Identification of soybean peptide leginsulin variants in different cultivars and their insulin-like activities

Tsutomu Hashidume,Sohei Ito,Ayaka Mochizuki,Yasuaki Kawarasaki,Noriyuki Miyoshi,Keisuke Ito,Taiken Sakano

doi:10.1038/s41598-018-35331-5

Abstract

We have recently reported that green soybean cultivar, echigomidori, and not the yellow cultivar, fukuyutaka, is a rich source of hormone-like peptide leginsulin consisting of 37 amino acids (Leg_1_37, PDB 1JU8A) and its C-terminal glycine deletant, Leg_1_36. Green soybean is mature, but the color of the seedcoat and cotyledon remains green. Therefore, in this study, we examined the leginsulin content in different varieties of 11 colored soybeans (including green, yellow, red, brown and black) and edamame (immature soybean). Profile analysis of soybean constituents by LC-MS showed that Leg_1 (36 + 37) detected as a prominent peak in 3 green and 1 yellow soybean cultivar was the strongest contributor in principal component analysis, indicating Leg_1 is the most characteristic feature for distinguishing soybean cultivars. However, smaller amounts of leginsulin-like peptides, defined as Leg_2 and Leg_3, were detected in other samples. The cDNA sequences and LC-MS/MS analyses revealed that Leg_2 was a homologue of Leg_1 with three amino acid substitutions derived from SNPs, while Leg_3 was a Leg_1/Leg_2 paralog. Expression levels of Leg_1 were markedly higher than Leg_2 and Leg_3. Additionally, in glucose uptake assay, purified TRX-His-tag fused recombinant Leg_1_37 prepared by bacterial expression showed stronger insulin-like activities than other variants including Leg_2, Leg_3, and their Gly deletants in myotube-like differentiated L6 and C2C12 cells. These results suggest that dietary consumption of soybean seed, especially including a higher amount of Leg_1_37, could be useful for lowering of blood glucose.

Highlights

IntroductionLeginsulin (PDB 1JU8A), a peptide consisting of 37 amino acids (leginsulin variant 1 consists of 37 amino acids; Leg_1_37) and its C-terminal glycine deletion form (Leg_1_36), have been found in radicles of germinated soybean (Glycine max) seeds as a high affinity molecule for 43 kDa basic 7 S globulin protein (Bg)[1]
Leginsulin (PDB 1JU8A), a peptide consisting of 37 amino acids and its C-terminal glycine deletion form (Leg_1_36), have been found in radicles of germinated soybean (Glycine max) seeds as a high affinity molecule for 43 kDa basic 7 S globulin protein (Bg)[1]
We have previously performed LC-MS analysis of green soybean seed extract to detect leginsulin, which consists of 37 amino acids and corresponds to PDB 1JU8A (Leg_1_37) and its C-terminal glycine deletant (Leg_1_36)[11]

Summary

Introduction

Leginsulin (PDB 1JU8A), a peptide consisting of 37 amino acids (leginsulin variant 1 consists of 37 amino acids; Leg_1_37) and its C-terminal glycine deletion form (Leg_1_36), have been found in radicles of germinated soybean (Glycine max) seeds as a high affinity molecule for 43 kDa basic 7 S globulin protein (Bg)[1]. One of the homologous peptide, PA1bF, sharing 59% sequence identity with Leg_1 (Fig. 1), which shows strong insecticidal activity in several insect pests[9,10]. These findings indicate that the discriminative bioactivities of leginsulin and its homologue would be rather depending on the variation of their amino acid sequences. Six recombinant leginsulin (36 or 37 amino acid peptide of Leg_1~3, respectively) were prepared in this study, which showed insulin-like activity in myotube-like differentiated rat L6 and mouse C2C12 cells

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Conclusion