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Core fucosylation, catalysed by α-1, 6 fucosyltransferase (FUT8), regulates growth factor receptors in immune function. Although core fucose regulates many immune cell types, few reports confront the association between core fucose activity and an innate immune reaction. Here, we have investigated the function of core fucose in macrophages in vivo and in vitro using Fut8-deficient mice and cells. Following lipopolysaccharide (LPS) stimulation, inflammatory cytokine production in Fut8-deficient (Fut8-/-) macrophages was suppressed in both in vivo and in vitro experiments. Because LPS is recognized by Toll-like receptor 4 (TLR4), which induces the signalling cascade, TLR4 signalling was assumed to be impaired in Fut8-/- cells. Flow cytometry analyses revealed, however, that a lack of core fucose reduced the expression of, not TLR4, but CD14, which is necessary for TLR4 endocytosis. Because CD14 is necessary for TLR2 signalling, the immune response of TLR2 was also impaired in Fut8-/- macrophages. Moreover, in the dextran sodium sulphate (DSS)-induced murine colitis model, the mice grafted with Fut8-/- bone marrow cells exhibited higher resistance to inflammation than those grafted with Fut8+/+ bone marrow cells. These findings indicate that core fucose is essential for CD14-dependent TLR4 and TLR2 signalling in murine macrophage activity, leading to DSS-induced experimental colitis., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Eukaryotic typical 2-Cys type peroxiredoxin (Prx) is inactivated by hyperoxidation of the peroxidatic cysteine to a sulphinic acid in a catalytic cycle-dependent manner. This inactivation process has been well documented for cytosolic isoforms of Prx. However, such a hyperoxidative inactivation has not fully been investigated in Prx-4, a secretable endoplasmic reticulum-resident isoform, in spite of being a typical 2-Cys type, and details of this process are reported herein. As has been observed in many peroxiredoxins, the peroxidase activity of Prx-4 was almost completely inhibited in the reaction with t-butyl hydroperoxide. On the other hand, when H(2)O(2) was used as the substrate, the peroxidase activity significantly remained after oxidative damage. In spite of these different consequences, mass spectrometric analyses indicated that both reactions resulted in the same oxidative damage, i.e. sulphinic acid formation at the peroxidatic cysteine, suggesting that another cysteine in the active site confers the peroxidase activity. As suggested by the analyses using cysteine-substituted mutants sulphinic acid formation at the peroxidatic cysteine may play a role in the development of the possible alternative mechanism, thereby sustaining the peroxidase activity that prefers H(2)O(2).

The alpha2,6-sialylated blood group type 2H (ST2H) antigen (Fucalpha1-2(NeuAcalpha2-6)Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-Cer) is a fucoganglioside found in human colon cancer tissues. To elucidate an enzyme responsible for the ST2H antigen formation, we screened some partially purified candidate enzymes, alpha2,6-sialyltransferases, ST6Gal I and ST6Gal II, and alpha1,2-fucosyltransferases, FUT1 and FUT2 for their activities towards pyridylaminated type 2H (Fucalpha1-2Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-PA) or LS-tetrasaccharide c (LST-c: NeuAcalpha2-6Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-PA) as acceptor substrates. Here we show the ST6Gal I transfers NeuAc from the donor CMP-NeuAc to the terminal Gal of PA-type 2H, which formed the ST2H antigen, but the others could not synthesize it. Using a recombinant ST6Gal I, enzymatic reactions with two types of acceptors, PA-type 2H and PA-lacto-N-neotetraose (LNnT), were kinetically analysed. On the basis of catalytic efficiency (V(max)/K(m)), the specificity of ST6Gal I towards the PA-type 2H was estimated to be 42 times lower than that for PA-LNnT. The overexpression of ST6Gal I in human colon cancer DLD-1 cells effectively resulted in the ST2H antigen formation, as judged by LC-ESI-IT-MS. Many lines of evidence suggest the up-regulation of ST6Gal I in human colon cancer specimens. Collectively, these findings indicate that ST6Gal I is responsible for ST2H antigen biosynthesis in human colon cancer cells.

alpha1,6-Fucosylation plays key roles in many biological functions, as evidenced by the study of alpha1,6-fucosyltransferase (Fut8) knockout (Fut8(-/-)) mice. Phenotypically, Fut8(-/-) mice exhibit emphysema-like changes in the lung, and severe growth retardation. Fut8(-/-) cells also show marked dysregulation of the TGF-beta1 receptor, EGF receptor, integrin activation and intracellular signalling, all of which can be rescued by reintroduction of Fut8. The results of the present study demonstrated that vascular endothelial growth factor receptor-2 (VEGFR-2) expression was significantly suppressed in Fut8(-/-) mice, suggesting that Fut8 was required for VEGFR-2 expression. The expression of VEGFR-2 mRNA and protein was consistently down-regulated by knockdown of the Fut8 gene with small interference RNA in A549 cells, as well as in TGP49 cells, suggesting that suppression occurs at the level of transcription. In contrast, the expression level of ceramide, an inducer of cell apoptosis, was increased in the lungs of Fut8(-/-) mice. The terminal transferase dUTP nick end-labelling (TUNEL) assay was used to identify apoptotic cells. The number of TUNEL-positive septal epithelia and endothelia cells was significantly increased in the alveolar septa of lungs from Fut8(-/-) mice when in comparison with lungs from wild-type mice. It is well known that, in emphysema, ceramide expression can be greatly enhanced by blockade of the VEGFR-2. Thus, suppression of VEGFR-2 expression may provide a novel explanation for the emphysema-like changes in Fut8(-/-) mice.

3-Nitrotyrosine (NTyr) is considered as a biomarker of the generation of reactive nitrogen species (RNS). However, it is still difficult to determine its concentration in biological samples. To develop a reliable and high-throughput method, we optimized the conditions for high performance liquid chromatography and electrochemical detection (HPLC-ECD). The best separation of NTyr was achieved using a highly acidic mobile phase (pH 2.5). The concentration of protein-bound NTyr in plasma protein was 593.6 +/- 53.8 fmol/mg in rats treated with lipopolysaccharide (LPS) and 114.4 +/- 27.6 fmol/mg in control. After intravenous administration of in vitro-nitrated plasma protein, NTyr concentration decreased; the half-life was 63.4 +/- 16.8 h. Consistently, protein-bound NTyr concentration in plasma after LPS treatment declined gradually, but was detectable for 1 week. Our protocol is reproducible and suitable for analysing multiple clinical samples to study RNS production in vivo.

alpha1,6-Fucosyltransferase (Fut8) catalyzes the transfer of a fucose residue from GDP-fucose to the innermost N-acetylglucosamine residue of N-glycans. Here we report that the loss of core fucosylation impairs the function of low-density lipoprotein (LDL) receptor-related protein-1 (LRP-1), a multifunctional scavenger and signaling receptor, resulting in a reduction in the endocytosis of insulin like growth factor (IGF)-binding protein-3 (IGFBP-3) in the cells derived from Fut8-null (Fut8-/-) mice. The reduced endocytosis was restored by the re-introduction of Fut8. Serum levels of IGFBP-3 were markedly upregulated in Fut8-/- mice. These data clearly indicate that core fucosylation is crucial for the scavenging activity of LRP-1 in vivo.

3-Deoxyglucosone (3-DG), a dicarbonyl compound produced by glycation, plays a role in the modification and cross-linking of long-lived proteins. We synthesized [3H]3-DG from [3H]glucose and developed an internalization assay system using HPLC to examine its cellular metabolism. When smooth muscle cells or human umbilical vein endothelial cells were incubated with [3H]3-DG, it was found that [3H]3-DG was internalized by cells in a time dependent manner. The rate of internalization was reduced when the cells were incubated at 4 degrees C or treated with phenylarsine oxide (PAO). By monitoring [3H]3-DG taken up by cells, it was confirmed that 3-DG is reduced to 3-deoxyfructose (3-DF) and that this reaction was inhibited by an aldo-keto reductase inhibitor (ARI). The presence of 3-DG led to an increase in reactive oxygen species levels in the cells and subsequent apoptosis, and the effect was enhanced by pretreatment with ARI. These results suggest that 3-DG is internalized by cells and reduced to 3-DF by aldo-keto reductases, and that the internalized 3-DG is responsible for the production of intracellular oxidative stress.

Mammalian sulfoglycolipids are comprised of two major classes of compounds, sulfatide (SO(3)-3Gal-ceramide) and seminolipid (SO(3)-3Gal-alkylacylglycerol). Sulfatide is present in relatively high levels in myelin, and seminolipid is present in testis. The sulfation of these sulfoglycolipids is catalyzed by a common enzyme, cerebroside sulfotransferase (CST). Disruption of the Cst gene in mice revealed that sulfatide and seminolipid are essential for, respectively, myelin formation and spermatogenesis. The present study describes the generation of a recombinant single-chain variable fragment (scFv) antibody against sulfoglycolipid, for use in the functional analysis of sulfoglycolipids in living cells. A positive hybridoma producing anti-sulfoglycolipid IgG3, referred to as DI8, was initially obtained by immunizing CST-null mice with an isolated sulfatide. The DI8 monoclonal antibody was found to bind specifically to sulfoglycolipids with the terminal 3-O-sulfated galactose structure, as evidenced by ELISA and thin-layer chromatogram-immunostaining. The antibody stained seminolipid on the cell surface of spermatogenic cells of wild-type testis, but it did not react with any cells in the seminiferous tubules of CST-null testis. Total RNA was extracted from this hybridoma, and cDNAs that encode the variable regions of the heavy and light chains of IgG3 were obtained by RT-PCR. These DNA fragments were linked through a DNA linker coding (Gly(4)Ser)(3), and the recombinant scFv fragment was then inserted into a phagemid vector pCANTAB 5E. The scFv antibody that was displayed at the tip of the M13 phage in the form of a g3p fusion protein bound to sulfatide. Furthermore, a soluble form of the scFv antibody was also found to bind to the sulfoglycolipids in ELISA.

Glycoprotein-3-sulfotransferase (GP3ST) is a key enzyme in downregulating the expression of Galalpha1,3Galbeta1,4GlcNAc-R (the alpha-Gal epitope), via enzymatic competition with an alpha1,3 galactosyltransferase (alpha1,3GT), such as alpha2,6 sialyltransferase (alpha2,6ST). In this study, we report the dominance of GP3ST over alpha1,3GT using transfected pig endothelial cell (PEC) lines. The introduction of the GP3ST gene into PEC suppresses its antigenicity with respect to normal human pooled serum (NHS), including the alpha-Gal epitope and the Hanganutziu-Deicher (H-D) antigen, and, in addition, reduces the susceptibility to NHS in complement-mediated cell lysis. Western and lectin blot analyses of the products of parental PEC and its transfectants indicated that proteins smaller than 66 kDa have a diminished reactivity with NHS and the IB4 lectin. The levels of the alpha-Gal epitope in neutral glycosphingolipids were also decreased in the GP3ST transfectants as detected in thin layer chromatography by immunostaining. These data indicate that GP3ST is very effective in reducing xenoepitope levels.

Cleavage of an asparagine-linked sugar chain by hydrogen peroxide (H2O2) and a copper salt was investigated. Incubation of a 2-aminopyridine (PA)-labeled biantennary sugar chain, GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-2Manalpha1-3)Manbeta1-4GlcNAcbeta1-4GlcNAc-PA, with H2O2 and Cu2+ led to formation of four major degradation products. Reversed phase high performance liquid chromatographic analysis coupled with glycosidase digestion indicated that the sugar chain is not randomly degraded but specifically degraded at a GlcNAc residue. Treatment with either of H2O2 or copper alone did not cleave nor degrade the sugar chain to any extent. Electron spin resonance (ESR) spectra obtained using a spin trap reagent were consistent with the generation of OH* or an OH*-like radical by the H2O2/copper salt mixture. The addition of ascorbic acid enhanced this radical generation as well as the degradation of the sugar chain. It was also found that H2O2/Cu2+ destroys the N-acetyl group of the monosaccharide GlcNAc, as judged by a decrease in the ultraviolet absorption spectrum of this group. On the other hand, replacement of copper by Fe2+ caused no cleavage of the sugar chain, although comparable levels of the same radical species were generated. Furthermore, spectrophotometric analysis showed that a GlcNAc-containing sugar chain coordinates to copper but not to iron, and, thus, the coordination appears to play an essential role in the degradation of the sugar chain. These findings suggest that coordination of copper ions to GlcNAc residues localizes the generation of a radical, which cleaves the glycosidic linkage, possibly involving alteration of the N-acetyl group, thereby allowing the GlcNAc-specific cleavage.

The present study was undertaken in an attempt to clarify the pathway by which hyperosmotic stress induces HB-EGF gene expression in rat aortic smooth muscle cells (RASMC). Hyperosmotic stress induced by a high concentration of glucose or mannitol resulted in an increase in HB-EGF mRNA level in a dose- and time-dependent manner. HB-EGF induction was blocked by curcumin, a c-jun/fos antisense oligonucleotide and a dominant-negative mutant of JNK1. Electrophoretic mobility shift assay also showed the involvement of AP-1 in HB-EGF gene expression by glucose. In addition, hyperosmotic stress induced rapid phosphorylation of Pyk2 in RASMC. TPA and calcium chelating agents (BAPTA-AM and EGTA) blocked Pyk2 phosphorylation and HB-EGF gene expression. Furthermore, HB-EGF gene expression and JNK activation by hyperosmotic stress were sensitive to PP2, an Src kinase-specific inhibitor. These findings indicate that hyperosmotic stress activates JNK via calcium-Pyk2 signaling cascades, which in turn induce HB-EGF gene expression.

Mammalian thioredoxin reductase [EC 1.6.4.5], a homodimeric flavoprotein, has a marked similarity to glutathione reductase. The two cysteines in the N-terminal FAD domain (-Cys59-x-x-x-x-Cys64-) and histidine (His472) are conserved between them at corresponding positions, but the mammalian thioredoxin reductase contains a C-terminal extension of selenocysteine (Sec or U) at the penultimate position and a preceding cysteine (-Gly-Cys497-Sec498-Gly). Introduction of mutations into the cloned rat thioredoxin reductase gene revealed that residues Cys59, Cys64, His472, Cys497, and Sec498, as well as the sequence of Cys497 and Sec498 were essential for thioredoxin-reducing activity. To analyze the catalytic mechanism of the mammalian thioredoxin reductase, the wild-type, U498C, U498S, C59S, and C64S were overproduced in a baculovirus/insect cell system and purified. The wild-type thioredoxin reductase produced in this system, designated as WT, was found to lack the Sec residue and to terminate at Cys497. A Sec-containing thioredoxin reductase, which was purified from COS-1 cells transfected with the wild-type cDNA, was designated as SecWT and was used as an authentic enzyme. Among mutant enzymes, only U498C retained a slight thioredoxin-reducing activity at about three orders magnitude lower than SecWT. WT, U498C, and U498S showed some 5,5'-dithiobis(2-nitrobenzoic acid)-reducing activity and transhydrogenase activity, and C59S and C64S had substantially no such activities. These data and spectral analyses of these enzymes suggest that Cys59 and Cys64 at the N-terminus, in conjunction with His472, function as primary acceptors for electrons from NADPH via FAD, and that the electrons are then transferred to Cys497-Sec498 at the C-terminus for the reduction of oxidized thioredoxin in the mammalian thioredoxin reductase.

The kinetic basis of the donor substrate specificity of beta1, 4-N-acetylglucosaminyltransferase III (GnT-III) was investigated using a purified recombinant enzyme. The enzyme also transfers GalNAc and Glc moieties from their respective UDP-sugars to an acceptor at rates of 0.1-0.2% of that for GlcNAc, but Gal is not transferred at a detectable rate. Kinetic analyses revealed that these inefficient transfers, which are associated with the specificity of the enzyme, are due to the much lower V(max) values, whereas the K(m) values for UDP-GalNAc and UDP-Glc differ only slightly from that for UDP-GlcNAc. It was also found that various other nucleotide-Glc derivatives bind to the enzyme with comparable affinities to those of UDP-GlcNAc and UDP-Glc, although the derivatives do not serve as glycosyl donors. Thus, GnT-III does not appear to distinguish UDP-GlcNAc from other structurally similar nucleotide-sugars by specific binding in the ground state. These findings suggest that the specificity of GnT-III toward the nucleotide-sugar is determined during the catalytic process. This type of specificity may be efficient in preventing a possible mistransfer when other nucleotide-sugars are present in excess over the true donor.

Neural- and thymus-derived activator for ErbB kinases (NTAK) is a recently described member of the neuregulin family that binds directly to ErbB3 and ErbB4 and transactivates ErbB2. Rat NTAK has at least five alternative-spliced isoforms: alpha1, alpha2a, alpha2b, beta, and gamma. In order to understand their biological properties, this study focused on the NTAK alpha2a and beta isoforms, which have different EGF-like domains. The effect of these isoforms on cell growth and tyrosine phosphorylation in human breast cancer cells, MDA-MB-453 and T47D, was examined using the recombinant proteins. In terms of cell growth, NTAKalpha2a and NTAKbeta preferentially stimulate T47D cells and MDA-MB-453 cells, respectively, in a dose-dependent manner. Although both NTAKs induce the highest level of tyrosine phosphorylation of ErbB2, NTAKalpha2a and NTAKbeta preferentially induce ErbB3 and ErbB4 phosphorylation, respectively. Thus, NTAKalpha2a and NTAKbeta stimulate cell growth in different ways, by means of different combinations of receptors.

Peroxiredoxins (PRxs) play a role in protecting protein free thiol groups against oxidative damage and thioredoxin-dependent peroxidase activity. This report describes the characteristics of the fourth member of the mammalian PRxs, PRx IV. Rat PRx IV produced in Sf21 cells by a baculovirus expression system has two bands with different electrophoretic mobilities, 31 and 27 kDa [Matsumoto et al. (1999) FEBS Lett. 443, 246-250]. The 27-kDa PRx IV lacks the NH(2)-terminal 36 amino acids which correspond to a predicted leader peptide, which is required for secretion from cells. Thus, the 31-kDa form is probably a precursor form, and the 27-kDa form, a secretable form which is enzymatically active. Pulse-chase experiments of PRx IV-transfected COS-1 cells showed that PRx IV is processed within 10 min and released from cells. The secretable form contains both reduced and oxidized forms. The reduced form binds to both a heparin affinity column and human umbilical vein endothelial cells, while the oxidized form does not. The equilibrium dissociation constants, K(D), for heparin and heparan sulfate as judged by surface plasmon resonance experiments were 19 and 870 nM, respectively. The secretable form corresponds to the major bands found in most tissues, as evidenced by immunoblot analysis. Within cells, secretable form was largely localized on the endoplasmic reticulum, as judged by colocalization with calreticulin. Moreover, PRx IV has glutathione-dependent peroxidase activity in addition to thioredoxin-dependent activity. These data indicate that PRx IV is a secretable protein and may exert its protective function against oxidative damage by scavenging reactive oxygen species in the extracellular space.

The effect of remodeling of a glycoantigen such as the alpha-Gal epitope, Galalpha1,3Galbeta1,4GlcNAc-R, by the introduction of glycosyltransferase genes on natural killer (NK) cell-mediated direct cytotoxicity was investigated using human peripheral blood mononuclear cells (PBMC) or an NK-like cell line, YT cells, as an effector, and swine endothelial cells (SEC) as a target. Several SEC transfectants were established by transfection with the genes for beta1,4-N-acetylglucosaminyltransferase III, alpha2, 3-sialyltransferase and alpha1,2-fucosyltransferase. These transfections led to dramatic reductions in both direct and indirect NK cell-mediated cytotoxicity, by 72-94% in the case of PBMC and 27-72% in that of YT cells, in addition to an effective reduction in xenoantigenicity, which is substantially caused by the alpha-Gal epitope, to human natural antibodies. The NK cell-mediated direct cytotoxicity was remarkably blocked by an anti-alpha-Gal epitope monoclonal antibody or GSI lectin which preferentially binds to the epitope. Furthermore, treatment of the parental cells with alpha-galactosidase resulted in a significant reduction in cytotoxicity. These results suggest that the alpha-Gal epitope is involved not only in hyperacute rejection and acute vascular rejection, but also in NK cell-mediated direct cytotoxicity. Thus, the genetic remodeling of the alpha-Gal epitope and probably other glycoantigens as well can be expected to represent a new approach for overcoming not only indirect but also direct immunity to xenografts.

To determine the role of the polyol metabolizing pathway under hyperglycemic conditions, the effects of aldose reductase (AR) on the cellular functions of pancreatic beta-cells were examined. Stable transfectants of rat AR cDNA were obtained with a pancreatic beta-cell line, HIT, in which a negligible amount of AR was originally expressed. Overproduction of AR triggered DNA fragmentation, as judged with the TUNEL method and agarose gel electrophoresis. Morphological analysis by electron microscopy also clearly showed apoptosis of the AR-overexpressing HIT cells. Induction by interleukin-1beta of gene expression such as those of an inducible form of nitric oxide synthase (NOS-II) and Mn-superoxide dismutase (Mn-SOD), was much lower in the transfectants than in the control cells, while the expression of constitutively expressed genes such as those for Cu,Zn-superoxide dismutase and insulin was not changed. The susceptibility to interleukin-1beta stimulation of the expression of the NOS II and Mn-SOD genes was due to suppressed NF-kappaB activity, which is essential for the expression of these genes. In addition, the intracellular NADPH/NADP+ ratio was considerably lower in the AR-transfected cells than in control cells. Thus, the overexpression of AR in pancreatic beta-cells induced apoptosis that may be caused by a redox imbalance.

A human histiocytic lymphoma cell line, U-937 cells, secretes several vascular endothelial cell growth inhibitors including leukemia inhibitory factor, oncostatin M, tumor necrosis factor-alpha, and transforming growth factor-beta1. Characterization of partially purified fractions from the conditioned media of phorbol ester-treated U-937 cells suggested the existence of unknown endothelial growth inhibitors. Using a combination of copper affinity, heparin affinity, cation exchange, and reversed phase liquid chromatographies, a growth inhibitor for endothelial cells was purified to homogeneity from conditioned media. The purified growth inhibitor migrated as a 65 kDa band on SDS-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. Microsequencing analyses of the tryptic fragments of the growth inhibitor and a BLAST search analysis revealed a unique sequence showing no homology to known proteins. The purified protein inhibited endothelial cell growth in a dose-dependent manner, but had no effect on smooth muscle cell growth. The factor also blocked endothelial cell growth induced by both fibroblast growth factor-2 and vascular endothelial growth factor, and was additively effective in inhibiting the growth of endothelial cells by U-937 cell-derived endothelial cell growth inhibitors. Thus, this factor appears to be a novel inhibitor with specificity for vascular endothelial cells, and is tentatively called endothelial cell inhibitor (ECI) in this study.

2,3-Bisphosphoglycerate mutase (BPGM) [EC 5.4.2.4] is a multifunctional enzyme that catalyzes both the synthesis and the degradation of 2,3-diphosphoglycerate (2,3-DPG) and contains three types of activities in that it functions as a 2,3-DPG synthetase, a phosphoglycerate mutase and a 2,3-DPG phosphatase. In humans, BPGM occurs only in erythrocytes and plays a pivotal role in the dissociation of oxygen from hemoglobin via 2,3-DPG. The present study shows that the specific activity of BPGM in erythrocytes of diabetic patients is decreased, compared to normal controls as judged by 2,3-DPG synthetase activity and immunoreactive contents. To understand the mechanism by which the enzyme is inactivated, the enzyme was purified from pooled erythrocytes from diabetic patients and subjected to a boronate affinity column. The flow through fraction was active while the bound fraction was completely inactive. The bound fraction was reactive to an anti-hexitollysine antibody, indicating that the enzyme had undergone glycation and inactivation. The primary glycated site of the enzyme was found to be Lys158 as judged by amino acid sequencing and the reactivity with an anti-hexitollysine IgG, after reverse-phase HPLC of the lysyl-endopeptidase-digested peptides. Extensive glycation of recombinant BPGM in vitro indicated that the glycation sites were Lys2, Lys4, Lys17, Lys42, Lys158, and Lys196. From these results, the loss of enzymatic activity appears to be due to the glycation of Lys158 which may be located in the vicinity of the substrate binding site.

Wild-type and several mutant human manganese superoxide dismutases (Mn-SODs) were produced in a baculovirus/insect cell system and characterized. The enzymatic activity of a homogenate of Sf21 cells, infected with baculovirus carrying wild-type Mn-SOD and grown in the conventional medium, was indistinguishable from that of control cells, but was augmented by supplementation with Mn2+. The protein produced was largely imported into the mitochondria, as judged from the enrichment in the mitochondrial fraction, the mobility of the protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the results of N-terminal processing, which was confirmed by sequencing of the purified enzyme. However, a significant amount of precursor was also detected by an antibody raised against the human Mn-SOD signal peptide. While both Mn2+ and Fe3+ stimulated Mn-SOD accumulation within mitochondria, the active form was produced in the presence of submillimolar Mn2+ only. Amino acid substitutions at a signal peptide-cleavage site, His-Ser-Leu4 to Pro-Met-Va14, in the mature Mn-SOD prevented the processing of the precursor protein, and thus resulted in the accumulation of the precursor protein within mitochondria, as judged on immunostaining with an anti-Mn-SOD antibody. Mutant Mn-SODs with a truncated signal peptide or carboxyl region (8, 13, and 42 amino acid residues in the mature form) were barely solubilized, even with a nonionic detergent, and exhibited no activity, suggesting inappropriate folding of these mutant SODs. They were also susceptible to proteolytic degradation, while the wild-type and precursor forms were resistant. Thus, the baculovirus/insect cell expression system appears to be adequate for the analysis of mitochondrial import using intact cells as well as for the large scale production of active Mn-SOD.

The glycation reaction (Maillard reaction) plays a major role in diabetic complications, since some reaction intermediates are responsible for the modification and cross-linking of long-lived proteins, resulting, in turn, in a deterioration of normal cell function. The reaction intermediates include methylglyoxal (MG) and 3-deoxyglucosone (3-DG), both of which are cytotoxic dicarbonyl compounds and are elevated during hyperglycemia. Aldehyde reductase (ALR) catalyzes the reduction of both compounds. To examine the intracellular role of ALR in the diabetic complications of neural cells, its gene was overexpressed in rat pheochromocytoma PC12 cells, which normally express a low level of ALR. Western blot analysis showed that ALR protein in the ALR gene-transfected cells was more than twice as much as in the control cells. In the parental cells, cytotoxicity, including apoptotic cell death, which was determined by fluorescent microscopy using the fluorescent DNA binding dye Hoechst 33258, was observed at 100 microM MG. In the ALR gene-transfected cells, the cytotoxicity of both MG and 3-DG and apoptotic cell death were decreased. This suggests that intracellular ALR protects neural cells from the cytotoxicity of 3-DG or MG, and that neural cells, which normally express a low level of ALR, might be susceptible to diabetic complications caused by intermediate products of the Maillard reaction, such as 3-DG and MG.

Human vascular endothelial cells play a pivotal role in atherosclerotic changes but are resistant to apoptotic inducers such as Fas ligand and it has been difficult to induce apoptosis. We developed an experimental model for the apoptosis in the endothelial cells by using snake venom treatment. Snake venom was found to generate intracellular reactive oxygen species (ROS) in the endothelial cells, which leads to apoptosis as judged by electron microscopy as well as by DNA cleavage. Buthionine sulfoximine (BSO) and diethyldithiocarbamate (DDC) accelerated the apoptosis, indicating intracellular glutathione and superoxide levels play a critical role. Pretreatment with tumor necrosis factor (TNF) or phorbol ester (TPA), which increases the Mn-SOD level, prevented the apoptosis. These data suggest that intracellular ROS enhances apoptosis whereas several anti-oxidants are protective in human endothelial cells. The induction of apoptosis by ROS of endothelial cells may be related to initiation of atherosclerotic changes.

A novel member of the epidermal growth factor (EGF) family, the neural- and thymus-derived activator for ErbB kinases (NTAK), has been purified and cloned. Five alternative spliced isoforms have been detected in the rat adrenal pheochromocytoma cell line, PC-12 cells. The rat NTAK alpha2a isoform exhibits 94% identity in its primary sequence with the human NTAK alpha isoform. In vivo, NTAK is only expressed in the brain of rat E11.5 embryos, and in the brain and thymus of adult rats. The soluble 46 kDa form binds directly to ErbB3 and B4, but not to ErbB1 or B2. NTAK, however, transactivates ErbB1 and B2 via heterodimerization with ErbB3 or B4. NTAK stimulates the differentiation of MDA-MB-453 cells and competitively inhibits the binding of [125I]neuregulin to these cells. In addition to these neuregulin-like properties, NTAK exhibits limited structural homology to neuregulins in the immunoglobulin (Ig)-like, EGF-like, and hydrophobic domains. Thus, NTAK appears to be a new member of the EGF family displaying neuregulin properties.

Heparin/heparan-sulfate proteoglycan (HSPG) binds to heparin-binding epidermal growth factor-like growth factor (HB-EGF) through its heparin-binding domain (HBD), which consists of 21 amino acid residues (P21). The CD9 antigen also interacts with a membrane-anchored form of HB-EGF (proHB-EGF) and enhances its juxtacrine activity. The CD9 antigen potentiates the juxtacrine activity of both proHB-EGF and proamphiregulin, but has no effect on proTGF-alpha. While both HB-EGF and amphiregulin contain an HBD, TGF-alpha does not. This suggests that the HBD of HB-EGF is also involved in CD9 antigen binding. Mutant CHO cells which lack HSPG recovered their capacity to bind to immobilized P21 when transfected with CD9 antigen cDNA. This binding was competitively inhibited by heparin in a dose-dependent manner. The interactions between synthetic peptides corresponding to the extracellular domain of CD9 antigen and the immobilized P21 were analyzed with surface plasmon resonance. The 119VIKEVQEFYKDTYNKLKTKD138 sequence of the CD9 antigen is thought to represent the binding site for HB-EGF. The k(D) values for heparin/P21 and 119V-D138/P21 were (2.82+/-0.10) x 10(-8) M and (3.71+/-0.71) x 10(-5) M, respectively. These results suggest that the 119V-D138 sequence of the CD9 antigen is the site which interacts with the HBD and may play an essential role in the upregulation of the juxtacrine activity of proHB-EGF.

GDP-L-Fuc:N-acetyl-beta-D-glucosaminide:alpha1-6 fucosyltransferase (alpha1-6 FucT), which catalyzes the transfer of fucose from GDP-Fuc to N-linked type complex glycopeptides, was purified from a culture supernatant of human gastric cancer cell line MKN45. The purification procedures included chromatographies on Q-Sepharose Fast Flow, synthetic GDP-hexanolamine-Sepharose, and GnGn-bi-Asn-Sepharose columns. SDS-PAGE of the purified enzyme gave a major band corresponding to an apparent molecular mass of 60 kDa. The enzyme was recovered in a 12% final yield with an approximately 4,600-fold increase in specific activity. The pH optimum was 7.5, and the enzyme was fully active in the presence of 5 mM EDTA and did not require divalent cations, Mg2+ and Ca2+. Oligonucleotide primers designed from partial amino acid sequences were used to amplify and clone alpha1-6 FucT cDNA from a cDNA library of MKN45 cells. The cDNA encodes 575 amino acids in length, and contains the predicted N-terminal and internal amino acid sequences derived on lysyl endopeptidase digestion. The homology to porcine brain alpha1-6 FucT is 92.2% at the nucleotide level and 95.7% at the amino acid level. No putative N-glycosylation sites were found in the predicted amino acid sequence of the human MKN45 cell enzyme or that of porcine brain. Thus, the enzyme is distinct from other fucosyltransferases which catalyze alpha1-2, alpha1-3, and alpha1-4 fucose addition.

An assay method for GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha 1-6fucosyltransferase (alpha 1-6FucT; EC 2.4.1.68) activity has been developed, involving a fluorescent pyridylaminated substrate. A glycopeptide derived from bovine gamma-globulin was coupled with 4-(2-pyridylamino)butylamine (PABA) through the peptide bond, and the following substrate was obtained. [equation: see text] The substrate and guanosine diphospho-fucopyranoside (GDP-Fuc) were incubated with a crude enzyme extract for 2 h, and then the enzymatic product was separated by reversed phase HPLC. Quantitation of the product involved measurement of the fluorescence intensity of the fucosylated pyridylaminated sugar. The structures of both synthesized GnGn-bi-Asn-PABA (substrate), and synthesized GnGnF-bi-Asn-PABA (product) were analyzed by 1H NMR. The enzymatic product was also analyzed by 1H NMR and was found to have alpha 1-6fucose at the reducing end GlcNAc. This method is highly specific for alpha 1-6FucT and is applicable for various experiments, including purification and cell culture ones.

gamma-Glutamyl transpeptidase possesses two histidine residues at positions 383 and 505 which are conserved in all mammalian and bacterial species. In order to elucidate the functions of these residues, we prepared mutants in which these residues were replaced by Ala. Kinetic analysis of the hydrolysis of L-gamma-glutamyl-p-nitroanilide indicated that substitution at His-383 decreased the Vmax value to 14% of that of the wild type, but had no effect on Vmax/K(m). In reactions involving glycylglycine as the acceptor substrate, the Vmax value of this mutant decreased to 38% with little alteration of Vmax/K(m) for L-gamma-glutamyl-p-nitroanilide as a gamma-glutamyl donor, but with a significant reduction of Vmax/K(m) for the acceptor. These results show that this substitution causes impairment of the step in which the free enzyme is regenerated from the gamma-glutamyl enzyme by water or an acceptor substrate. On the other hand, replacement of His-505 resulted in a decrease of the Vmax value for transpeptidation to about 10% of that of the wild type despite no substantial effect on the Vmax value for the hydrolysis reaction. However, this substitution did not affect Vmax/K(m) for the acceptor on transpeptidation. Thus, the formation of a non-productive enzyme-substrate complex with the acceptor substrate would decrease the Vmax value on transpeptidation. These results suggest that His-383 plays an important catalytic role in facilitating the degradation of the gamma-glutamyl-enzyme through hydrolysis or transfer of the gamma-glutamyl moiety to an acceptor. It was also shown that His-505 is important in the formation of a complex of the gamma-glutamyl enzyme with the acceptor substrate even though it plays no critical role in the catalysis. Although the pH-dependence profile and the van't Hoff plot for the ionic group responsible for enzyme activity were consistent with the requirement of a histidine residue, neither of the conserved histidines could be assigned as such an ionic group. This suggests that another histidine residue(s) might play an essential role in the enzyme function.

Selenium-dependent glutathione peroxidase (GPx) plays a protective role in oxidative stress-induced apoptosis. In this study, we demonstrated that MDBK cells, a bovine renal epithelial cell line, exhibited internucleosomal DNA fragmentation characteristic of apoptotic cell death under selenium-deficient conditions with lower doses of hydrogen peroxide (H2O2) than under selenium-supplemented ones. This was due to a decreased amount of GPx in the cells under selenium-deficient conditions, because other antioxidative enzyme activities were not affected by the selenium supplementation. Cumene hydroperoxide also induced DNA fragmentation in selenium-deficient cells but no ladder formation was observed. Flow cytometric analysis showed that selenium-deficient cells were less capable of scavenging intracellular peroxides after exposure to exogenous H2O2 than selenium-supplemented ones. In contrast, there was no difference in viability between selenium-supplemented and non-supplemented cells in cell survival after exposure to menadione, which activates the electron transport system and increases intracellular superoxide radicals. Clofibrate, a peroxisomal proliferator and an inducer of catalase (CAT), partially protected both Se-deficient and Se-supplemented cells from exogenous H202. We concluded that selenium-deficient cells were more easily brought to apoptotic cell death by peroxides, but not by superoxide radicals, than selenium-supplemented ones and that CAT could compensate for the depletion of GPx to a certain degree by scavenging H2O2.

Nitric oxide (NO) is synthesized from L-arginine by three isoforms of NO synthase (NOS). It is essential to suppress the function of the inducible isoform (macNOS) for amelioration of some inflammatory diseases in which the cytotoxic effect of NO is involved. S-Ethylsiothiourea (S-EIU) was reported to be a potent and specific inhibitor of macNOS. We also confirmed that it rather specifically inhibited the activity of the purified macNOS and the formation of nitrite by RAW264.7 cells compared to NG-monomethyl-L-arginine (L-NMA) and NG-nitro-L-arginine (L-NNA), the other isoforms being less effective. S-EIU suppressed the release of nitrite and lactate dehydrogenase from rat vascular smooth muscle cells treated with interleukin-1 beta and forskolin more potently than L-NMA or L-NNA. S-EIU also slightly suppressed internucleosomal DNA cleavage in pancreatic beta-cells induced by NO produced by macNOS. Intravenous administration of either S-EIU at 0.1 mg/kg/min or L-NMA at 1 mg/kg/min increased the blood pressure but decreased the heart rate in normal rabbits, while aminoguanidine at 1 mg/kg/min affected neither cardiovascular function. These inhibitors at these doses caused recovery of the blood pressure in lipopolysaccharide-treated rabbits that exhibited lowered blood pressure similar to that in the case of septic shock. Although S-EIU seemed not to be an adequate inhibitor for therapeutic use in vivo due to its side effects on cardiovascular functions, it is one of the most potent inhibitors of macNOS among reported inhibitors in vitro.

Nitric oxide synthase (NOS) has been purified over 6,500-fold with a 3.4% yield from rat colorectum with 2',5'-ADP-Sepharose, DEAE cellulose, and gel filtration. The purified enzyme gave a single band corresponding to an apparent molecular mass of 160 Dka on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When assayed in the requisite presence of L-arginine, CaCl2, NADPH, calmodulin, tetrahydro-L-biopterin, and FAD, the purified enzyme exhibited a specific activity of 328 nmol/min/mg L-citrulline formed and an apparent Km for L-arginine of 2.9 microM. Amino acid sequencing of 12 peptides revealed identical sequences to that of the neuronal type enzyme except for two altered amino acid residues. When partial reverse transcription-polymerase chain reaction of RNA from rat colorectum and cerebellum was performed using primers designed according to the amino acid sequences determined, these amino acid changes were found in both cDNA fragments, indicating the identity of the colorectal enzyme to the cerebellar one. A polyclonal antibody raised against NOS purified from rat cerebellum cross-reacted with the NOS from colorectum but not that from IFN-gamma stimulated macrophage-derived cells, RAW 264.7. Immunohistochemical analysis of the colorectum using this specific antibody indicated that Auerbach's plexus is strongly immunoreactive, supporting the hypothesis that NO is an inhibitory transmitter for non-adrenergic and non-cholinergic nerves in the colorectum.

UDP-N-acetylglucosamine:beta-D-mannoside beta 1,4 N-acetylglucosaminyltransferase III (GnT-III) [EC 2.4.1.144] catalyzes the addition of N-acetylglucosamine in beta 1-4 linkage to the beta-linked mannose of the trimannosyl core of N-linked sugar chains to produce a bisecting GlcNAc residue. We have isolated six independent cDNA clones of human GnT-III from a fetal liver cDNA library. The cDNA sequence has an open reading frame that predicts a protein of 531 amino acids. The homology to rat GnT-III is 86% at the nucleotide level and is 91% at the amino acid level. The amino-terminal transmembrane domain and the catalytic domain are well conserved in the two species. Human GnT-III has a deletion of four amino acids in the "neck" region and several differences in the COOH-terminal region compared with the rat sequence. Using one of the human cDNA clones as the probe, two overlapping genomic clones have been isolated from a human cosmid library. The GnT-III gene has been mapped to chromosome 22q.13.1 using fluorescence in situ hybridization.

A beta 1-6N-acetylglucosaminyltransferase (GnT-V) [EC 2.4.1.155] which catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to alpha-D-6-mannoside has been purified up to 20,000-fold from the cultured supernatant of the QG small lung cancer cell line with a 37% yield. The isolation procedure included chromatography on phenyl-Sepharose, hydroxylapatite, UDP-hexanolamine Sepharose, and a biantennary sugar substrate (GnGn-bi-Asn) coupled to activated CH-Sepharose 4B. Sodium dodecyl sulfate gel electrophoresis under non-reducing conditions showed a single band of 73 kDa. Under reducing conditions, however, an additional component of 60 kDa was seen. Peptide mapping analysis indicated that both of these proteins were essentially identical, indicating that the 60-kDa component is probably a proteolytically cleaved form of the 73-kDa protein. Studies on the activity of the enzyme toward a variety of pyridylaminated sugars showed that the enzyme is most active toward triantennary (GnGnGn-tri-PA) and biantennary (GnGn-bi-PA) sugars. The Km values for GnGn-bi-PA and UDP-GlcNAc were 133 microM and 3.5 mM, respectively. These studies represent the first report of the enzymatic properties of a highly purified human GnT-V.

Activator protein for galactosylceramide sulfatase (GSase) was purified from human liver. The activator has an approximate molecular weight of 22,000, is glycoprotein in nature, and is most probably a trimer consisting of an 8,000 dalton monomer. Monospecific rabbit antiserum raised against the activator strongly inhibited the activity of the activator. In the presence of a 10-fold or more excess of galactosylceramide sulfate (GS) on a molar basis, GS binding to the GSase activator occurred, and was saturated at an equimolar ratio. Binding studies on the GSase activator were conducted using affinity chromatography on derivatives of GS as ligands, and gel filtration of mixtures containing glycolipids and the activator. A "GS-acid" derivative, which was prepared by oxidative cleavage of sphingosine moiety in GS, and a sulfonamide derivative of GS as ligands still retained affinity for the GSase activator, while a hydrophobic ligands, an aminohexyl group did not bind completely the activator. A ligand of "galactosylceramide-acid" had weak affinity for GSase activator. These results suggest that the sulfate group and one of the two hydrocarbon chains in GS are not essential for the binding of the activator. The affinity of galactosylceramide for the GSase activator was confirmed by the detection of the lipid-protein complex on gel filtration. The activator weakly stimulated porcine GM1-beta-galactosidase activity.

beta-N-Acetylhexosaminidase (hexosaminidase) I, which has an intermediate charge character between those of hexosaminidases A(alpha beta 2) and B[beta beta)2), was purified 1,500-fold from human placenta by procedures including chromatographies on concanavalin A (Con A)-Sepharose and an immunoadsorbent column. The isolated hexosaminidase I was heat-stable, and antigenically cross-reactive to anti-beta chain-IgG but not to anti-alpha chain-IgG. The results of substrate specificity experiments using 3H-labeled natural substrates indicated that the hexosaminidase I hydrolyzed Gb4Cer to Gb3Cer but not GM2 to GM3. The tryptic peptide map of the hexosaminidase I was similar to that of hexosaminidase B, though some differences were observed. The hexosaminidase I after treatment with neuraminidase or endo-beta-N-acetylglucosaminidase H was partly converted to less acidic forms. Treatment of the hexosaminidase I with acid phosphatase did not change the charge character. Therefore hexosaminidase I is an acidic variant form of hexosaminidase B, possibly resulting from sialylation and the presence of phosphodiester bonds at the carbohydrate moiety.