Fig 1.
Agarose gel electrophoresis of Mo-CBP3 cDNA fragments amplified by PCR.
A. 3′ RACE products (lane 1). B. 5′ RACE products amplified using 3 distinct gene-specific primers (lanes 1, 2 and 3). Lane M (A and B): molecular markers.
Table 1.
General features of the cDNA sequences encoding Mo-CBP3.
Fig 2.
The cDNA sequence and deduced amino acid sequence of preproMo-CBP3–3.
The deduced amino acid sequence of the preproMo-CBP3–3 is shown below the cDNA sequence. Numbers for the first nucleotide and the last amino acid residue in each row are shown on the left and right, respectively. The N-terminal signal peptide, as predicted by the SignalP 4.1 program [27], is shaded in gray. The N-terminal sequence of the large chain of Mo-CBP3, as determined by Edman degradation, is underlined with a dashed line. The stop codon is indicated by an asterisk. The poly(A) signal nearest to the poly(A) tail is boxed, and two other upstream poly(A) signals are underlined.
Fig 3.
Predicted secondary structures of the 5′ UTR and 3′ UTR sequences of the Mo-CBP3 mRNAs.
A. The predicted MFE secondary structures of the entire 5′ UTR and the first 32 nt of the CDS of the Mo-CBP3–2, Mo-CBP3–3 and Mo-CBP3–4 mRNAs are shown. B. The predicted MFE secondary structures of the entire 3′ UTRs of the Mo-CBP3–2, Mo-CBP3–3 and Mo-CBP3–4 mRNAs are shown. Heat color gradation from blue to red represents the base-pairing probability from 0 to 1.
Fig 4.
Unrooted neighbor-joining (NJ) tree depicting the phylogenetic relationship of Mo-CBP3–3 with representative members of the prolamin superfamily.
The amino acid sequences of the small and large chains of Mo-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.
Fig 5.
Multiple alignment of the amino acid sequences of the Mo-CBP3 precursors with proMabinlin-II.
The amino acid sequences of the precursors of Mo-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 [93], 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 Mo-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 [94].
Fig 6.
Tricine-SDS-polyacrylamide gel electrophoresis of Mo-CBP3.
Mo-CBP3 was purified from M. oleifera seeds using affinity chromatography on a chitin matrix followed by cation exchange chromatography as described previously [8]. Protein bands were resolved by tricine-SDS-PAGE (17.5% polyacrylamide) and stained with Coomassie Brilliant Blue as described in the methods section. Lane 1: molecular weight markers. Lane 2: Mo-CBP3 treated with β-mercaptoethanol (10 μg per lane).
Table 2.
Amino acid sequences of Mo-CBP3 peptides identified by LC-ESI-MS/MS from in-gel tryptic digestions of protein bands separated by tricine-SDS-PAGE.
Table 3.
Amino acid sequences of Mo-CBP3 peptides identified by LC-ESI-MS/MS from in-solution tryptic digestions of the purified protein.
Fig 7.
Alignments of the amino acid sequences of the small (A) and large (B) chains of Mo-CBP3–3 to the corresponding sequences of representative 2S albumins.
The sequences of the small and large chains of Mo-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 (Capparis masaikai; P30233), Sesa1 (Arabidopsis thaliana; P15457), Napin-2 (Brassica napus; P01090), Sin a 1 (Sinapis alba; P15322), Napin-1A (B. napus; P24565), Ric c 3 (Ricinus communis; P01089), Ber e 1 (Bertholletia excelsa; P04403), Ric c 1 (R. communis; P01089), and Gm2S-1 (Glycine max; 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 [93], 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 [94].
Fig 8.
Multiple alignment of the amino acid sequences of a segment of the Mo-CBP3 precursors with the polypeptide chains of MO2X and cMoL.
The amino acid sequences of a segment of the precursors of Mo-CBP3 were aligned to the primary structures of MO2X (GenBank accession number P24303) and cMoL [65] 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 [93], respectively, are also indicated. The linker peptide C-terminal end (LPCTE), the large chain and the C-terminal extension (CTE) of the Mo-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 Mo-CBP3, as identified by Edman degradation, is indicated by a blue triangle. The numbers of the Mo-CBP3 residues relative to Met1 are shown on the right side of each sequence. The alignment was edited using the program ALINE [94].