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

Abstract

The 11S storage globulin of white lupin seeds binds to a metal affinity chromatography matrix. Two unusual stretches of contiguous histidine residues, reminiscent of the multiple histidines forming metal binding motifs, at the C-terminal end of 11S globulin acidic chains were hypothesized as candidate elements responsible for the binding capacity. To prove this, the protein was incubated with a lupin seed endopeptidase previously shown to cleave at twin arginine motifs, recurrent in the sequence region of interest. Upon incubation with this enzyme, the loss of metal binding capacity paralleled that of the anti-his-tag reactive polypeptides. The recovered small proteolytic fragment was analyzed by mass spectrometry and N-terminal sequencing and found to correspond to the 24-mer region cleaved off at twin arginine residues and containing the natural his-tag-like region. Similarly, when lupin seeds were germinated for a few days, the his-tag containing 11S globulin chain was converted to a form devoid of such region, suggesting that this mechanism is a part of the natural degradatory process of the protein. The hypothesis that the ordered and controlled dismantling of storage proteins may generate peptide fragments with potential functional roles in plant ontogenesis is presented and discussed.

Cysteine-containing peptides are produced by sequential clipping, but not released, from lupin 11S storage globulin during early germination

“…It has been previously shown that the first step of the lupin legumin degradation is the selective removal of a peptide of about 3 kDa located at the surface of the protein, due to the action of a highly specific protease cutting at a double arginine sequence. The core of this peptide is formed by two consecutive stretches, the first made of six histidine residues and the second of nine glutamic acids [7]. The loss of this peptide apparently did not cause major structural variations in the compactness of the legumin, as evidenced by the chromatographic determination shown in Figure 1B.…”

“…Due to its peculiar amino acid composition, this peptide is likely located at the surface of the native protein, being charged negatively at physiological pH. It has been postulated that it may be involved in metal binding, acting as a carrier for divalent ions [7], once it is released from the "parental" protein.…”

“…Studies based on sequence alignment reveal that most plant cupins contain a single metal-binding site, characterized by two conserved histidines and a glutamic acid, able to bind manganese ions [15]. Although legumins are not the most important proteins with regard to metal-binding capacity, the lupin one presents some molecular features that indicate potential metal-binding sites other that the mentioned histidine/glutamate-rich peptide [7]. Lupin legumin (Figure 3) shows a conserved cupin metal-binding site located close to one cysteine in the major subunit, in which the second hystidine is substitute by a lysine.…”

“…Purification of lupin 11S globulin (legumin) from mature and germinated seeds was performed using a previously described procedure based on anion exchange chromatography separation [7]. To prevent hydrolysis during the procedure, proteins were extracted in the presence of a protease inhibitors cocktail.…”

A peptide that regulates metalation and function of theArabidopsisethylene receptor

“…3e). It is possible that the three arginines (amino acids 10-12) may represent a cleavage site ( 37, 38 ) which would produce the observed shorter GFP fusion protein detected in co-IP experiments (Fig. 3e).…”