New molecular features of cowpea bean (Vigna unguiculata, l. Walp) ß-vignin.

Cowpea seed ß-vignin, a vicilin-like globulin, proved to exert various health favourable effects, including blood cholesterol reduction in animal models. The need of a simple scalable enrichment procedure for further studies for tailored applications of this seed protein is crucial. A chromatography-independent fractionation method allowing to obtain a protein preparation with a high degree of homogeneity was used. Further purification was pursued to deep the molecular characterisation of ß-vignin. The results showed: (i) differing glycosylation patterns of the two constituent polypeptides, in agreement with amino acid sequence features; (ii) the seed accumulation of a gene product never identified before; (iii) metal binding capacity of native protein, a property observed only in few other legume seed vicilins.

Structural basis of the lack of endo-glucanase inhibitory activity of Lupinus albus γ-conglutin.

Lupin γ-conglutin and soybean BG7S are two legume seed proteins strongly similar to plant endo-ß-glucanases inhibitors acting against fungal GH11 and GH12 glycoside hydrolase. However these proteins lack inhibitory activity. Here we describe the conversion of lupin γ-conglutin to an active inhibitor of endo-ß-glucanases belonging to GH11 family. A set of γ-conglutin mutants was designed and expressed in Pichia pastoris, along with the wild-type protein. Unexpectedly, this latter was able to inhibit a GH11 enzyme, but not GH12, whereas the mutants were able to modulate the inhibition capacity. In lupin, γ-conglutin is naturally cleaved in two subunits, whereas in P. pastoris it is not. The lack of proteolytic cleavage is one of the reasons at the basis of the inhibitory activity of recombinant γ-conglutin. The results provide new insights about structural features at the basis of the lack of inhibitory activity of wild-type γ-conglutin and its legume homologues.

Identification in lupin seed of a serine-endopeptidase activity cleaving between twin arginine pairs and causing limited proteolysis of seed storage proteins.

The occurrence of twin-arginine motifs (-R-R-) in the amino acid sequences of animal pro-proteins frequently defines the cleavage site(s) for their structural/functional maturation. No information is available on the presence and possible biological meaning of these motifs in the seed storage proteins. In this work, a novel endopeptidase activity with cleavage specificity to twin-arginine pairs has been detected in mature dry Lupinus albus seeds. The endopeptidase was tested with a number of endogenous and exogenous protein substrates, which were selected according to the presence of one or more twin-arginine residue motifs in their amino acid sequences. The observed hydrolysis patterns were limited and highly specific. Partial proteolysis led to stable polypeptide fragments that were characterized by 1- and 2-D electrophoresis. Selected polypeptides were submitted to N-terminal amino acid sequencing and mass spectrometry analyses. These approaches, supported by bioinformatic analysis of the available sequences, allowed the conclusion that the polypeptide cleavage events had occurred at the peptide bonds comprised between twin-arginine residue pairs with all tested protein substrates. The endopeptidase activity was inhibited by 4-(2-AminoEthyl)Benzene-Sulphonyl Fluoride hydrochloride (AEBSF), leupeptin, and serine proteinase protein inhibitors, while it was not affected by pepstatin, trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane (E64), and ethylenediaminetetraacetic acid (EDTA), thus qualifying the Arg-Arg cleaving enzyme as a serine endopeptidase. The structural features of storage proteins from lupin and other legume seeds strongly support the hypothesis that the occurrence of an endopeptidase activity cleaving -R-R- bonds may be functional to facilitate their degradation at germination and possibly generate polypeptide fragments with specific biological activity.

Heterologous expression and purification of the soybean 7S globulin α' subunit extension region: in vitro evidence of its involvement in cell cholesterol homeostasis.

In a previous paper, the biological activity of a 216-amino acid recombinant truncated form of the soybean 7S globulin α' subunit, known to control cholesterol and triglyceride homeostasis, was described. In this work, a shorter version of the polypeptide chain, spanning 142 amino acid residues from the N-terminus and thus exclusively including the so-called extension region, was cloned and overexpressed in Pichia pastoris. The yield of the recombinant polypeptide, which was termed α'E, was 8-fold greater than the previous truncated version. The α'E polypeptide was purified by simple conventional biochemical techniques to make it available for biological assays. Human hepatoma cell lines (Hep G2) were used to monitor the uptake and degradation of labeled low-density lipoproteins (LDL), according to an established procedure. The LDL uptake (+86%) and degradation (+94%) by cells tested at the highest α'E dose (2 µM) were similar to those found in cells incubated with 1 µM simvastatin, a potent inhibitor of cholesterol biosynthesis. Additionally, the cell response to α'E was found to be dose-dependent. The present findings strongly suggest that this recombinant polypeptide, or a fragment thereof, is the molecular determinant for cholesterol homeostasis and open new prospects for understanding the mechanism involved in this biological response, as a gateway to its utilization in lipid-lowering therapies.

Cloning, yeast expression, purification and biological activity of a truncated form of the soybean 7S globulin alpha' subunit involved in Hep G2 cell cholesterol homeostasis.

A truncated form of alpha' chain (talpha'), the soybean 7S globulin subunit previously demonstrated to be active in controlling the cholesterol and triglyceride homeostasis in in vitro and in vivo models, was cloned and expressed in the yeast Pichia pastoris. The recombinant polypeptide spanned 216 amino acid residues from the N-terminal side and included the N-terminal extension region of the soybean subunit. The talpha' polypeptide was purified by conventional biochemical techniques, and its potential to modulate the activity of low-density lipoprotein (LDL) receptor was evaluated in a human hepatoma cell line (Hep G2) by monitoring the uptake and degradation of labeled LDL. The LDL uptake (+192%) and degradation (+143%) by cells tested at the highest talpha' dose (8 microM) were similar to those found in cells incubated with 1 microM simvastatin, a potent inhibitor of cholesterol biosynthesis. The cell response to talpha' was found to be dose dependent. The use of a recombinant polypeptide ruled out the involvement of any other soybean component. These findings open new prospects in the studies aimed at identifying soybean regulatory (poly)peptide(s) and the mechanism involved in this biological response, as a gateway to their utilization for the management of human health.

Identification and characterization of a Bowman-Birk inhibitor active towards trypsin but not chymotrypsin in Lupinus albus seeds.

The paper describes the purification, structural characterization and inhibitory properties of a trypsin inhibitor from Lupinus albus L., a leguminous plant believed to be devoid of any protease inhibitor. The protein has been isolated by a newly set-up procedure and characterized by direct amino acid sequencing, MALDI-TOF mass spectroscopy and circular dichroism. Inhibitory properties toward bovine trypsin and chymotrypsin, as well as its thermal and pH stabilities, have been also assessed. The inhibitor is 63 amino acid long (Mr 6858; pI 8.22) and it is capable to inhibit two trypsin molecules simultaneously, with a Kd of 4.2+/-0.4 nM, but not chymotrypsin. BLAST search against UniProtKB/TrEMBL database indicates that the inhibitor belongs to the Bowman-Birk inhibitor (BBI) family. The interest in these serine-protease inhibitors arises from the ability to prevent or suppress carcinogen-induced transformation, as shown in various in vitro and in vivo model systems.

Inhibitory properties and solution structure of a potent Bowman-Birk protease inhibitor from lentil (Lens culinaris, L) seeds.

Bowman-Birk serine protease inhibitors are a family of small plant proteins, whose physiological role has not been ascertained as yet, while chemopreventive anticarcinogenic properties have repeatedly been claimed. In this work we present data on the isolation of a lentil (Lens culinaris, L., var. Macrosperma) seed trypsin inhibitor (LCTI) and its functional and structural characterization. LCTI is a 7448 Da double-headed trypsin/chymotrypsin inhibitor with dissociation constants equal to 0.54 nM and 7.25 nM for the two proteases, respectively. The inhibitor is, however, hydrolysed by trypsin in a few minutes timescale, leading to a dramatic loss of its affinity for the enzyme. This is due to a substantial difference in the kon and k*on values (1.1 microM-1.s-1 vs. 0.002 microM-1.s-1), respectively, for the intact and modified inhibitor. A similar behaviour was not observed with chymotrypsin. The twenty best NMR structures concurrently showed a canonical Bowman-Birk inhibitor (BBI) conformation with two antipodal beta-hairpins containing the inhibitory domains. The tertiary structure is stabilized by ion pairs and hydrogen bonds involving the side chain and backbone of Asp10-Asp26-Arg28 and Asp36-Asp52 residues. At physiological pH, the final structure results in an asymmetric distribution of opposite charges with a negative electrostatic potential, centred on the C-terminus, and a highly positive potential, surrounding the antitryptic domain. The segment 53-55 lacks the anchoring capacity found in analogous BBIs, thus rendering the protein susceptible to hydrolysis. The inhibitory properties of LCTI, related to the simultaneous presence of two key amino acids (Gln18 and His54), render the molecule unusual within the natural Bowman-Birk inhibitor family.