Unrooted neighbor-joining (NJ) tree depicting the phylogenetic relationship of <i>Mo</i>-CBP₃–3 with representative members of the prolamin superfamily.

<p>The amino acid sequences of the small and large chains of <i>Mo</i>-CBP₃–3 (this work) were concatenated and aligned to the corresponding sequences of representative non-specific type 1 LTP (nsLTP1), type 2 LTP (nsLTP2), alpha-amylase inhibitors and 2S albumins. The evolutionary distances were computed using the Poisson correction method, and the units are the number of amino acid substitutions per site. The percentages of replicate trees in which the associated sequences clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The PDB codes or GenBank accession numbers of the sequences used are shown.</p

Multiple alignment of the amino acid sequences of a segment of the <i>Mo</i>-CBP₃ precursors with the polypeptide chains of MO2X and cMoL.

<p>The amino acid sequences of a segment of the precursors of <i>Mo</i>-CBP₃ were aligned to the primary structures of MO2X (GenBank accession number P24303) and cMoL [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119871#pone.0119871.ref065" target="_blank">65</a>] using Clustal Omega. Positions containing the same residue in at least 4 sequences are shaded and the Cys residues are highlighted in yellow. Sites containing residues with side chains that have strongly (:) or weakly (.) similar properties, scoring > 0.5 and ≤ 0.5 in the Gonnet PAM 250 matrix [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119871#pone.0119871.ref093" target="_blank">93</a>], respectively, are also indicated. The linker peptide C-terminal end (LPCTE), the large chain and the C-terminal extension (CTE) of the <i>Mo</i>-CBP₃ precursors are labeled. The putative processing site of the CTE is indicated by a red triangle, whereas the N-terminal residue of the large chain of <i>Mo</i>-CBP₃, as identified by Edman degradation, is indicated by a blue triangle. The numbers of the <i>Mo</i>-CBP₃ residues relative to Met¹ are shown on the right side of each sequence. The alignment was edited using the program ALINE [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119871#pone.0119871.ref094" target="_blank">94</a>].</p

General features of the cDNA sequences encoding <i>Mo</i>-CBP₃.

<p>¹ Partial 3′ UTR sequence</p><p>² Not determined</p><p>General features of the cDNA sequences encoding <i>Mo</i>-CBP₃.</p

Amino acid sequences of <i>Mo</i>-CBP₃ peptides identified by LC-ESI-MS/MS from in-gel tryptic digestions of protein bands separated by tricine-SDS-PAGE.

<p>¹Score-values calculated by Mascot [Score = −10 × Log (<i>p</i>)] express the probability <i>p</i> that a match of calculated and experimental mass is by chance; a score of 30, for example, accounts for <i>p</i> ≤ 0.001</p><p>²The numbers before and after each sequence indicate the residue positions (relative to Met¹) in the corresponding preprosequences (the accession numbers of these sequences are shown in the last column); underlined residues are modified as shown in the column on the right</p><p>³Small and large chains are indicated by S and L, respectively, and these chains correspond to the protein bands with apparent molecular masses of 4.1 and 8.1 kDa as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119871#pone.0119871.g006" target="_blank">Fig. 6</a>; sequence coverage is the percentage of the corresponding chains covered by matching peptides and is indicated in parenthesis</p><p>Amino acid sequences of <i>Mo</i>-CBP₃ peptides identified by LC-ESI-MS/MS from in-gel tryptic digestions of protein bands separated by tricine-SDS-PAGE.</p

Tricine-SDS-polyacrylamide gel electrophoresis of <i>Mo</i>-CBP₃.

<p><i>Mo</i>-CBP₃ was purified from <i>M</i>. <i>oleifera</i> seeds using affinity chromatography on a chitin matrix followed by cation exchange chromatography as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119871#pone.0119871.ref008" target="_blank">8</a>]. Protein bands were resolved by tricine-SDS-PAGE (17.5% polyacrylamide) and stained with Coomassie Brilliant Blue as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119871#sec002" target="_blank">methods</a> section. Lane 1: molecular weight markers. Lane 2: <i>Mo</i>-CBP₃ treated with β-mercaptoethanol (10 μg per lane).</p

Predicted secondary structures of the 5′ UTR and 3′ UTR sequences of the <i>Mo-CBP</i>₃ mRNAs.

<p>A. The predicted MFE secondary structures of the entire 5′ UTR and the first 32 nt of the CDS of the <i>Mo-CBP</i>_<i>3</i>–<i>2</i>, <i>Mo-CBP</i>_<i>3</i>–<i>3</i> and <i>Mo-CBP</i>_<i>3</i>–<i>4</i> mRNAs are shown. B. The predicted MFE secondary structures of the entire 3′ UTRs of the <i>Mo-CBP</i>_<i>3</i>–<i>2</i>, <i>Mo-CBP</i>_<i>3</i>–<i>3</i> and <i>Mo-CBP</i>_<i>3</i>–<i>4</i> mRNAs are shown. Heat color gradation from blue to red represents the base-pairing probability from 0 to 1.</p

Amino acid sequences of <i>Mo</i>-CBP₃ peptides identified by LC-ESI-MS/MS from in-solution tryptic digestions of the purified protein.

<p>¹Score-values calculated by Mascot [Score = −10 × Log (<i>p</i>)] express the probability <i>p</i> that a match of calculated and experimental mass is by chance; a score of 30, for example, accounts for <i>p</i> ≤ 0.001</p><p>²The numbers before and after each sequence indicate the residue positions (relative to Met¹) in the corresponding preprosequences (the accession numbers of these sequences are shown in the last column); underlined residues are modified as shown in the column on the right</p><p>³Small and large chains are indicated by S and L, respectively, and these chains correspond to the protein bands with apparent molecular masses of 4.1 and 8.1 kDa as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119871#pone.0119871.g006" target="_blank">Fig. 6</a>; sequence coverage is the percentage of the corresponding chains covered by matching peptides and is indicated in parenthesis</p><p>Amino acid sequences of <i>Mo</i>-CBP₃ peptides identified by LC-ESI-MS/MS from in-solution tryptic digestions of the purified protein.</p

Multiple alignment of the amino acid sequences of the <i>Mo</i>-CBP₃ precursors with proMabinlin-II.

<p>The amino acid sequences of the precursors of <i>Mo</i>-CBP₃ were aligned to the sequence of proMabinlin-II (GenBank accession number P30233) using Clustal Omega. Positions containing the same residue in at least 3 sequences are shaded, and the Cys residues are highlighted in yellow. Sites containing residues with side chains that have strongly (:) or weakly (.) similar properties, scoring > 0.5 and ≤ 0.5 in the Gonnet PAM 250 matrix [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119871#pone.0119871.ref093" target="_blank">93</a>], respectively, are also indicated. The α-helices of the small and large chains of Mabinlin-II (PDB code 2DS2) are shown as cylinders. The N-terminal extension (NTE), the linker peptide (LP) and the C-terminal extension (CTE) of the proMabinlin-II are labeled. The processing sites in the proMabinlin-II sequence are indicated by red triangles, whereas the N-terminal residue of the large chain of <i>Mo</i>-CBP₃, as identified by Edman degradation, is indicated by a blue triangle. The numbers of the residues relative to Met¹ are shown on the right side of each sequence. The alignment was edited using the program ALINE [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119871#pone.0119871.ref094" target="_blank">94</a>].</p

Structural properties of <i>Mo</i>-CBP₃.

<p>(A) Circular dichroism spectra (Far-UV) of <i>Mo</i>-CBP₃ (2.22 mM) in 20 mM sodium phosphate buffer, pH 7.0, using a rectangular quartz cuvette with a 0.1 cm path length. (B) Denaturing polyacrilamide gel electrophoresis (SDS-PAGE - 15% acrylamide gel) of <i>Mo-</i>CBP₃. Molecular mass standards are shown (in kDa) on the left; Lanes 1 and 2, <i>Mo-</i>CBP₃ (20 µg) in reducing (4 kDa and 8 kDa subunits) and non-reducing conditions (18 kDa), respectively.</p

Alignments of the amino acid sequences of the small (A) and large (B) chains of <i>Mo</i>-CBP₃–3 to the corresponding sequences of representative 2S albumins.

<p>The sequences of the small and large chains of <i>Mo</i>-CBP₃–3 were deduced from the cognate cDNA (this work). All the other sequences were retrieved from the GenBank database and are as follows (source species and sequence accession numbers are shown in parenthesis): Mabinlin-II (<i>Capparis masaikai</i>; P30233), Sesa1 (<i>Arabidopsis thaliana</i>; P15457), Napin-2 (<i>Brassica napus</i>; P01090), Sin a 1 (<i>Sinapis alba</i>; P15322), Napin-1A (<i>B</i>. <i>napus</i>; P24565), Ric c 3 (<i>Ricinus communis</i>; P01089), Ber e 1 (<i>Bertholletia excelsa</i>; P04403), Ric c 1 (<i>R</i>. <i>communis</i>; P01089), and Gm2S-1 (<i>Glycine max</i>; P19594). The alignments were performed using the program Clustal Omega. Positions containing the same residue in at least 6 sequences are shaded, and the Cys residues are highlighted in yellow. Sites containing residues with side chains that have strongly (:) or weakly (.) similar properties, scoring > 0.5 and ≤ 0.5 in the Gonnet PAM 250 matrix [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119871#pone.0119871.ref093" target="_blank">93</a>], respectively, are also indicated. The numbers of the residues relative to Met¹ are shown on the right side of each sequence. The alignment was rendered using the program ALINE [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119871#pone.0119871.ref094" target="_blank">94</a>].</p