Precursor genes of Bowman-Birk-type serine proteinase inhibitors comprise multiple inhibitory domains to promote diversity

Proteinase inhibitors are important for the regulation of the activity of enzymes essential for the survival and maintenance of all organisms, and they may hold medicinal and agricultural value. Hyacinthus orientalis L. serine protease inhibitors (HOSPIs), belonging to the Bowman–Birk type inhibitor (BBI) family, have strong inhibitory activities against mammalian serine proteinases. This study explored the relationship between gene structure and multiple isoinhibitor production of these diversified BBIs by analyzing sequences of HOSPI precursor genes. Genomic DNA of H. orientalis roots was obtained and fragmented using 13 specific restriction enzymes, which were amplified by inverse and nested polymerase chain reactions, cloned into the pBluescript II SK (+) vector, and directly sequenced using specific primers. HOSPI gene and protein expression were assessed by quantitative real-time PCR and western blot, respectively. Proteinase inhibitory activity of hyacinth bulb extracts was evaluated by fluorescein isothiocyanate-labeled casein. Four distinct HOSPI precursor genes were identified, encoding 2–4 different HOSPI domains that were surrounded by additional sequences (named head, linker, and tail sequences) and some introns. Moreover, 3′ splicing of the linker sequence may occur through introns inserted between linker sequences. HOSPI gene and protein expression was higher during the stem elongation and the flowering periods. These results indicate that gene duplication of the HOSPI precursor as a single set, including tandem repeated HOSPI domains, leads to diversity and effective production of mature HOSPIs by posttranslational processing. These findings shed light on the diversity of proteinase inhibitors. •Herein, Hyacinthus orientalis serine protease inhibitors (HOSPIs) were evaluated.•HOSPI precursors have coding regions that contain 4–2 different inhibitory domains.•Additional head, linker, and tail sequences connect these HOSPI domains.•Introns may occur between the linkers, introducing alternative splicing sites to HOSPI.•These findings shed light into the diversity of protease inhibitors diversity.