Iwao Ohkubo

Transcriptional regulation of the legumain gene by p53 in HCT116 cells

Legumain (EC 3.4.22.34) is an asparaginyl endopeptidase. Strong legumain activity was observed in the mouse kidney, and legumain was found to be highly expressed in tumors. We previously reported that bovine kidney annexin A2 was co-purified with legumain and that legumain cleaved the N-terminal region of annexin A2 at an Asn residue in vitro and in vivo. In this study, we found a p53-binding site in intron 1 of the human legumain gene using computational analysis. To determine whether transcription of the legumain gene is regulated by p53, HCT116 cells were transfected with p53 siRNA and the effect of knockdown of p53 expression on legumain expression was examined. The results showed that expression levels of both legumain mRNA and protein were decreased in the siRNA-treated cells. Furthermore, enzyme activity of legumain was also increased by doxorubicin and its activity was reduced by knockdown of p53 in HCT116 cells. These results suggest that legumain expression and its enzyme activity are regulated by p53.

Improvement of blood glucose levels and obesity in mice given aronia juice by inhibition of dipeptidyl peptidase IV and α-glucosidase

Aronia berries have many potential effects on health. Previous human studies have shown that aronia juice may be useful for treatment of obesity disorders. Recently, we have reported that aronia juice has an inhibitory effect on dipeptidyl peptidase (DPP IV) activity and that the DPP IV inhibitor in aronia juice was identified as cyanidin 3,5-diglucoside. In this study, we found that body weights and blood glucose levels were reduced in diabetes model KK-Ay mice given aronia juice. We also found that weights of white adipose tissues were reduced in KK-Ay mice given aronia juice. Furthermore, levels of DPP IV activity in the serum and liver from KK-Ay mice were lower than those in the serum and liver from C57BL/6JmsSlc mice. Interestingly, although levels of DPP IV activity were not changed in the serum and liver from aronia-juice-administered KK-Ay mice, levels of DPP IV activity were increased in those from aronia-juice-administered C57BL/6JmsSlc mice. Furthermore, α-glucosidase activity was inhibited in the upper region of the small intestine from aronia-juice-administered KK-Ay mice but not in the lower region. Inhibition of α-glucosidase activity in the upper portion of the small intestine induced a reduction of glucose-dependent insulinotropic polypeptide (GIP) level. The results suggest that DPP IV activity in diabetic mice is inhibited by aronia juice, that the GIP level in the upper region of the small intestine is reduced by inhibition of α-glucosidase activity and that weights of adipose tissues are reduced by aronia juice.

Identification and characterization of a dipeptidyl peptidase IV inhibitor from aronia juice.

Aronia berries have many potential effects on health, including an antioxidant effect, effect for antimutagenesis, hepatoprotection and cardioprotection, an antidiabetic effect and inhibition of cancer cell proliferation. Previous human studies have shown that aronia juice may be useful for treatment of obesity disorders. In this study, we found that aronia juice has an inhibitory effect against dipeptidyl peptidase IV (DPP IV) (EC 3.4.14.5). DPP IV is a peptidase that cleaves the N-terminal region of incretins such as glucagon-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). Inactivation of incretins by DPP IV induces reduction of insulin secretion. Furthermore, we identified that cyanidin 3, 5-diglucoside as the DPP IV inhibitor in aronia juice. DPP IV was inhibited more strongly by cyanidin 3, 5-diglucoside than by cyanidin and cyanidin 3-glucoside. The results suggest that DPP IV is inhibited by cyanidin 3, 5-diglucoside present in aronia juice. The antidiabetic effect of aronia juice may be mediated through DPP IV inhibition by cyanidin 3, 5-diglucoside.

Knockdown of legumain inhibits cleavage of annexin A2 in the mouse kidney

Legumain (EC 3.4.22.34) is an asparaginyl endopeptidase. Strong legumain activity was observed in the mouse kidney, and legumain was highly expressed in tumors. We previously reported that bovine kidney annexin A2 was co-purified with legumain and that legumain cleaved the N-terminal region of annexin A2 at an Asn residue in vitro. In this study, to determine whether annexin A2 is cleaved by legumain in vivo, siRNA-lipoplex targeting mouse legumain was injected into mouse tail veins. Mouse kidneys were then isolated and the effect of knockdown of legumain expression on annexin A2 cleavage was examined. The results showed that both legumain mRNA and protein expression levels were decreased in the siRNA-treated mouse kidneys and that legumain activity toward a synthetic substrate, Z-Ala-Ala-Asn-MCA, was decreased by about 40% in the kidney but not in the liver or spleen. Furthermore, cleavage of annexin A2 at the N-terminal region was decreased in the mouse kidney that had been treated with the legumain siRNA-lipoplex. These results suggest that legumain siRNA was delivered to the kidney by using LipoTrust and that the reduced legumain expression inhibited legumain-induced degradation of annexin A2 in vivo.

Purification, molecular cloning and functional characterization of swine phosphatidylethanolamine-binding protein 4 from seminal plasma

Phosphatidylethanolamine-binding proteins (PEBPs) are found in various species and have multiple functions. In this study, we purified the swine homolog of human PEBP4 (sPEBP4) from swine seminal plasma, cloned the sPEBP4 cDNA and functionally characterized this protein. The molecular mass of the purified protein was calculated to be 25 kDa by SDS-polyacrylamide gel electrophoresis under reducing conditions. The full-length cDNA of sPEBP4 contains 815 bp with an open reading frame of 669 bp that encodes a protein 222 residues in length. sPEBP4 contains a putative phosphatidylethanolamine-binding domain between residues 79 and 195; however, this domain did not show lipid binding activity. The overall amino acid sequence identity of PEBP4s from swine, human, mouse, bovine and canine ranges between 56.1% and 82.4%. Immunohistochemical staining and western blotting analysis showed that sPEBP4 is secreted from epithelial cells in the epididymis to the seminal plasma. To explore the role of sPEBP4 in the seminal plasma, we tested the effect of sPEBP4 on swine sperm motility. Sperms suspended in phosphate-buffered saline began to swim after the addition of purified sPEBP4, but not when swine serum albumin was added, indicating that sPEBP4 promotes sperm motility.

Serum DJ-1 level is positively associated with improvements in some aspects of metabolic syndrome in Japanese women through lifestyle intervention

DJ-1 is a protein that is associated with Parkinson disease and cancer, and the reduction of DJ-1 function and expression is also thought to be a cause of diabetes and hypertension. However, little is known about the association between the plasma concentration of DJ-1 and risk of metabolic syndrome. We hypothesized that a lifestyle intervention would increase serum DJ-1 and that up-regulated DJ-1 functions will result in the prevention of metabolic syndrome. The objective of our study is to examine whether the level of serum DJ-1 is associated with the risk of metabolic syndrome. Therefore, to reveal the association between DJ-1 and metabolic syndrome, this study investigated lifestyle intervention in a control group (n = 37) and intervention group (n = 45). The results showed that body mass index, body fat ratio, waist-hip ratio, waist circumference, blood pressure, and plasma glucose level were improved in the intervention group, as compared with those in the control group. Furthermore, serum levels of DJ-1 were increased in the intervention group, when compared with those in the control group. These results suggest that serum DJ-1 is increased by lifestyle intervention and that increased serum DJ-1 prevents metabolic syndrome. Thus, the level of serum DJ-1 will become one of the indexes for the risk of metabolic syndrome.

Domain 5 of high molecular weight kininogen inhibits collagen-mediated cancer cell adhesion and invasion in association with α-actinin-4

High molecular weight kininogen (HK) is a plasma glycoprotein with multiple functions, including the regulation of coagulation. We previously demonstrated that domain 5 (D5(H)), a functional domain of HK, and its derived peptides played an important role in the vitronectin-mediated suppression of cancer cell adhesion and invasion. However, the underlying mechanisms of the D5(H)-mediated suppressive effects remain to be elucidated. Here, we showed that D5(H) and its derivatives inhibited the collagen-mediated cell adhesion and invasion of human osteosarcoma MG63 cells. Using purified D5(H) fused to glutathione-S-transferase (GST) and D5(H)-derived peptides for column chromatography, an actin-binding protein, α-actinin-4, was identified as a binding protein of D5(H) with high-affinity for P-5m, a core octapeptide of D5(H). Immunofluorescence microscopy demonstrated that D5(H) co-localized with α-actinin-4 inside MG63 cells. In addition, exogenous GST-D5(H) added to the culture media was transported into MG63 cells, although GST alone as a control was not. As α-actinin-4 regulates actin polymerization necessary for cell adhesion and is related to the integrin-dependent attachment of cells to the extracellular matrix, our results suggest that D5(H) may modulate cell adhesion and invasion together with actinin-4.

Rapid and Simple Purification of Lysozyme from the Egg Shell Membrane

Lysozyme (EC 3.2.1.17) is a hydrolytic enzyme that cleaves the β-(1,4)-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan, a major bacterial cell wall polymer. In the food industry, lysozyme is used as an additive mainly in the production of wine and beer. Lysozyme was found to be localized in the egg shell membrane. In this study, we found that lysozyme was easily purified from the egg shell membrane and that the enzyme also had antibacterial activity. Furthermore, we found that the antibacterial activity of purified lysozyme from the egg shell membrane was lower than that of purified lysozyme from the egg white at alkaline pH. The method for rapid purification of lysozyme developed in this study should contribute to the food industry.

Protease activity of legumain is inhibited by an increase of cystatin E/M in the DJ-1-knockout mouse spleen, cerebrum and heart

Legumain (EC 3.4.22.34) is an asparaginyl endopeptidase. Legumain activity has been detected in various mouse tissues including the kidney, spleen and epididymis. Legumain is overexpressed in the majority of human solid tumors and transcription of the legumain gene is regulated by the p53 tumor suppressor in HCT116 cells. The legumain activity is also increased under acid conditions in Alzheimers disease brains. DJ-1/PARK7, a cancer- and Parkinsons disease-associated protein, works as a coactivator to various transcription factors, including the androgen receptor, p53, PSF, Nrf2, SREBP and RREB1. Recently, we found that legumain expression, activation and cleavage of annexin A2 are regulated by DJ-1 through p53. In this study, we found that the expression levels of legumain mRNA were increased in the cerebrum, kidney, spleen, heart, lung, epididymis, stomach, small intestine and pancreas from DJ-1-knockout mice, although legumain activity levels were decreased in the cerebrum, spleen and heart from DJ-1-knockout mice. Furthermore, we found that cystatin E/M expression was increased in the spleen, cerebrum and heart from DJ-1-knockout mice. These results suggest that reduction of legumain activity is caused by an increase of cystatin E/M expression in the spleen, cerebrum and heart from DJ-1-knockout mice.