Yoshihiko Sakaguchi

S100C/A11 is a key mediator of Ca2+-induced growth inhibition of human epidermal keratinocytes

An increase in extracellular Ca2+ induces growth arrest and differentiation of human keratinocytes in culture. We examined possible involvement of S100C/A11 in this growth regulation. On exposure of the cells to high Ca2+, S100C/A11 was specifically phosphorylated at 10Thr and 94Ser. Phosphorylation facilitated the binding of S100C/A11 to nucleolin, resulting in nuclear translocation of S100C/A11. In nuclei, S100C/A11 liberated Sp1/3 from nucleolin. The resulting free Sp1/3 transcriptionally activated p21CIP1/WAF1, a representative negative regulator of cell growth. Introduction of anti-S100C/A11 antibody into the cells largely abolished the growth inhibition induced by Ca2+ and the induction of p21CIP1/WAF1. In the human epidermis, S100C/A11 was detected in nuclei of differentiating cells in the suprabasal layers, but not in nuclei of proliferating cells in the basal layer. These results indicate that S100C/A11 is a key mediator of the Ca2+-induced growth inhibition of human keratinocytes in culture, and that it may be possibly involved in the growth regulation in vivo as well.

Clostridium botulinum type A haemagglutinin-positive progenitor toxin (HA+-PTX) binds to oligosaccharides containing Galβ1-4GlcNAc through one subcomponent of haemagglutinin (HA1)

Haemagglutinin (HA) activity of Clostridium botulinum type A 19S and 16S toxins (HA-positive progenitor toxin; HA(+)-PTX) was characterized. HA titres against human erythrocytes of HA(+)-PTX were inhibited by the addition of lactose, D-galactose, N-acetyl-D-galactosamine and D-fucose to the reaction mixtures. A direct glycolipid binding test demonstrated that type A HA(+)-PTX strongly bound to paragloboside and some neutral glycolipids, but did not bind to gangliosides. Type A HA(+)-PTX also bound to asialoglycoproteins (asialofetuin, neuraminidase-treated transferrin), but not to sialoglycoproteins (fetuin, transferrin). Although glycopeptidase F treatment of asialofetuin abolished the binding of HA(+)-PTX, endo-alpha-N-acetylgalactosaminidase treatment did not. Thus these results can be interpreted as indicating that type A HA(+)-PTX detects and binds to Gal beta 1-4GlcNAc in paragloboside and the N-linked oligosaccharides of glycoproteins. Regardless of neuraminidase treatment, type A HA(+)-PTX bound to glycophorin A which is a major sialoglycoprotein on the surface of erythrocytes. Both native glycophorin A and neuraminidase-treated glycophorin A inhibited the binding of erythrocytes to type A HA(+)-PTX. Since the N:-linked oligosaccharide of glycophorin A is di-branched and more than 50% of this sugar chain is monosialylated, type A HA(+)-PTX probably bound to the unsialylated branch of the N-linked oligosaccharide of glycophorin A and agglutinated erythrocytes. One subcomponent of HA, designated HA1, did not agglutinate native erythrocytes, although it did bind to erythrocytes, paragloboside and asialoglycoproteins in a manner quite similar to that of HA(+)-PTX. These results indicate that type A HA(+)-PTX binds to oligosaccharides through HA1.

Truncation of Annexin A1 Is a Regulatory Lever for Linking Epidermal Growth Factor Signaling with Cytosolic Phospholipase A2 in Normal and Malignant Squamous Epithelial Cells

Regulation of cell growth and apoptosis is one of the pleiotropic functions of annexin A1 (ANXA1). Although previous reports on the overexpression of ANXA1 in many human cancers and on growth suppression and/or induction of apoptosis by ANXA1 may indicate the tumor-suppressive nature of ANXA1, molecular mechanisms of the function of ANXA1 remain largely unknown. Here we provide evidence that ANXA1 mechanistically links the epidermal growth factor-triggered growth signal pathway with cytosolic phospholipase A2 (cPLA2), an initiator enzyme of the arachidonic acid cascade, through interaction with S100A11 in normal human keratinocytes (NHK). Ca2+-dependent binding of S100A11 to ANXA1 facilitated the binding of the latter to cPLA2, resulting in inhibition of cPLA2 activity, which is essential for the growth of NHK. On exposure of NHK to epidermal growth factor, ANXA1 was cleaved solely at Trp12, and this cleavage was executed by cathepsin D. In squamous cancer cells, this pathway was shown to be constitutively activated. The newly found mechanistic intersection may be a promising target for establishing new measures against human cancer and other cell growth disorders.

Tubulin homolog TubZ in a phage-encoded partition system

Partition systems are responsible for the process whereby large and essential plasmids are accurately positioned to daughter cells during bacterial division. They are typically made of three components: a centromere-like DNA zone, an adaptor protein, and an assembling protein that is either a Walker-box ATPase (type I) or an actin-like ATPase (type II). A recently described type III segregation system has a tubulin/FtsZ-like protein, called TubZ, for plasmid movement. Here, we present the 2.3 Å structure and dynamic assembly of a TubZ tubulin homolog from a bacteriophage and unravel the Clostridium botulinum phage c-st type III partition system. Using biochemical and biophysical approaches, we prove that a gene upstream from tubZ encodes the partner TubR and localize the centromeric region (tubS), both of which are essential for anchoring phage DNA to the motile TubZ filaments. Finally, we describe a conserved fourth component, TubY, which modulates the TubZ-R-S complex interaction.

Involvement of Interferon Regulatory Factor 1 and S100C/A11 in Growth Inhibition by Transforming Growth Factor β1 in Human Hepatocellular Carcinoma Cells

Growth inhibition by transforming growth factor (TGF)-β1 has been attributed to the induction of cyclin-dependent kinase inhibitors, among which p21/Waf1 plays a major role in many biological contexts. In the present study, two new intracellular mediators for the induction of p21/Waf1 by TGF-β1 were identified in a human hepatocellular carcinoma cell line (JHH-5) expressing mutant-type p53. After addition of TGF-β1 to JHH-5 cells, a marked increase of the p21/Waf1 expression preceded the inhibition of DNA synthesis. Expression of IFN regulatory factor (IRF)-1, a known transacting factor for p21/Waf1 promoter, was elevated just before or in parallel with the increase of p21/Waf1. Transduction of antisense IRF-1 inhibited the increase in p21/Waf1 in JHH-5 cells treated with TGF-β1 and partially released the cells from the growth arrest by TGF-β1. Expression of S100C/A11, a member of the Ca2+-binding S100 protein family, also markedly increased after addition of TGF-β1. S100C/A11 protein was translocated to and accumulated in nuclei of TGF-β1-treated JHH-5 cells, where p21/Waf1 was concomitantly accumulated. When a recombinant S100C/A11 protein was introduced into nuclei of JHH-5 cells, DNA synthesis was markedly inhibited in a dose-dependent manner in the absence of TGF-β1. Prior transfection of p21/Waf1-targeted small interfering RNA efficiently blocked decrease of DNA synthesis in JHH-5 cells caused by TAT-S100C/A11 or TGF-β1 and markedly inhibited expression of p21/Waf1 protein in the cells. These results indicate that IRF-1 and S100C/A11 mediate growth inhibition by TGF-β1 via induction of p21/Waf1.

C Terminal Half Fragment (50 kDa) of Heavy Chain Components of Clostridium botulinum Type C and D Neurotoxins Can Be Used as an Effective Vaccine

Recombinant whole heavy chains (H, 100 kDa) and their N‐terminal (Hn, 50 kDa) and C‐terminal (Hc, 50 kDa) half fragments of Clostridium botulinum type C and D neurotoxins were expressed as glutathione S‐transferase (GST) fusion proteins in Escherichia coli. GST eliminated‐preparations of H (10 μg), Hn (5 μg), Hc (5 μg), or a mixture of Hn (5 μg) and Hc (5 μg) of types C and D were mixed with an equal volume of adjuvant, and then were twice injected into mice subcutaneously. After immunization, the mice were challenged with up to 106 the minimum lethal doses (MLD)/0.5 ml of C or D toxin, the type of which was same as that of the immunogens. All of the mice immunized with antigens except for Hn survived against 105 to 106 MLD/0.5 ml of the toxins, but the mice immunized with Hn were killed by 100 MLD/0.5 ml. The mice immunized with a mixture of C‐Hc and D‐Hc, each 5 μg, also showed a high level of resistance against both C and D toxins. Antibody levels immunized with GST fused‐ or GST eliminated‐preparation were quite similar. These results indicate that recombinant GST‐fused Hc can be used as a safe and effective vaccine for type C and D botulism in animals. It also became clear that one time inoculation with a large amount of C‐Hc or D‐Hc, 100 μg, is useful for vaccine trials in mice.

Transcriptional regulation of a brown adipocyte-specific gene, UCP1, by KLF11 and KLF15

Several growth factors and transcription factors have been reported to play important roles in brown adipocyte differentiation and modulation of thermogenic gene expression, especially the expression of UCP1. In this study, we focused on KLF11 and KLF15, which were expressed highly in brown adipose tissue. Our data demonstrated that KLF11 and KLF15 interacted directly with the UCP1 promoter using GC-box and GT-boxes, respectively. Co-transfection of KLF11 and KLF15 in the mesenchymal stem cell line muBM3.1 during brown adipocyte differentiation enhanced the expression level of UCP1. KLF11, but not KLF15, was essential for UCP1 expression during brown adipocyte differentiation of muBM3.1.

Molecular Analysis of an Extrachromosomal Element Containing the C2 Toxin Gene Discovered in Clostridium botulinum Type C

Clostridium botulinum cultures are classified into seven types, types A to G, based on the antigenicity of the neurotoxins produced. Of these seven types, only types C and D produce C2 toxin in addition to the neurotoxin. The C2 toxin consists of two components designated C2I and C2II. The genes encoding the C2 toxin components have been cloned, and it has been stated that they might be on the cell chromosome. The present study confirmed by using pulsed-field gel electrophoresis and subsequent Southern hybridization that these genes are on a large plasmid. The complete nucleotide sequence of this plasmid was determined by using a combination of inverse PCR and primer walking. The sequence was 106,981 bp long and contained 123 potential open reading frames, including the c2I and c2II genes. The 57 products of these open reading frames had sequences similar to those of well-known proteins. It was speculated that 9 these 57 gene products were related to DNA replication, 2 were responsible for the two-component regulatory system, and 3 were sigma factors. In addition, a total of 20 genes encoding proteins related to diverse processes in purine catabolism were found in two regions. In these regions, there were 9 and 11 genes rarely found in plasmids, indicating that this plasmid plays an important role in purine catabolism, as well as in C2 toxin production.

Molecular properties of each subcomponent in Clostridium botulinum type B haemagglutinin complex

The role of each subcomponent of Clostridium botulinum serotype B haemagglutinin (HA), which is one component of 16S toxin, and consists of four subcomponents (HA1, 2, 3a, and 3b), was investigated. In order to identify the subcomponent contributing to the stability of a neurotoxin in the gastro-intestinal tract, each recombinant HA (rHA) subcomponent was incubated with gastro-intestinal proteases. Although rHA1 and rHA3 were stable to these proteases except for specific cleavage, rHA2 was not. Anti-free whole HA serum reacted with neither rHA2 nor HA2 in 16S toxin on both Western blot and ELISA, while anti-rHA2 serum reacted with both rHA2 and HA2 in 16S toxin on Western blots, although it did not react with 16S toxin in ELISA. Binding or haemagglutination activity against erythrocytes was found in rHA1 and rHA3, but not in rHA2. In addition, only HA1 bound to the intestinal section. These results indicate that the HA (and 16S toxin) complex is assembled in the way that HA1 and HA3 (HA3a plus HA3b) encase HA2, followed by modification with trypsin-like bacterial protease, leading to the conclusion that HA1 and HA3 act as protective factors for the neurotoxin and as attachment factors to host cells.

Characterisation of monoclonal antibodies against haemagglutinin associated with Clostridium botulinum type C neurotoxin

Of 11 monoclonal antibodies (MAbs) prepared against the non-toxic component of type C Clostridium botulinum 16S toxin to clarify the function of the non-toxic component, seven recognised HA1, three recognised HA3b and one recognised HA2. Results of epitope mapping indicated that three of the seven anti-HA1 MAbs recognised the region between amino acid residues 121 and 140 and four recognised the three-dimensional structure of HA1. Three anti-HA3b MAbs recognised different regions between (approximately) amino acids 405-430, 180-270 and 275-297. The ability of these MAbs to interfere with binding of 16S toxin or non-toxic component, HA1 or HA3b to erythrocytes and to intestine tissue sections of guinea-pig was observed. MAbs against HA3b and HA2 did not inhibit 16S toxin binding to either erythrocytes or epithelial cells, whereas some MAbs against HA1 did inhibit binding. The seven anti-HA1 MAbs can be classified into four groups based on their binding inhibition activities. The anti-HA1 MAbs that inhibited the binding of 16S toxin to the epithelial cells also neutralised or reduced the oral toxicity in mice, indicating that HA may play an important role in the absorption of the 16S toxin from the small intestine.