Takayasu Motoyama

Analyzing a dipeptide library to identify human dipeptidyl peptidase IV inhibitor

Human dipeptidyl peptidase IV (hDPPIV) inhibitors provide an effective strategy for the treatment of type 2 diabetes. Because certain peptides are known to act as hDPPIV inhibitors, a dataset of possible peptides with their inhibition intensities will facilitate the development of functional food for type 2 diabetes. In this study, we examined a total of 337 dipeptides with respect to their hDPPIV inhibitory effects. Amino acid residues at N-termini dominated their inhibition intensities. Particularly highly inhibitory dipeptides discovered included the following novel dipeptides: Thr-His, Asn-His, Val-Leu, Met-Leu, and Met-Met. Using our dataset, prime candidates contributing to the hDPPIV inhibitory effect of soy protein hydrolyzates were successfully identified. Possible dietary proteins potentially able to produce particularly highly hDPPIV inhibitory peptides are also discussed on the basis of the dataset.

Analysing the substrate multispecificity of a proton-coupled oligopeptide transporter using a dipeptide library

Peptide uptake systems that involve members of the proton-coupled oligopeptide transporter (POT) family are conserved across all organisms. POT proteins have characteristic substrate multispecificity, with which one transporter can recognize as many as 8,400 types of di/tripeptides and certain peptide-like drugs. Here we characterize the substrate multispecificity of Ptr2p, a major peptide transporter of Saccharomyces cerevisiae, using a dipeptide library. The affinities (Ki) of di/tripeptides toward Ptr2p show a wide distribution range from 48 mM to 0.020 mM. This substrate multispecificity indicates that POT family members have an important role in the preferential uptake of vital amino acids. In addition, we successfully establish high performance ligand affinity prediction models (97% accuracy) using our comprehensive dipeptide screening data in conjunction with simple property indices for describing ligand molecules. Our results provide an important clue to the development of highly absorbable peptides and their derivatives including peptide-like drugs.

Co-expression of soybean glycinins A1aB1b and A3B4 enhances their accumulation levels in transgenic rice seed

The soybean major storage protein glycinin is encoded by five genes, which are divided into two subfamilies. Expression of A3B4 glycinin in transgenic rice seed reached about 1.5% of total seed protein, even if expressed under the control of strong endosperm-specific promoters. In contrast, expression of A1aB1b glycinin reached about 4% of total seed protein. Co-expression of the two proteins doubled accumulation levels of both A1aB1b and A3B4 glycinins. This increase can be largely accounted for by their aggregation with rice glutelins, self-assembly and inter-glycinin interactions, resulting in the enrichment of globulin and glutelin fractions and a concomitant reduction of the prolamin fraction. Immunoelectron microscopy indicated that the synthesized A1aB1b glycinin was predominantly deposited in protein body-II (PB-II) storage vacuoles, whereas A3B4 glycinin is targeted to both PB-II and endoplasmic reticulum (ER)-derived protein body-I (PB-I) storage structures. Co-expression with A1aB1b facilitated targeting of A3B4 glycinin into PB-II by sequestration with A1aB1b, resulting in an increase in the accumulation of A3B4 glycinin.

α′ Subunit of soybean β-conglycinin forms complex with rice glutelin via a disulphide bond in transgenic rice seeds.

The α′ and β subunits of soybean β-conglycinin were expressed in rice seeds in order to improve the nutritional and physiological properties of rice as a food. The α′ subunit accumulated in rice seeds at a higher level than the β subunit, but no detectable difference in mRNA transcription level between subunits was observed. Sequential extraction results indicate that the α′ subunit formed one or more disulphide bonds with glutelin. Electron microscopic analysis showed that the α′ subunit and the β subunit were transported to PB-II together with glutelin. In mature transgenic seeds, the β subunit accumulated in low electron density regions in the periphery of PB-II, whereas the α′ subunit accumulated together with glutelin in high-density regions of the periphery. The subcellular localization of mutated α′ subunits lacking one cysteine residue in the N-terminal mature region (α′ΔCys1) or five cysteine residues in the pro and N-terminal mature regions (α′ΔCys5) were also examined. Low-density regions were formed in PB-II in mature seeds of transgenic rice expressing α′ΔCys 5 and α′ΔCys1. α′ΔCys5 was localized only in the low-density regions, whereas α′ΔCys1 was found in both low- and high-density regions. These results suggest that the α′ subunit could make a complex via one or more disulphide bonds with glutelin and accumulate together in PB-II of transgenic rice seeds.

Systematic analysis of a dipeptide library for inhibitor development using human dipeptidyl peptidase IV produced by a Saccharomyces cerevisiae expression system

The inhibition of human dipeptidyl peptidase IV/CD26 (hDPPIV) is an accepted treatment for type 2 diabetes. In this study, an extracellular production system of hDPPIV using Saccharomyces cerevisiae was established to facilitate the screening of hDPPIV inhibitors. As dipeptides that mimic the hDPPIV substrate are candidate inhibitors of this protein, X-Ala or X-Pro dipeptides (in which X represents any amino acid) were tested systematically. Based on the results obtained in the first screening, a second screening was performed for Trp-X dipeptides. To elucidate the manner via which the physicochemical features at the P(1) and P(2) positions contributed to the hDPPIV inhibitory effect, correlations between the inhibitory activity of dipeptides and 13 amino acid indices were analyzed. The most effective inhibitory dipeptide was Trp-Pro (K(i)=0.04 mM). The mode of inhibition of hDPPIV by dipeptides was explained well by some amino acid indices and by the structure of the substrate-binding site of hDPPIV. The information obtained from the systematic analysis of a dipeptide library provides important clues for the development of hDPPIV targeting drugs and functional foods for type 2 diabetes.

Development of transgenic rice containing a mutated β subunit of soybean β-conglycinin for enhanced phagocytosis-stimulating activity.

Improving the nutraceutical value of rice would positively impact the health and well-being of rice consumers worldwide. Based on the three-dimensional structure of soybean beta-conglycinin, we designed a beta subunit with a strong phagocytosis-stimulating activity (mbeta subunit). Here, we describe the genetic modification and production of rice seeds containing the mbeta subunit as part of our aim to develop a food material that promotes human health. The mbeta subunit folded correctly and was accumulated in the protein body II of rice seeds at a level similar to wild-type beta subunit. Mutant beta subunit purified from transgenic rice seeds exhibited high phagocytosis-stimulating activity, demonstrating its potential value in enhancing the nutritional value of rice.

Co-expression of α′ and β subunits of β-conglycinin in rice seeds and its effect on the accumulation behavior of the expressed proteins

A transgenic rice that produces both the α′ and β subunits of β-conglycinin has been developed through the crossing of two types of transgenic rice. Although the accumulation level of the α′ subunit in the α′β-transgenic rice was slightly lower than that in the transgenic rice producing only the α′ subunit, the accumulation level of the β subunit in the α′β-transgenic rice was about 60% higher than that in the transgenic rice producing only the β subunit. Results from sequential extraction and gel-filtration experiments indicated that part of the β subunit formed heterotrimers with the α′ subunit in a similar manner as in soybean seeds and that the heterotrimers interacted with glutelin via cysteine residues. These results imply that the accumulation level of the β subunit in the α′β-transgenic rice increases by an indirect interaction with glutelin. Immunoelectron microscopy revealed that the α′ and β subunits are localized in a low electron-dense region of protein body-II (PB-II) and that α′ homotrimers in the α′β-transgenic rice seeds seem to accumulate outside of this low electron-dense region.

Improvement of glucose and lipid metabolism via mung bean protein consumption: clinical trials of GLUCODIA™ isolated mung bean protein in the USA and Canada

The aim of the present study was to confirm the effects of a commercially available mung bean protein isolate (GLUCODIA™) on glucose and lipid metabolism. The main component of GLUCODIA™ is 8S globulin, which constitutes 80 % of the total protein. The overall structure of this protein closely resembles soyabean β-conglycinin, which accounts for 20 % of total soya protein (soya protein isolate; SPI). Many physiological beneficial effects of β-conglycinin have been reported. GLUCODIA™ is expected to produce beneficial effects with fewer intakes than SPI. We conducted two independent double-blind, placebo-controlled clinical studies. In the first (preliminary dose decision trial) study, mung bean protein was shown to exert physiological beneficial effects when 3·0 g were ingested per d. In the second (main clinical trial) study, mung bean protein isolate did not lower plasma glucose levels, although the mean insulin level decreased with consumption of mung bean protein. The homeostatic model assessment of insulin resistance (HOMA-IR) values significantly decreased with mung bean protein. The mean TAG level significantly decreased with consumption of mung bean protein isolate. A significant increase in serum adiponectin levels and improvement in liver function enzymes were observed. These findings suggest that GLUCODIA™ could be useful in the prevention of insulin resistance and visceral fat accumulation, which are known to trigger the metabolic syndrome, and in the prevention of liver function decline.

Dietary mung bean protein reduces high-fat diet-induced weight gain by modulating host bile acid metabolism in a gut microbiota-dependent manner

The 8-globulin-rich mung bean protein (MPI) suppresses hepatic lipogenesis in rodent models and reduces fasting plasma glucose and insulin levels in obese adults. However, its effects on mitigating high fat diet (HFD)-induced obesity and the mechanism underlying these effects remain to be elucidated. Herein, we examined the metabolic phenotype, intestinal bile acid (BA) pool, and gut microbiota of conventionally raised (CONV-R) male C57BL/6 mice and germ-free (GF) mice that were randomized to receive either regular HFD or HFD containing mung bean protein isolate (MPI) instead of the dairy protein present in regular HFD. MPI intake significantly reduced HFD-induced weight gain and adipose tissue accumulation, and attenuated hepatic steatosis. Enhancement in the secretion of intestinal glucagon-like peptide-1 (GLP-1) and an enlarged cecal and fecal BA pool of dramatically elevated secondary/primary BA ratio were observed in mice that had consumed MPI. These effects were abolished in GF mice, indicating that the effects were dependent upon the presence of the microbiota. As revealed by 16S rRNA gene sequence analysis, MPI intake also elicited dramatic changes in the gut microbiome, such as an expansion of taxa belonging to the phylum Bacteroidetes along with a reduced abundance of the Firmicutes.

Seed Storage Proteins; Strategies for Developing Crops Promoting Human Health

Plant seeds contain high amounts of storage proteins. These are classified on basis of their solubility as water-soluble albumins, salt-soluble globulins, alcohol-soluble prolamins, and acidor alkaline-soluble glutelins (Osborne 1924, Utsumi, 1992). The compositions of seed storage proteins differ among plant species. For examples, monocot seeds contain mainly glutelins and prolamins (Ogawa et al., 1987; Li & Okita, 1993; Cagampang et al., 1966), whereas legume seeds contain mainly 7S/11S globulins (Utsumi, 1992). Rice is the staple food of approximately half of the population of the world. The major seed storage proteins of rice are glutelins and prolamin, similarly to the other monocots. The seed storage proteins present in rice, however, offer little significant benefit to human physiology. Therefore, improving the nutritional and physiological values of rice would be of benefit to the health of considerable numbers of people. A candidate protein that might be of interest in the context of improving the physiological values of rice is β-conglycinin. The seed storage protein of soybean, β-conglycinin, lowers plasma cholesterol and triglyceride levels in humans (Sirtori et al., 1995; Aoyama et al., 2001). Moreover, βconglycinin increases adiponectin levels and improves glucose tolerance (Tachibana et al., 2010). The α’ subunit of β-conglycinin has LDL-cholesterol-lowering activity (Sirtori and Lovati, 2001) and contains a phagocytosis-stimulating peptide (Tsuruki et al., 2003). Therefore, development of rice that can accumulate β-conglycinin should produce a staple food with several important physiological benefits to human health. β-Conglycinin has the trimeric structure common to 7S globulins of other plant species and is composed of three subunits, α, α’ and β. The α and α’ subunits contain an N-terminal extension in addition to a core region common to all the subunits (Maruyama et al., 1998, 2001 & 2004). The β subunit consists of only the core domain. The α and α’ subunits and the β subunit are synthesized on polysomes as preproand pre-forms, respectively. The signal peptides are co-translationally removed, the polypeptides are N-glycosylated with highmannose glycans and assemble into trimers in the ER (Yamauchi & Yamagishi, 1979; Utsumi, 1992). They are transported from the ER to the protein storage vacuoles through the Golgi apparatus (Mori et al., 2004). The pro regions of the α and α’ subunits are