Hideyuki Ihara

Multiple potential regulatory sites of TLR4 activation induced by LPS as revealed by novel inhibitory human TLR4 mAbs.

Recognition of LPS by the toll-like receptor 4 (TLR4)/MD-2 complex is a trigger of innate immune defense against bacterial invasion. However, excessive immune activation by this receptor complex causes septic shock and autoimmunity. Manipulation of TLR4 signaling represents a potential therapy that would avoid the detrimental consequences of unnecessary immune responses. In this study, we established two novel mAbs that inhibit LPS-induced human TLR4 activation. HT52 and HT4 mAbs inhibited LPS-induced nuclear factor-κB activation in TLR4/MD-2-expressing Ba/F3-transfected cells and cytokine production and up-regulation of CD86 in the human cell line U373 and PBMCs. These inhibitory activities were stronger than that of HTA125 mAb, which we previously reported. Immunofluorescent and biochemical studies using TLR4 deletion mutants revealed that HT52 and HT4 recognized spatially distinct regions on TLR4 irrespective of MD-2 association. The HT52 and HTA125 epitopes were localized within aa 50-190, while the HT4 epitope was formed only by the full length of TLR4. In addition, we demonstrated that HT52 and HT4 failed to compete with LPS for binding to TLR4/MD-2 but inhibited LPS-induced TLR4 internalization. Inhibitory activities were not due to the interaction with the Fcγ receptor CD32. Our finding that binding of mAbs to at least two distinct regions on TLR4 inhibits LPS-dependent activation provides a novel method for manipulating TLR4 activation and also a rationale for designing drugs targeted to TLR4.

Fucosylation of chitooligosaccharides by human α1,6-fucosyltransferase requires a nonreducing terminal chitotriose unit as a minimal structure

FUT8, a eukaryotic alpha1,6-fucosyltransferase, catalyzes the transfer of a fucosyl residue from guanine nucleotide diphosphate-beta-l-fucose to the innermost GlcNAc of an asparagine-linked oligosaccharide (N-glycan). The catalytic domain of FUT8 is structurally similar to that of NodZ, a bacterial alpha1,6-fucosyltransferase, which acts on a chitooligosaccharide in the synthesis of Nod factor. While the substrate specificities for the nucleotide sugar and the N-glycan have been determined, it is not known whether FUT8 is able to fucosylate other sugar chains such as chitooligosaccharides. The present study was conducted to investigate the action of FUT8 on chitooligosaccharides that are not generally thought to be a substrate in mammals, and the results indicate that FUT8 is able to fucosylate such structures in a manner comparable to NodZ. Surprisingly, structural analyses of the fucosylated products by high performance liquid chromatography, mass spectrometry and nuclear magnetic resonance indicated that FUT8 does not utilize the reducing terminal GlcNAc for fucose transfer but shows a preference for the third GlcNAc residue from the nonreducing terminus of the acceptor. These findings suggest that FUT8 catalyzes the fucosylation of chitooligosaccharide analogous to NodZ, but that a nonreducing terminal chitotriose structure is required for the reaction. The substrate recognition by which FUT8 selects the position to fucosylate might be distinct from that for NodZ and could be due to structural factor requirements which are inherent in FUT8.

Reduced Surface Expression of TLR4 by a V254I Point Mutation Accounts for the Low Lipopolysaccharide Responder Phenotype of BALB/c B Cells

LPS is recognized by TLR4 and radioprotective 105 kDa in B cells. Susceptibility to LPS in murine B cells is most closely linked to the locus containing the TLR4 gene. However, the molecular mechanism underlying genetic control of LPS sensitivity by this locus has not been fully elucidated. In this study, we revealed that C57BL/6 (B6) B cells respond to mAb-induced, TLR4-specific signals stronger than BALB/c (BALB) B cells, as assessed by proliferation and upregulation of CD69 and CD86. In contrast, BALB B cells were not hyporesponsive to agonistic anti–radioprotective 105 kDa mAb or the TLR9 agonist CpG. Although the level of TLR4 mRNA in BALB B cells was comparable with that in B6 B cells, surface TLR4 expression in BALB B cells was lower than that in B6 B cells. This lower surface expression of BALB TLR4 was also observed when HEK293 and Ba/F3 cells were transfected with a BALB TLR4 expression construct. We identified a V254I mutation as the responsible single nucleotide polymorphism for lower surface expression of BALB TLR4. Furthermore, cotransfection of myeloid differentiation factor-2 increased BALB TLR4 expression, although it was still lower than B6 TLR4 expression. In concordance with reduced expression, Ba/F3 cells transfected with BALB TLR4 and myeloid differentiation factor-2 were hyporesponsive compared with those with B6 TLR4, as assessed by LPS-induced NF-κB activation. In conclusion, we revealed that LPS sensitivity is genetically controlled by the level of surface TLR4 expression on B cells. A V254I mutation accounts for the LPS hyporesponsive phenotype of BALB B cells.

Bidirectional N-acetylglucosamine transfer mediated by β-1,4-N-acetylglucosaminyltransferase III

beta-1,4-N-Acetylglucosaminyltransferase III (GnT-III) catalyzes the formation of the bisecting GlcNAc and plays a regulatory role in the biosynthesis of the N-linked oligosaccharide. In this study, we examined whether the glycosyl transfer catalyzed by GnT-III is reversible, and, in addition, investigated the equilibrium of the GnT-III-catalyzed reaction. Incubation of the agalactosyl-bisected biantennary oligosaccharide with GnT-III in the presence of the sufficiently high concentration of uridine diphosphate (UDP) resulted in conversion of the bisected oligosaccharide into the nonbisected one. This reaction was accompanied by the stoichiometric formation of UDP-GlcNAc, which appeared to result from the transfer of GlcNAc from the oligosaccharide to UDP. Thus, these results indicate that GnT-III is capable of perceivably catalyzing the reverse reaction in vitro, as found in some glycosyltransferases. When the equilibrium of the reaction was kinetically analyzed, it was found that the state of the equilibrium is greatly displaced toward the formation of the bisecting GlcNAc. In terms of free energy change, as estimated, the reaction by GnT-III can be comparable to the hydrolysis of ATP. Although GnT-III catalyzes bidirectional transfer of GlcNAc between the oligosaccharide and UDP, the removal of the bisecting GlcNAc is unlikely in vivo, due to the displacement of the equilibrium. It is known that equilibria of certain glycosyltransferase reactions are not biased as greatly as the case of GnT-III, and thus it seems likely that there are a variety of equilibrium states in glycosyltransferase reactions. In living cells, the assembly of oligosaccharides could be regulated by not only rate control but also equilibrium control.

Measurement of peroxiredoxin-4 serum levels in rat tissue and its use as a potential marker for hepatic disease

Peroxiredoxin (Prx)-4, a secretable endoplasmic reticulum (ER)-resident isoform of the mammalian Prx family, functions as a thioredoxin-dependent peroxidase. It is acknowledged that Prx-4 plays a role in the detoxification of hydrogen peroxide, and potentially other peroxides, which may be generated during the oxidative folding of proteins and oxidative stress in the ER. The present study was undertaken in order to specifically quantify the tissue levels of Prx-4. To accomplish this, an enzyme-linked immunosorbent assay was developed using a specific polyclonal antibody produced by immunizing a rabbit with native recombinant rat Prx-4 protein. The assay was used to detect Prx-4 in the range of 0.1 and 10 ng/ml, and to investigate tissue distribution in rats. Using this immunoassay, we found that the serum levels of Prx-4 were substantially lower in asymptomatic Long-Evans Cinnamon rats, a rat model of Wilsons disease, compared to normal rats. In addition, the treatment of rat hepatoma cells with N-acetylcysteine led to a significant increase in the release of Prx-4 protein into the medium; thus, it appears likely that the secretion of Prx-4 is associated with the redox state within cells. These results suggest that serum Prx-4 has potential for use as a biomarker for hepatic oxidative stress.

Expression of N-terminally truncated forms of rat peroxiredoxin-4 in insect cells

Peroxiredoxins (Prxs), a family of thioredoxin-dependent peroxidases, are highly conserved in many organisms and function in detoxifying reactive oxygen species as well as other cellular processes. Six members of the Prx family are known in mammals, i.e., Prx-1 through -6. Among these proteins, only Prx-4 appears to contain a signal peptide that serves for localization in the endoplasmic reticulum, membrane translocation and secretion into the extracellular space, as demonstrated in a previous study using a baculovirus-insect cell system. The present study was conducted to determine whether the signal peptide-truncated mutant of rat Prx-4 is expressed as an enzymatically active form and is produced in large amounts. Two N-terminally truncated mutants were prepared by deletion of only the signal peptide and the larger region encompassing both the signal and the unique extension to Prx-4. These mutants were successfully produced within Spodoptera frugiperda 21 cells by infection with the recombinant baculoviruses, rather than by extracellular secretion. Both mutants were efficiently purified to homogeneity by two column chromatography steps. Biochemical characterization of the purified proteins showed that the truncated enzymes are enzymatically active and form an oligomeric structure, as reported for the mammalian Prx family. The findings also suggest that the unique extension plays a role in the regulation of non-covalent oligomerization. More than 4 mg of the purified proteins can be obtained from cells grown in monolayer cultures in twenty 75 cm(2) tissue culture flasks. The procedures described in this study permit recombinant Prx-4 to be prepared more efficiently and easily for purposes of crystallization and antibody preparation.

Cloning, expression and characterization of Bombyx mori α1,6-fucosyltransferase.

Although core α1,6-fucosylation is commonly observed in N-glycans of both vertebrates and invertebrates, the responsible enzyme, α1,6-fucosyltransferase, has been much less characterized in invertebrates compared to vertebrates. To investigate the functions of α1,6-fucosyltransferase in insects, we cloned the cDNA for the α1,6-fucosyltransferase from Bombyx mori (Bmα1,6FucT) and characterized the recombinant enzyme prepared using insect cell lines. The coding region of Bmα1,6FucT consists of 1737bp that code for 578 amino acids of the deduced amino acid sequence, showing significant similarity to other α1,6-fucosyltransferases. Enzyme activity assays demonstrated that Bmα1,6FucT is enzymatically active in spite of being less active compared to the human enzyme. The findings also indicate that Bmα1,6FucT, unlike human enzyme, is N-glycosylated and forms a disulfide-bonded homodimer. These findings contribute to a better understanding of roles of α1,6-fucosylation in invertebrates and also to the development of the more efficient engineering of N-glycosylation of recombinant glycoproteins in insect cells.

Difucosylation of chitooligosaccharides by eukaryote and prokaryote α1,6-fucosyltransferases.

BACKGROUNDnThe synthesis of eukaryotic N-glycans and the rhizobia Nod factor both involve α1,6-fucosylation. These fucosylations are catalyzed by eukaryotic α1,6-fucosyltransferase, FUT8, and rhizobial enzyme, NodZ. The two enzymes have similar enzymatic properties and structures but display different acceptor specificities: FUT8 and NodZ prefer N-glycan and chitooligosaccharide, respectively. This study was conducted to examine the fucosylation of chitooligosaccharides by FUT8 and NodZ and to characterize the resulting difucosylated chitooligosaccharides in terms of their resistance to hydrolysis by glycosidases.nnnMETHODSnThe issue of whether FUT8 or NodZ catalyzes the further fucosylation of chitooligosaccharides that had first been monofucosylated by the other. The oligosaccharide products from the successive reactions were analyzed by normal-phase high performance liquid chromatography, mass spectrometry and nuclear magnetic resonance. The effect of difucosylation on sensitivity to glycosidase digestion was also investigated.nnnRESULTSnBoth FUT8 and NodZ are able to further fucosylate the monofucosylated chitooligosaccharides. Structural analyses of the resulting oligosaccharides showed that the reducing terminal GlcNAc residue and the third GlcNAc residue from the non-reducing end are fucosylated via α1,6-linkages. The difucosylation protected the oligosaccharides from extensive degradation to GlcNAc by hexosamidase and lysozyme, and also even from defucosylation by fucosidase.nnnCONCLUSIONSnThe sequential actions of FUT8 and NodZ on common substrates effectively produce site-specific-difucosylated chitooligosaccharides. This modification confers protection to the oligosaccharides against various glycosidases.nnnGENERAL SIGNIFICANCEnThe action of a combination of eukaryotic and bacterial α1,6-fucosyltransferases on chitooligosaccharides results in the formation of difucosylated products, which serves to stabilize chitooligosaccharides against the action of glycosidases.

MD-2-dependent human Toll-like receptor 4 monoclonal antibodies detect extracellular association of Toll-like receptor 4 with extrinsic soluble MD-2 on the cell surface.

MD-2 is essential for lipopolysaccharide (LPS) recognition of Toll-like receptor 4 (TLR4) but not for cell surface expression. The TLR4/MD-2 complex is formed intracellularly through co-expression. Extracellular complex formation remains a matter for debate because of the aggregative nature of secreted MD-2 in the absence of TLR4 co-expression. We demonstrated extracellular complex formation using three independent monoclonal antibodies (mAbs), all of which are specific for complexed TLR4 but unreactive with free TLR4 and MD-2. These mAbs bound to TLR4-expressing Ba/F3 cells only when co-cultured with MD-2-secreting Chinese hamster ovary cells or incubated with conditioned medium from these cells. All three mAbs bound the extracellularly formed complex indistinguishably from the intracellularly formed complex in titration studies. In addition, we demonstrated that two mAbs lost their affinity for TLR4/MD-2 on LPS stimulation, suggesting that these mAbs bound to conformation-sensitive epitopes. This was also found when the extracellularly formed complex was stimulated with LPS. Additionally, we showed that cell surface TLR4 and extrinsically secreted MD-2 are capable of forming the functional complex extracellularly, indicating an additional or alternative pathway for the complex formation.

An Assay for α 1,6-Fucosyltransferase (FUT8) Activity Based on the HPLC Separation of a Reaction Product with Fluorescence Detection

N-Glycans with an α-fucose unit linked to the 6-position of the innermost GlcNAc are widely distributed among the animal kingdom, from worms and insects to human. This α1,6-linked fucosyl residue, frequently referred to as a core fucose, is formed via the action of an α1,6-fucosyltransferase, the mammalian ortholog which is systematically called FUT8. In mammals, it is well known that the extent of core-fucosylation in cellular and secreted glycoproteins varies, e.g., according to differentiation and carcinogenesis of the cells. This chapter describes a method for the sensitive and quantitative assay of FUT8 activity using a fluorescence-labeled oligosaccharyl asparagine derivative as the glycosyl acceptor substrate.