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

• Published in: Plant physiology and biochemistry Vol. 57; pp. 45 - 53
• Main Authors: De Paola, Domenico, Blanco, Emanuela, Pierri, Ciro Leonardo, Sonnante, Gabriella
• Format: Journal Article
• Language: English
• Published: Paris Elsevier Masson SAS 01-08-2012; Elsevier
• Subjects: Biological and medical sciences; Bowman–Birk isoforms; Comparative modeling; Fundamental and applied biological sciences. Psychology; Grass pea; Inhibitory properties; Lathyrus - genetics; Lathyrus - metabolism; Lathyrus sativus; Phylogeny; Pichia - genetics; Pichia - metabolism; Plant physiology and development; Plant Proteins - classification; Plant Proteins - genetics; Plant Proteins - metabolism; Proteolytic enzymes; Reactive site; Recombinant proteins; Trypsin Inhibitor, Bowman-Birk Soybean - classification; Trypsin Inhibitor, Bowman-Birk Soybean - genetics; Trypsin Inhibitor, Bowman-Birk Soybean - metabolism; HLE; Comparative modeling; Reactive site; NR; BApNA; SAAVpNA; Recombinant proteins; RDA; LCTI; DMEM; BBIs; MD; MSA; PIs; Grass pea; BMG; gDNA; BMGY; BMM; Proteolytic enzymes; BMMY; BTEE; FBBS; Bowman–Birk isoforms; APNE; SAAPLpNA; Inhibitory properties; Site; Properties; Modeling; Characterization; Genetic code; Dicotyledones; Angiospermae; Recombinant protein; Enzyme; Lathyrus sativus; Bowman-Birk isoforms; Variant; Grain legume; Peptidases; Leguminosae; Plant physiology; Hydrolases; Isolation; Spermatophyta; Comparative study
• ISSN: 0981-9428; 1873-2690
• DOI: 10.1016/j.plaphy.2012.05.001

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 P1. A more variable second binding loop was observed allowing to identify three groups based on the identity of residue P1: 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 P1 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.
► We describe a set of full length Bowman–Birk sequences from grass pea. ► Most variation is observed at the second active site. ► Recombinant BBIs are inhibitors of trypsin; three of them also of chymotrypsin, and one inhibits elastase. ► Modeling and docking deeply analyze interactions between BBIs and proteases.