding to the protein isolated from lentil seeds, and its C-terminal unprocessed form. In order to understand the implications of particular C-terminal amino acid residues for the specificity and potency of inhibition of key target proteases, the two forms were expressed in the methylotrophic yeast Pichia pastoris. After purification, recombinant molecules were analysed by MALDI-TOF mass spectrometry, and their inhibitory activities evaluated, by means of enzymatic assays using specific substrates for trypsin or chymotrypsin. Ki both for trypsin and chymotrypsin were comparable to other Ki observed for BBI proteins. The ability of lentil trypsin/chymotrypsin BBI to modulate the viability of human colorectal adenocarcinoma HT29 cells in vitro was assessed.
Bowman-Birk inhibitors from lentil: heterologous expression, characterization and anti-tumoral properties
Ceci LR;Siciliano RA;Pignone D;Sonnante G
2008
Abstract
The Bowman-Birk inhibitors (BBIs) represent the most widespread class of serine proteinase inhibitors, and are widely found in legume seeds as well as in other legume organs or other plant families. BBIs are generally double-headed and their inhibitory domains are associated primarily with inhibition of the digestive enzymes, trypsin and chymotrypsin. The BBI trypsin inhibitor site has the ability to inhibit animal digestive enzymes and has been associated with the negative effect on bioavailability of dietary proteins and protein digestibility. The role of the trypsin proteinase inhibitors in the plant seems to be related to plant defence from attacks by insects, pathogens and other predators. On the other hand, many reports highlight the involvement of BBI chymotrypsin inhibitor site to prevent or suppress carcinogen-induced transformation in vitro and carcinogenesis in animal model systems. As a result, soybean extracts enriched in BBI have attained investigational new drug status with the US Food and Drug Administration and is being studied in the prevention of cancer. Two BBI gene classes have been reported in lentil, one coding for a trypsin/trypsin inhibitor, the other encoding a trypsin/chymotrypsin inhibitor, the sequence of the latter one being incomplete at the 3' end. In the present study, we report on the isolation of a complete cDNA sequence coding for lentil trypsin/chymotrypsin BBI. Two forms of the inhibitor were identified: a mature form, corresponding to the protein isolated from lentil seeds, and its C-terminal unprocessed form. In order to understand the implications of particular C-terminal amino acid residues for the specificity and potency of inhibition of key target proteases, the two forms were expressed in the methylotrophic yeast Pichia pastoris. After purification, recombinant molecules were analysed by MALDI-TOF mass spectrometry, and their inhibitory activities evaluated, by means of enzymatic assays using specific substrates for trypsin or chymotrypsin. Ki both for trypsin and chymotrypsin were comparable to other Ki observed for BBI proteins. The ability of lentil trypsin/chymotrypsin BBI to modulate the viability of human colorectal adenocarcinoma HT29 cells in vitro was assessed.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
