Isolation and characterization of novel variants of BBI coding genes from the legume Lathyrus sativus.

A pool of twelve cDNA sequences coding for Bowman-Birk inhibitors (BBIs) was identified in the legume grass pea (Lathyrus sativus L.). The corresponding amino acid sequences showed a canonical first anti-trypsin domain, predicted according to the identity of the determinant residue P(1). A more variable second binding loop was observed allowing to identify three groups based on the identity of residue P(1): two groups (Ls_BBI_1 and Ls_BBI_2) carried a second reactive site specific for chymotrypsin, while a third group (Ls_BBI_3) was predicted to inhibit elastase. A fourth variant carrying an Asp in the P(1) position of the second reactive site was identified only from genomic DNA. A phylogenetic tree constructed using grass pea BBIs with their homologs from other legume species revealed grouping based on taxonomy and on specificity of the reactive sites. Five BBI sequences, representing five different second reactive sites, were heterologously expressed in the yeast Pichia pastoris. The recombinant proteins demonstrated to be active against trypsin, while three of them were also active against chymotrypsin, and one against human leukocyte elastase. Comparative modeling and protein docking were used to further investigate interactions between two grass pea BBI isoforms and their target proteases. Thus two reliable 3D models have been proposed, representing two potential ternary complexes, each constituted of an inhibitor and its target enzymes.

Novel alleles among soybean Bowman-Birk proteinase inhibitor gene families.

Trypsin inhibitors have been found in various animals, plants and microorganisms. There were two types of trypsin inhibitors in soybean including Bowman-Birk protease inhibitors (BBI) and Kunitz inhibitors (KTI). The different BBI genes from wild soybean (G. soja) and cultivated soybean (G. max) formed a multigene family. We constructed a cDNA library of cultivar 'SuiNong 14' seed at the R7 growth stage using the SMART Kit. Seventeen contigs or singletons were highly homologous to soybean protease inhibitors. Contigs of 5, 35, 8 and 9 were highly homologous to BBI family members BBI-A1, BBI-A2, BBI-C and BBI-D, respectively. Sequence analyses showed there were novel allelic variations among the 4 BBI members in SuiNong 14. Based on the comparison of soybean seed cDNA libraries from different developmental stages, it was apparent that the expression of trypsin inhibitors increased during seed development in soybean. Phylogenetic analysis of BBI gene sequences among dicotyledonous and monocotyledonous plants demonstrated that these genes shared a common progenitor.

Molecular evolution of Bowman-Birk type proteinase inhibitors in flowering plants.

The Bowman-Birk family (BBI) of proteinase inhibitors is probably the most studied family of plant inhibitors. We describe the primary structure and the gene expression profile of 14 putative BBIs from the sugarcane expressed sequence tag database and show how we used these newly discovered sequences together with 87 previously described BBI sequences from the GenBank database to construct phylogenetic trees for the BBI family. Phylogenetic analysis revealed that BBI-type inhibitors from monocotyledonous and dicotyledonous plants could be clearly separated into different groups, while the overall topology of the BBI tree suggests a different pattern of evolution for BBI families in flowering plants. We also found that BBI proteinase inhibitors from dicotyledonous plants were well conserved, accumulating only slight differences during their evolution. In addition, we found that BBIs from monocotyledonous plants were highly variable, indicating an interesting process of evolution based on internal gene duplications and mutation events.

On a Bowman-Birk family proteinase inhibitor from Erythrina variegata seeds.

A Bowman-Birk family proteinase inhibitor (EBI) was isolated from the seeds of Erythrina variegata. The protein was purified by ion-exchange column chromatography on DEAE-cellulose followed by gel filtration on Sephadex G-75. The stoichiometry with trypsin was estimated to be 1:1, while that with chymotrypsin was not obvious, as determined from the titration patterns of its inhibitory activities. The complete amino acid sequence of EBI was determined by sequencing tryptic and chymotryptic peptides. The EBI protein consists of 61 amino acid residues, which is the shortest among the Bowman-Birk family inhibitors sequenced to date, and has a M(r) of 6,689. Comparison of this sequence with those of other leguminous Bowman-Birk family inhibitors revealed that EBI could be classified as a group II inhibitor, showing the best homology (67%) to the Bowman-Birk proteinase inhibitor from soybeans.

The Bowman-Birk inhibitor. Trypsin- and chymotrypsin-inhibitor from soybeans.

Four decades of studies on the isolation, characterization, properties, structure, function and possible uses of the Bowman-Birk trypsin- and chymotrypsin-inhibitor from soybeans are reviewed. Starting from Bowman's Acetone Insoluble factor, designated Ai, AA and SBTIAA, the Bowman-Birk inhibitor (BBI) was found to be a protein molecule consisting of a chain of 71 amino acids cross linked by 7 disulfide bonds, with a tendency to self-associate. BBI possesses two independent sites of inhibition, one at Lys 16-Ser 17 against trypsin and the other at Leu 43-Ser 44 against chymotrypsin. It forms a 1:1 complex with either trypsin or chymotrypsin and a ternary complex with both enzymes. Ingestion of BBI by rats, chicks or quails affects the size and protein biosynthesis of the pancreas. Establishment of the full covalent structure of BBI revealed a high homology in the sequences around the two inhibitory sites, suggesting evolutionary gene duplication from a single-headed ancestral inhibitor. Scission of BBI by CNBr followed by pepsin results in two active fragments, one that inhibits trypsin and the other, chymotrypsin. Replacements and substitutions in the reactive sites result in changes in inhibitory activity and in specificity of inhibition. Conformation studies, labeling of BBI with a photoreactive reagent, chemical synthesis of cyclic peptides that include inhibitory sites, in vitro synthesis of BBI, and species specificity regarding the inhibited enzymes are described. The significance of BBI as a prototype of a family of inhibitors present in all legume seeds is discussed.