Identification in Lupin Seed of a Serine-Endopeptidase Activity Cleaving between Twin Arginine Pairs and Causing Limited Proteolysis of Seed Storage Proteins

Abstract

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.

Isolation of a thiol-dependent serine protease in peanut and investigation of its role in the complement and the allergic reaction

“…In our case, the active 30 kDa protease corresponds to an intermediate size between the 38.9 kDa (with propeptide) of the cysteine protease papain from Carica papaya (O'Hara et al, 1995) and the 24.3 kDa of the serine protease trypsin (Mitsuru et al, 1986). Recently, a serine endopeptidase activity was associated to ␥-conglutin enriched fractions of Lupus alba (lupine) which is also a member of the Fabaceae family (Magni et al, 2012). The ␥-conglutin is a lupin non-storage protein composed of two subunits of 30 and 16 kDa respectively.…”

Proteolysis of the peanut allergen Ara h 1 by an endogenous aspartic protease

“…Our suggested site for initiation of proteolysis in the linker region between the two cupin domains was also a cleavage site for or early mobilization of 7S globulins of soybean (Seo et al 2001), common bean (Zakharov et al 2004), pea (Gatehouse et al 1983), and lupin (Magni et al 2012).The similarities in cleavage sites, however, do not extend to the type of proteolytic enzyme that catalyzes the reactions, which were legumain-like or papainlike cysteine proteases in soybean and common bean and a serine protease in the lupin.…”

Proteolytic Cleavage at Twin Arginine Residues Affects Structural and Functional Transitions of Lupin Seed 11S Storage Globulin

“…Whether such specific cleavages take place in the plant kingdom, as they do in mammalian cells, leading to the liberation of biologically active peptides is not known yet, though the hypothesis of the role of adjacent basic residues as endopeptidase prominent recognition sites in seed proteins has already been put forward [ 3 ] and it is definitely intriguing. Several legume seed storage proteins of both the 7S and 11S globulin families contain twin arginine motifs in their amino acid sequences [ 1 ], and the existence of a -R-R- endopeptidase further supports this hypothesis.…”

“…Despite a long history of investigations on legume seed storage proteins, our knowledge of the structural and functional features of this class of proteins is still incomplete and research continues to reveal new and intriguing properties. Among them, the frequent presence of twin and multiple arginine residues in the amino acid sequence of several storage proteins has been proposed as a cleavage-prone site for selective degradation [ 1 ]. A white lupin seed endopeptidase with specificity for peptide bonds containing double arginine (-R-R-) residues and active in vitro on selected storage proteins has been identified [ 1 ].…”

“…Among them, the frequent presence of twin and multiple arginine residues in the amino acid sequence of several storage proteins has been proposed as a cleavage-prone site for selective degradation [ 1 ]. A white lupin seed endopeptidase with specificity for peptide bonds containing double arginine (-R-R-) residues and active in vitro on selected storage proteins has been identified [ 1 ]. Although this activity is quite unusual in plants, in that it resembles the pro-protein conversion of some animal peptide hormones and secretory proteins [ 2 ], no attempts to investigate the potential structural and functional effects of this specific hydrolytic activity on seed storage proteins have been undertaken so far.…”