Ikuko Kakizaki

A hyaluronan synthase suppressor, 4‐methylumbelliferone, inhibits liver metastasis of melanoma cells

4‐Methylumbelliferone (MU) inhibits the cell surface hyaluronan (HA) formation, and that such inhibition results in suppression of adhesion and locomotion of cultured melanoma cells. Here, we examine the effect of MU on melanoma cell metastasis in vivo. MU‐treated melanoma cells showed both decreased cell surface HA formation and suppression of liver metastasis after injection into the mice. Oral administration of MU to mice decreased tissue HA content. These HA knock‐down mice displayed suppressed liver metastasis. Thus, both cell surface HA of melanoma cells and recipient liver HA can promote liver metastasis, indicating that MU has potential as an anti‐metastatic agent.

CDNA AND DEDUCED AMINO ACID SEQUENCE OF HUMAN PK-120, A PLASMA KALLIKREIN-SENSITIVE GLYCOPROTEIN

PK‐120 is a substrate for plasma kallikrein (PK), recently purified from human plasma. Here we have established the cDNA sequence for human PK‐120 mRNA. The deduced amino sequence of PK‐120 revealed that it consists of 902 amino acid residues with a calculated mass of 116,423 Da. The putative cleavage sites by PK have been proposed, suggesting that PK‐120 may be a precursor of a bioactive peptide. Most interestingly, PK‐120 showed significant sequence identities to heavy chains (HCs) of the inter‐α‐trypsin inhibitor (ITI) superfamily.PK-120 is a substrate for plasma kallikrein (PK), recently purified from human plasma. Here we have established the cDNA sequence for human PK-120 mRNA. The deduced amino sequence of PK-120 revealed that it consists of 902 amino acid residues with a calculated mass of 116,423 Da. The putative cleavage sites by PK have been proposed, suggesting that PK-120 may be a precursor of a bioactive peptide. Most interestingly, PK-120 showed significant sequence identities to heavy chains (HCs) of the inter-alpha-trypsin inhibitor (ITI) superfamily.

Activation of mouse Pi-class glutathione S-transferase gene by Nrf2 (NF-E2-related factor 2) and androgen

The Pi-class glutathione S-transferases (GSTs) play pivotal roles in the detoxification of xenobiotics, carcinogenesis and drug resistance. The mechanisms of regulation of these genes during drug induction and carcinogenesis are yet to be elucidated. Recently, Nrf2 (NF-E2-related factor 2; a bZip-type transcription factor) knockout mice were shown to display impaired induction of Pi-class GST genes by drugs. It is known that the mouse Pi-class GST gene GST-P1 is expressed predominantly in the male liver, and is regulated by androgen. To determine whether Nrf2 and the androgen receptor regulate GST-P1 directly, we analysed the molecular mechanism of activation of this gene by these factors. The promoter of the GST-P1 gene was activated markedly by Nrf2 in transient transfection analyses. Gel mobility shift assay and footprinting analyses revealed three Nrf2 binding sites: one at the proximal and two at distal elements, located at positions -59, -915 and -937 from the cap site. The fifth intron of the GST-P1 gene contains the androgen-responsive region. Multiple androgen receptor binding sites are clustered within a 500 bp region of this intron. The whole fragment contains a minimum of seven androgen receptor binding sites, which collectively display strong androgen-dependent enhancer activity. However, on division into small fragments containing two or three elements each, individual enhancer activities were dramatically decreased. This suggests that multiple elements work synergistically as a strong androgen-responsive enhancer. Our findings indicate that Nrf2 and the androgen receptor directly bind to and activate the mouse GST-P1 gene.

4-methylumbelliferone, a hyaluronan synthase suppressor, enhances the anticancer activity of gemcitabine in human pancreatic cancer cells

Hyaluronan (HA) is a ubiquitous, major component of the pericellular matrix and is necessary for various physiological processes. It plays a very important role in biological barriers. We previously reported that 4-methylumbelliferone (MU) inhibits HA synthesis and pericellular HA matrix formation in cultured human skin fibroblasts, Streptococcus equi FM100, and B16F10 melanoma cells. We hypothesized that MU-mediated inhibition of HA synthesis and pericellular HA matrix formation would increase the efficacy of anticancer drugs. We have already demonstrated in vitro, using a sandwich binding protein assay and a particle exclusion assay, that MU inhibits HA synthesis and formation of the pericellular HA matrix, respectively, in human KP1-NL pancreatic cancer cells. AlamarBlue assay revealed that the anticancer effect of gemcitabine in KP1-NL cells was increased by pretreatment with MU. In vivo simultaneous administration of MU and gemcitabine to tumor-bearing mice with severe combined immunodeficiency disease (SCID) decreased the size of the primary and metastatic tumors more than did gemcitabine alone. These data strongly suggest that a combination of MU and gemcitabine is effective against human pancreatic cancer cells. MU may have potential as a chemosensitizer and may provide us with a new anticancer strategy.

Mechanism for the hydrolysis of hyaluronan oligosaccharides by bovine testicular hyaluronidase

Synthetic hyaluronan oligosaccharides with defined structures and their pyridylaminated derivatives were used to investigate the mechanism of hydrolysis of hyaluronan by bovine testicular hyaluronidase. The products of the hydrolysis were analyzed by HPLC and ion‐spray mass spectroscopy (MS). It was confirmed that the minimum substrate for bovine testicular hyaluronidase is the hyaluronan hexasaccharide, even though it is a poor substrate that is barely cleaved, even on prolonged incubation. When hyaluronan octasaccharide was the substrate, increasing amounts of tetrasaccharide and hexasaccharide were produced with increasing time of incubation. Whereas disaccharide was not detectable in the reaction mixture by HPLC, MS analysis revealed trace amounts. The data suggest that the enzyme generates a disaccharide intermediate from hyaluronan oligosaccharide, the majority of which is transferred to the nonreducing ends of other oligosaccharides, only traces being released as free disaccharide. When hyaluronan octasaccharide, with an unsaturated glucuronic acid at the nonreducing end, was used as a substrate, only a tetrasaccharide was detected by HPLC. However, MS showed that the product was a mixture of equal amounts of two tetrasaccharides, one with and the other without the unsaturated glucuronic acid. This suggests that, in the case of substrates with a double bond at the nonreducing end, a tetrasaccharide is cleaved off instead of a disaccharide. The results of the experiments with pyridylaminated oligosaccharides were entirely consistent with these conclusions, and in addition showed the importance of the reducing end of the substrate for the enzyme to recognize the length of the saccharide.

Identification of proteoglycan from salmon nasal cartilage.

There has been no structural information about the core protein of salmon nasal cartilage proteoglycan although its physiological activities have been investigated. Internal amino acid sequencing using nano-LC/MS/MS revealed that the salmon proteoglycan was aggrecan. Primer walk sequencing based on the amino acid information determined that the salmon aggrecan cDNA is comprised of 4207bp nucleotides predicted to encode 1324 amino acids with a molecular mass of 143,276. It exhibited significant similarities to predicted pufferfish aggrecan, zebrafish similar to aggrecan, zebrafish aggrecan, bovine aggrecan and human aggrecan isoform 2 precursor; whose amino acid identities were 56%, 55%, 49%, 31% and 30%, respectively. Salmon cartilage aggrecan had globular domains G1, G2 and G3 as in mammalian aggrecans. Neither the putative keratan sulfate attachment domain enriched with serine, glutamic acid and proline, nor the putative chondroitin sulfate attachment domain with repeating amino acid sequence containing serine-glycine, found in mammalian aggrecans were observed in salmon, however, random serine-glycine (or glycine-serine) sequences predicted to the sugar chain attachment sites were observed. Based on cDNA analysis and amino acid analysis after β-elimination, the ratio of serine attached to sugar chains was calculated to be approximately 37.7% of total serine, that is, 46 of 123 serine residues.

Structural Interactions in Chondroitin 4-Sulfate Mediated Adherence of Plasmodium falciparum Infected Erythrocytes in Human Placenta during Pregnancy-Associated Malaria†

Infection with Plasmodium falciparum during pregnancy results in the adherence of infected red blood cells (IRBCs) in placenta, causing pregnancy-associated malaria with severe health complications in mothers and fetuses. The chondroitin 4-sulfate (C4S) chains of very low sulfated chondroitin sulfate proteoglycans (CSPGs) in placenta mediate the IRBC adherence. While it is known that partially sulfated but not fully sulfated C4S effectively binds IRBCs, structural interactions involved remain unclear and are incompletely understood. In this study, structurally defined C4S oligosaccharides of varying sulfate contents and sizes were evaluated for their ability to inhibit the binding of IRBCs from different P. falciparum strains to CSPG purified from placenta. The results clearly show that, with all parasite strains studied, dodecasaccharide is the minimal chain length required for the efficient adherence of IRBCs to CSPG and two 4-sulfated disaccharides within this minimal structural motif are sufficient for maximal binding. Together, these data demonstrate for the first time that the C4S structural requirement for IRBC adherence is parasite strain-independent. We also show that the carboxyl group on nonreducing end glucuronic acid in dodecasaccharide motif is important for IRBC binding. Thus, in oligosaccharides containing terminal 4,5-unsaturated glucuronic acid, the nonreducing end disaccharide moiety does not interact with IRBCs due to the altered spatial orientation of carboxyl group. In such C4S oligosaccharides, 14-mer but not 12-mer constitutes the minimal motif for inhibition of IRBC binding to placental CSPG. These data have important implications for the development and evaluation of therapeutics and vaccine for placental malaria.

Anti-inflammatory effect of proteoglycan and progesterone on human uterine cervical fibroblasts.

AIMS The aim of this study was to compare the anti-inflammatory effect of proteoglycan (PG) with that of progesterone (P) in the cultured fibroblasts from human uterine cervix. MAIN METHODS After obtaining informed consent, the cervix was collected from normal women undergoing total hysterectomy. The cervix was cultured until fibroblasts proliferated and had grown to confluence, then, the fibroblasts were stimulated by lipopolysaccharide (LPS) with or without PG, P and a combination of both; they were cultured for 24-48 h. The anti-inflammatory effects of PG and P were evaluated by the suppression of IL-6 or IL-8 secretion. The expression of the IL-6 or IL-8 gene and the expression of their protein were determined by real-time PCR, and ELISA, respectively. Activation of Toll-like receptor (TLR) 4 was evaluated by Western blotting. KEY FINDINGS LPS markedly enhanced gene and protein expression of IL-6 and IL-8 in human uterine cervical fibroblasts. The up-regulation of the IL-6 or IL-8 gene and protein expression by LPS was significantly suppressed with PG, P and a combination of both. Western blotting revealed that combination of PG and P showed more potent inhibition on LPS-stimulated TLR4 induction than that seen by each. SIGNIFICANCE This study showed that both PG and P have an inhibitory effect on LPS-induced inflammation. This anti-inflammatory effect of PG and P was augmented by co-administration of both, suggesting for the first time that PG has an anti-inflammatory effect on human uterine cervical fibroblasts.

Novel products in hyaluronan digested by bovine testicular hyaluronidase

When the products of hyaluronan (HA) digested by bovine testicular hyaluronidase (BTH) were analyzed by high-performance liquid chromatography (HPLC), minor peaks were detected just before the main even-numbered oligosaccharide peaks. The amount of each minor peak was dependent on the reaction conditions for transglycosylation, rather than hydrolysis, by the BTH. Mainly based on HPLC and MS analysis, each minor peak was found to correspond to its oligosaccharide with one N-acetyl group removed from the reducing terminal N-acetylglucosamine. Enzymatic studies showed that the N-deacetylation activity was closely related to reaction temperature, pH, and the concentration of NaCl contained in the buffer, and glycosaminoglycan types and chain lengths of substrates. These findings strongly suggest that the N-deacetylation reaction in minor peaks was due to a novel enzyme contaminant in the BTH, N-deacetylase, that carries out N-deacetylation at the reducing terminal N-acetylglucosamine of oligosaccharides and is dependent on HA hydrolysis by BTH.

A glycomic approach to proteoglycan with a two-dimensional polysaccharide chain map.

Glycosaminoglycan chains were liberated from proteoglycans (bovine lung, tracheal cartilage, and cerebrum) by successive digestion with actinase and with cellulase from Aspergillus niger, which has endo-beta-xylosidase activity. The glycosaminoglycan chains were fluorescence-labeled with 2-aminopyridine after digestion with Streptomyces hyaluronidase. The resulting pyridylamino-glycosaminoglycans, including heparan sulfate, chondroitin sulfate/dermatan sulfate, and heparin, were separated by high-performance liquid chromatography. Each separated fraction was analyzed by two types of high-performance liquid chromatography: gel-filtration chromatography and anion-exchange chromatography. The correlation between molecular weight and degree of sulfation could be shown on the two-dimensional polysaccharide chain map. Use of a commonly available cellulase with endo-beta-xylosidase activity together with the two-dimensional polysaccharide chain map allows easy analysis of various glycosaminoglycan chains and comprehensive comparison among the structures. These techniques will become useful tools in the further development of glycotechnology and glycome analysis.