Bowman-Birk inhibitors in lentil: Heterologous expression,functional characterisation and anti-proliferative properties in human colon cancer cells |
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Authors: | Pasqua Caccialupi Luigi R Ceci Rosa A Siciliano Domenico Pignone Alfonso Clemente Gabriella Sonnante |
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Affiliation: | 1. Institute of Plant Genetics, CNR, Via Amendola, 165/A, 70126 Bari, Italy;2. Institute for Biomembranes and Bioenergetics, CNR, Via Amendola, 165/A, 70126 Bari, Italy;3. Institute of Food Science, CNR, Via Roma, 64, 83100 Avellino, Italy;4. Estación Experimental del Zaidín, CSIC, Profesor Albareda 1, 18008 Granada, Spain |
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Abstract: | A full-length cDNA, encoding a Bowman-Birk protease inhibitor (BBI), was isolated from lentil immature seeds. The deduced amino acid sequence was longer than that of the BBI extracted from lentil seeds and contained two binding sites; the first inhibitory site inhibits trypsin whereas the second one inhibits chymotrypsin. In order to characterize this lentil BBI, a longer (complete) and its C-terminally processed (mature) form were heterologously expressed in the yeast Pichia pastoris. The recombinant BBI proteins proved to be active against trypsin and chymotrypsin, showing Ki values at nanomolar levels. Mass spectrometry analysis revealed that complete BBI was composed of an array of molecular masses, whereas mature BBI showed the presence of a major peak of the expected size. The effects of mature BBI on the growth of human colon adenocarcinoma HT29 and colonic fibroblast CCD-18Co cells were evaluated. Lentil BBI was able to inhibit the growth of such cells at concentrations higher than 19 μM, in a concentration-dependent manner; by contrast, the CCD18-Co cells were unaffected. These data broaden our knowledge of the beneficial biological activities of naturally-occurring BBI proteins and address the need for systematic evaluation of natural variants in order to design novel strategies in preventive medicine. |
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Keywords: | Bowman-Birk inhibitors Cell proliferation Colorectal cancer cells Heterologous expression Lentil Pichia Protease inhibitory activity |
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