CHOLESTEROL-LOWERING PROPERTY OF A CHROMATIN-BINDING
PEPTIDE DERIVED FROM SOY

ALFREDO F. GALVEZ ^{1, 2, *,} FU CHUNJIANG², JIM PORTER²,
MONTY KERLEY²

1 SOY LABS, LLC, FAIRFIELD, CA; PROJECT SCIENTIST, CENTER OF EXCELLENCE FOR NUTRITIONAL GENOMICS, UNIVERSITY OF CALIFORNIA AT DAVIS

2 DEPARTMENT OF ANIMAL SCIENCE, UNIVERSITY OF MISSOURI, COLUMBIA, MO * CORRESPONDING AUTHOR

ABSTRACT

The American Heart Association recently reported that soy protein lowers LDL cholesterol by only 3%, contrary to the FDA health claim on soy protein. We now report the discovery of a chromatin-binding peptide from soy that could very well be the active factor responsible for the LDL cholesterol-lowering effect attributed to soy proteins and help clarify clinical results summarized in the recent AHA report.

Lunasin is a recently discovered bioactive soy component with a novel chromatin binding property. Lunasin has been shown to significantly reduce the acetylation of histone H3 by the histone acetylase enzyme, PCAF (p300/CBP-associated factor) but not by p300 or HAT1. Transcriptional regulation of HMG CoA reductase and LDL receptor genes by Sterol Regulatory Element Binding Proteins (SREBP) in cholesterol-free media requires the selective recruitment of different co-regulatory factors, CREB/CBP for HMG-CoA reductase and SP1 for LDL receptor. CREB-binding protein (CBP) interacts with the activation domain of SREBP and has been shown to associate with PCAF to specifically acetylate histone H3. The acetylation of histone H3 by PCAF is required for SREBP-controlled transcriptional activation of HMG-CoA reductase and by inhibiting H3 acetylation, lunasin can potentially reduce its expression.

Cell culture of HepG2 liver cells shows that treatment with lunasin can indeed significantly reduce HMG-CoA reductase expression by 50% in cholesterol-free media. In contrast, LDL receptor expression has increased by 60%, which can be explained in part by the coordinate increase in expression of its co-transcriptional activator, Sp1. Also, the inhibition of HMG-CoA reductase expression by lunasin lowers intracellular cholesterol levels that keeps SREBP activated, contributing to the upregulation of LDL receptor expression. Results from RT-PCR experiments show the corresponding reduction of HMG-CoA reductase and the increase of LDL receptor mRNA transcript levels in lunasin-treated, cholesterol-free media, which has also been validated by real-time quantitative RT-PCR. Hence, these studies show that lunasin has the potential to reduce LDL and total cholesterol levels by directly inhibiting gene expression of HMG-CoA reductase, which reduces cholesterol biosynthesis, and by increasing LDL receptor expression, which enhances clearance of plasma LDL cholesterol. The presence of the lunasin peptide in soy protein preparations provides a plausible mechanism of action to explain the cholesterol-lowering effect attributed to soy protein and paves the way for optimizing soy protein ingredients to maximize its heart-healthy benefits.

Lunasin concentration in different soybean genotypes, commercial soy
protein, and isoflavone products.
Elvira Gonzalez de Mejia, Miguel Vásconez, Ben O. de Lumen, and Randall Nelson
J Agric Food Chem. 2004 Sep 22;52(19):5882-7.
Lunasin is a unique and novel cancer preventive peptide originally isolated from soy. Information on lunasin concentration of soybean cultivars and commercial soy proteins would be useful in developing lunasin-enriched cultivars and soy products. We report the development of an enzyme-linked immunosorbent assay (ELISA) method to identify lunasin and quantify the variations in concentration in 144 selected, diverse soybean accessions from the U.S. Department of Agriculture Soybean Germplasm Collection, several commercially available soy protein fractions and isoflavone-enriched products. With synthetic lunasin and monoclonal antibody, ELISA shows a linear concentration range of 24-72 ng/mL, good reproducibility, a detection limit of 8 ng/mL, and a recovery of 90% on spiked soy samples. Lunasin concentrations in the tested materials range from 0.10 to 1.33 g/100 g flour. Differences that exceeded 100% have been observed among accessions of similar maturity that were grown in the same environment, indicating that genetic differences in soybeans exist for lunasin. The mean of 23 major ancestral lines of U.S. cultivars is similar to the mean of 16 modern cultivars selected to represent the current diversity of the crop, but the highest values were found within the ancestral and exotic accessions. Soy protein concentrate, isolate, and hydrolyzate contain 2.81 ± 0.30, 3.75 ± 0.43, and 4.43 ± 0.59 g lunasin/100 g flour, respectively, while soy flour and soy flakes contain 1.24 ± 0.22 g lunasin/100 g flour. Isoflavone-enriched products contain very little or no lunasin. The relative mass (Mr) of lunasin in the samples is 5.45 ± 0.25 kDa. The wide range of lunasin concentrations within the Glycine max species indicates that the levels of this important bioactive peptide can be genetically manipulated. Furthermore, soy isolates and hydrolyzed soy proteins contain the highest concentrations of lunasin.

Characterization of lunasin isolated from soybean.
Hyung J. Jeong, Jae H. Park, Yi Lam, and Ben O. de Lumen
J Agric Food Chem. 2003 Dec 31;51(27):7901-6.
Lunasin is a novel and promising chemopreventive peptide from soybean. We have shown previously that lunasin suppresses transformation of mammalian cells caused by chemical carcinogens and inhibits skin carcinogenesis in mice when applied topically. Although the lunasin gene was cloned from soybean, all experiments carried out so far in our lab have used synthetic lunasin and therefore there is no detailed description of natural lunasin isolated from soybean. We report here the first characterization of soybean lunasin that includes definitive identification by mass peptide mapping, partial purification, and measurement of bioactivities of the various purified fractions and protein expression in the developing seed. The identity of lunasin in the seed extracts was established by Western blot analysis and mass spectrometric peptide mapping. All lunasin fractions partially purified by anion exchange and immunoaffinity column chromatography suppress colony formation induced by the ras-oncogene and inhibit core H3-histone acetylation. During seed development, lunasin peptide appears 5 weeks after flowering and persists in the mature seed. Western blot analysis of different soybean varieties and commercially available soy proteins shows the presence of the peptide in varying amounts. These results demonstrate the feasibility of producing large quantities of natural lunasin from soybean for animal and human studies.

Barley lunasin suppresses ras-induced colony formation and inhibits core
histone acetylation in mammalian cells.
Hyung J. Jeong, Yi Lam, and Ben O. de Lumen
J Agric Food Chem. 2002 Oct 9;50(21):5903-8.
Lunasin is a novel peptide originally identified in soybean that suppresses chemical carcinogen-induced transformation in mammalian cells and skin carcinogenesis in mice. Since the lunasin gene was cloned from soybean and the chemically synthesized form of the lunasin peptide has been used in experiments conducted so far, the isolation of lunasin from other natural sources and testing of its biological properties have not been carried out. We report here the isolation, purification, and biological assay of lunasin from barley, a newly found rich source of the peptide. The identity of lunasin was established by Western blot analysis and mass spectrometric peptide mapping of the in-gel tryptic digest of the putative protein band. Lunasin was partially purified with anion exchange and immunoaffinity chromatography. The crude and partially purified lunasin from barley suppressed colony formation in stably ras-transfected mouse fibroblast cells induced with IPTG. These fractions also inhibited histone acetylation in mouse fibroblast NIH 3T3 and human breast MCF-7 cells in the presence of the histone deacetylase inhibitor sodium butyrate.

A soybean cDNA encoding a chromatinbinding peptide inhibits
mitosis of mammalian cells
Galvez A.F. and de Lumen B.O.
Nature Biotechnology, 1999.17:495-500.
A soybean cDNA encoding the small subunit peptide of a cotyledon-specific 2S albumin (Gm2S-1) is thought to play a role in arresting mitosis during the DNA endoreduplication and cell expansion phase of seed development. The peptide (termed lunasin) contains the cell adhesion motif Arg-Gly-Asp (RGD) followed by eight aspartic acid residues at its C-terminal end. A chimeric gene encoding the lunasin peptide tagged with green fluorescent protein (GFP) arrested cell division, caused abnormal spindle fiber elongation, chromosomal fragmentation, and cell lysis when transiently transfected into murine embryo fibroblast, murine hepatoma, and human breast cancer cells. Deletion of the polyaspartyl end abolished the antimitotic effect. Subcellular localization of lunasin and immunobinding assay using synthetic peptides revealed the preferential adherence of lunasin to chromatin. Immunofluorescence showed that kinetochore proteins were displaced from the centromere in lunasin-transfected cells. These observations suggest that lunasin binds to the chromatin, leading to disruption of kinetochore formation and inhibition of mitosis.