The unique biosynthetic route from lupinus beta-conglutin gene to blad

Abstract

Background: During seed germination, beta-conglutin undergoes a major cycle of limited proteolysis in which many of its constituent subunits are processed into a 20 kDa polypeptide termed blad. Blad is the main component of a glycooligomer, accumulating exclusively in the cotyledons of Lupinus species, between days 4 and 12 after the onset of germination.

Principal findings: The sequence of the gene encoding beta-conglutin precursor (1791 nucleotides) is reported. This gene, which shares 44 to 57% similarity and 20 to 37% identity with other vicilin-like protein genes, includes several features in common with these globulins, but also specific hallmarks. Most notable is the presence of an ubiquitin interacting motif (UIM), which possibly links the unique catabolic route of beta-conglutin to the ubiquitin/proteasome proteolytic pathway.

Significance: Blad forms through a unique route from and is a stable intermediary product of its precursor, beta-conglutin, the major Lupinus seed storage protein. It is composed of 173 amino acid residues, is encoded by an intron-containing, internal fragment of the gene that codes for beta-conglutin precursor (nucleotides 394 to 913) and exhibits an isoelectric point of 9.6 and a molecular mass of 20,404.85 Da. Consistent with its role as a storage protein, blad contains an extremely high proportion of the nitrogen-rich amino acids.

Figure 1. Comparison of β-conglutin precursor with the vicilins from other legume species.

(A) Multiple sequence alignment of predicted amino acid sequences of Lupinus albus β-conglutin precursor with other legume vicilins. In the β-conglutin sequence, the small arrowhead designates the putative cleavage of the signal peptide; larger closed arrowheads denote the N- and C- terminals of blad; larger open arrowheads denote the beginning and the end of the UIM; potential N-glycosylation and phosphorylation sites are underlined by thick and thin lines, respectively. The asterisks indicate that the residues in column are identical in all sequences in the alignment; colons indicate conservative substitutions; full stops mean that semi-conserved substitutions are observed. Hyphens are inserted to optimize the alignments. LUPINUS- Lupinus albus; V.FABA- Vicia faba; LENS- Lens culinaris; PISUM- Pisum sativum; V.NARBORENSIS- Vicia narborensis, GLYCINE- Glycine max; PHASEOLUS- Phaseolus vulgaris; CANAVALIA- Canavalia gladiata; MEDICAGO- Medicago truncatula; JUGLANS- Juglans nigra; ARACHIS- Arachis hypogaea. (B) Phylogram of vicilin-like proteins with high homology to β-conglutin from Lupinus albus. The following proteins were included in the phylogenetic analyses: Vicia faba (vicilin precursor, CAA68525), Lens culinaris (allergen Len c, CAD87731), Pisum sativum (vicilin, CAA32239), Vicia narbonensis (vicilin precursor, CAA96514), Glycine max (alpha' subunit of beta-conglycinin, BAE02726), Phaseolus vulgaris (phaseolin, AAC04316), Canavalia gladiata (canavalin, CAA33172), Medicago truncatula (cupin, ABD28364), Juglans nigra (vicilin, AAM54366) and Arachis hypogaea (allergen Ara h 1, P43237).

Figure 2. One-dimensional, structural analysis of β-conglutin from Lupinus albus, Lupins angustifolius, Lupinus luteus and Lupinus mutabilis.

L. albus (A), L. angustifolius (B), L. luteus (C) and L. mutabilis (D) seeds were germinated for up to 10 days, and β-conglutin extracted, purified, analysed by one-dimensional SDS-PAGE and stained for total protein. Seeds were germinated for the number of days indicated on top of the gels. Fifty µg of protein were loaded in each lane. Molecular masses of standards are indicated in kDa.

Figure 3. Two-dimensional, structural analysis of β-conglutin and blad from Lupinus albus.

β-Conglutin (A,C) and the protein containing blad (B) were extracted, purified from the cotyledons of dry seeds and eight-days germinated seedlings, respectively, and subjected to two-dimensional electrophoresis. The gels were either stained for total protein (A,B) or transferred onto a membrane and probed with anti-blad antibodies (C). Two hundred µg of protein were used to prepare the 2D-gels (A,B) and 50 µg to prepare the 2D-blot (C). Lanes a: molecular mass standards (kDa) (precision protein standards prestained, broad range, Bio-Rad, in C). Lanes b: β-conglutin (A, 50 µg; C, 15 µg) or the native protein containing blad (B, 50 µg).

Figure 4. Agarose gel showing the products obtained when the mRNA fragment (lane 1) or the gene fragment (lane 2) were amplified using primers specific for both terminals of blad.

The size of markers is indicated on the left.