A possible physiological function and the tertiary structure of a 4-kDa peptide in legumes


  • Note: The nucleotide sequences reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession numbers AB052880 and AB052881. The structure reported in this paper has been submitted to the Protein Data Bank with accession number 1JU8, BMRB 5098.

    Note: As the capability of binding of insulin and the 4-kDa peptide to the 43-kDa protein is similar, in our previous paper we named the 4-kDa peptide as leginsulin. But the 4-kDa peptide is not insulin, and one must discriminate between them. To avoid confusion, we use 4-kDa peptide as the name of the peptide instead of leginsulin in this paper.

H. Hirano, Yokohama City University, Kihara Institute for Biological Research/Graduate School of Integrated Science, Totsuka, Yokohama, 244–0813 Japan. Fax: + 81 45 8201901, Tel.: + 81 45 8201904, E-mail: hirano@yokohama-cu.ac.jp


Previously, we isolated a 4-kDa peptide capable of binding to a 43-kDa receptor-like protein and stimulating protein kinase activity of the 43-kDa protein in soybean. Both of them were found to localize in the plasma membranes and cell walls. Here, we report the physiological effects of 4-kDa peptide expressed transiently in the cultured carrot and bird's-foot trefoil cells transfected with pBI 121 plasmid containing the 4-kDa peptide gene. At early developmental stage, the transgenic callus grew rapidly compared to the wild callus in both species. Cell proliferation of in vitro cultured nonembryogenic carrot callus was apparently affected with the 4-kDa peptide in the medium. Complementary DNAs encoding the 4-kDa peptide from mung bean and azuki bean were cloned by PCR and sequenced. The amino-acid sequences deduced from the nucleotide sequences are homologous among legume species, particularly, the sites of cysteine residues are highly conserved. This conserved sequence reflects the importance of intradisulfide bonds required for the 4-kDa peptide to perform its function. Three dimensional structure of the 4-kDa peptide determined by NMR spectroscopy suggests that this peptide is a T-knot scaffold containing three β-strands, and the specific binding activity to the 43-kDa protein and stimulatory effect on the protein phosphorylation could be attributed to the spatial arrangements of hydrophobic residues at the solvent-exposed surface of two-stranded β-sheet of 4-kDa peptide. The importance of these residues for the 4-kDa peptide to bind to the 43-kDa protein was indicated by site-directed mutagenesis. These results suggest that the 4-kDa peptide is a hormone-like peptide and the 43-kDa protein is involved in cellular signal transduction of the peptide.