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Biomedical subjects

Naoyuki Taniguchi

Publications and source records attributed to Naoyuki Taniguchi.

At least 73 records · Page 4Linked to original sources
PubMed2004

Skin graft of double transgenic pigs of N-acetylglucosaminyltransferase III (GnT-III) and DAF (CD55) genes survived in cynomolgus monkey for 31 days.

BACKGROUND: Our previous study reported that cynomolgus monkey did not hyperacutely reject a skin xenograft from a N-acetylglucosaminyltransferase III (GnT-III) transgenic pig. In the present study, we reported on the survival time of skin xenografts in GnT-III, DAF (CD55), and double (D/G) transgenic pigs, and the effect of FK506 thereon. MATERIAL AND METHODS: Skin from GnT-III, DAF and D/G transgenic pigs were transplanted to cynomolgus monkeys. Under general anesthesia, full thickness skin defects (1.5 x 1.5 cm each) were made on the back of the monkey. Pig abdominal skin, obtained using an electric dermatome, was cut into pieces and transplanted onto the monkey wounds and fixed. In addition, skins of GnT-III and D/G pigs were also transplanted to cynomolgus monkeys that had been treated intramuscularly with FK506 at a dose of 0.5 mg/kg/day for 14 days after transplantation. Grafts were observed and photographed each day and skin graft biopsies were done on days 3, 5, 7, 10, 11, 14, 21, 28 and 31 after transplantation. Graft rejection was assessed histologically, based on our previous criteria for skin allografts. RESULTS: Even in the immuno-suppressive drug free condition, skin xenografts of GnT-III, DAF and D/G transgenic pigs were not hyperacutely rejected in early phase after transplantation by the cynomolgus monkey. The pattern of these xenograft rejections was histologically similar to those for rat allograft rejections. Most of the GnT-III, DAF and D/G pig skin grafts remained nearly intact up to day 5, but slight lymphocyte infiltration was noted on day 7 (grade 1). On day 9, while the GnT-III skin showed moderate lymphocyte and eosinophilic infiltration, the DAF and D/G pig skin grafts showed complete epidermal separation (grade 3). On the other hand, in the case of cynomolgus monkeys treated with FK506, the GnT-III skin showed complete epidermal separation (grade 3) on day 21. In addition, one of the D/G skin graft was intact on day 21 and moderate lymphocyte infiltration and intraepidermal blister formation (grade 1) was finally seen on day 31. CONCLUSION: Our data show the possibility that both the DAF and GnT-III double transgenic pig skin xenografts can be used in place of human skin allografts in cases of severe burns.

Animals↗
PubMed2004

Role of N-glycans in growth factor signaling.

Secreted proteins and membrane proteins are frequently post-translationally modified by oligosaccharides. Therefore, many glycoproteins are involved in signal transduction. One example is growth factor receptors, which are membrane proteins that often contain oligosaccharides. The oligosaccharides in those growth factor receptors play crucial roles in receptor functions. An analysis of glycosyltransferase-transfectants revealed that the branching structures of oligosaccharide also serve as important determinants. For example, N-glycans of epidermal growth factor receptor (EGFR) are involved in receptor sorting, ligand binding and dimerization. The addition of a bisecting GlcNAc to N-glycans increases the endocytosis of EGFR. N-glycans of Trk, a high affinity nerve growth factor receptor, also affect its function. Thus, oligosaccharides play an important role in growth factor signaling.

Animals↗
PubMed2004

Regulation of integrin functions by N-glycans.

Integrins are cell surface transmembrane glycoproteins that function as adhesion receptors in cell-ECM interactions and link matrix proteins to the cytoskeleton. Integrins play an important role in cytoskeleton organization and in the transduction of intracellular signals, regulating various processes such as proliferation, differentiation, apoptosis, and cell migration. Although integrin-mediated adhesion is based on the binding of alpha and beta subunits to a defined peptide sequence, the strength of this binding is modulated by various factors including the status of glycosylation of integrin. Glycosylation reactions are catalyzed by the catalytic action of glycosyltransferases, such as N-acetylglucosaminyltransferase III, V and alpha1, 6 fucosyltransferase, etc., which catalyze the formation of glycosidic bonds. This review summarizes effects of the posttranslational modification of N-glycans of alpha3beta1 and alpha5beta1 integrins on their association, activation and biological functions, by using biochemical and genetic approaches.

Animals↗
PubMed2004

Biological roles of sulfoglycolipids and pathophysiology of their deficiency.

Mammalian sulfoglycolipids are comprised of two major members, sulfatide (SO(3)-3Gal-ceramide) and seminolipid (SO(3)-3Gal-alkylacylglycerol). Sulfatide is abundant in the myelin sheath and seminolipid is expressed on the spermatogenic cells. Cerebroside sulfotransferase (CST)-deficient mice generated by gene targeting completely lack sulfatide and seminolipid all over the body. CST-null mice manifest some neurological disorders due to myelin dysfunction, an aberrant enhancement of oligodendrocyte terminal differentiation, and an arrest of spermatogenesis, indicating that sulfation of glycolipids is essential for myelin formation and spermatogenesis. Moreover, CST-deficiency ameliorates L-selectin-dependent monocyte infiltration in the kidney after ureteral obstruction, an experimental model of renal interstitial inflammation, indicating that sulfatide is an endogenous ligand of L-selectin. Studies on the molecular mechanisms by which sulfoglycolipids participate in these biological processes are ongoing.

Animals↗
PubMed2004

Attachment of human colon cancer cells to vascular endothelium is enhanced by N-acetylglucosaminyltransferase V.

Expression of N-acetylglucosaminyltransferase V (GnT-V) in colon cancer has been shown to be related to hematogenous metastasis and poor prognosis. To investigate the mechanism by which cancer cells expressing GnT-V metastasize to distant organs, we established GnT-V-overexpressing DLD-1 and WiDr cells (human colon cancer cell lines) by transfecting them with a GnT-V expression vector. Attachment to endothelial cells expressing E-selectin was studied, and expression of the E-selectin ligand, sialyl Lewis x, in colon cancer cells was investigated. Both of the cell lines showed reduced adhesion to fibronectin as compared with mock transfectants. In contrast, attachment to human umbilical vein endothelial cells expressing E-selectin was significantly enhanced by GnT-V expression (p < 0.01). Sialyl Lewis x is a ligand for E-selectin and a marker for poor prognosis of colon cancer. Its synthesis in cells has been shown to involve GnT-V. We demonstrated that expression of sialyl Lewis x in colon cancer cells was induced by GnT-V expression. These results suggest that GnT-V induces sialyl Lewis x expression and leads colon cancer cells to metastasize by enhancing their ability to attach to vascular endothelium in distant organs, such as liver or lung. Inhibition of GnT-V activity may prevent metastasis in colon cancer patients with high sialyl Lewis x expression.

Biomarkers, Tumor↗
PubMed2003

Gamma-glutamyltranspeptidase stimulates receptor activator of nuclear factor-kappaB ligand expression independent of its enzymatic activity and serves as a pathological bone-resorbing factor.

A novel bone-resorbing factor was cloned using an expression cloning technique, which involved a Xenopus oocyte expression system and an assay for osteoclast formation. A candidate clone was isolated from a BW5147 mouse T-lymphoma cell cDNA library. Sequencing analysis identified the factor as gamma-glutamyltranspeptidase (GGT), which is an enzyme involved in glutathione metabolism. The addition of purified GGT protein to mouse bone marrow culture effectively induced formation of osteoclasts. An antibody against GGT inhibited osteoclast formation but not the enzymatic activity. We also demonstrated that an inactive form of GGT, the enzymatic activity of which had been blocked by chemical modification with a specific inhibitor, acivicin, supported osteoclast formation. These results indicate that GGT acts on osteoclast formation independent of its own enzymatic activity. Furthermore, both native GGT and inactive GGT stimulated the expression of the receptor activator of nuclear factor-kappaB ligand (RANKL) mRNA and protein from bone marrow stromal cells. This report is the first demonstration of a novel biological activity of GGT protein in a manner independent of its enzymatic activity.

Animals↗
PubMed2003

N-Acetylglucosaminyltransferase IX acts on the GlcNAc beta 1,2-Man alpha 1-Ser/Thr moiety, forming a 2,6-branched structure in brain O-mannosyl glycan.

Mammals contain O-linked mannose residues with 2-mono- and 2,6-di-substitutions by GlcNAc in brain glycoproteins. It has been demonstrated that the transfer of GlcNAc to the 2-OH position of the mannose residue is catalyzed by the enzyme, protein O-mannose beta1,2-N-acetylglucosaminyltransferase (POMGnT1), but the enzymatic basis of the transfer to the 6-OH position is unknown. We recently reported on a brain-specific beta1,6-N-acetylglucosaminyltransferase, GnT-IX, that catalyzes the transfer of GlcNAc to the 6-OH position of the mannose residue of GlcNAcbeta1,2-Manalpha on both the alpha1,3- and alpha1,6-linked mannose arms in the core structure of N-glycan (Inamori, K., Endo, T., Ide, Y., Fujii, S., Gu, J., Honke, K., and Taniguchi, N. (2003) J. Biol. Chem. 278, 43102-43109). Here we examined the issue of whether GnT-IX is able to act on the same sequence of the GlcNAcbeta1,2-Manalpha in O-mannosyl glycan. Using three synthetic Ser-linked mannose-containing saccharides, Manalpha1-Ser, GlcNAcbeta1,2-Manalpha1-Ser, and Galbeta1,4-GlcNAcbeta1,2-Manalpha1-Ser as acceptor substrates, the findings show that (14)C-labeled GlcNAc was incorporated only into GlcNAcbeta1,2-Manalpha1-Ser after separation by thin layer chromatography. To simplify the assay, high performance liquid chromatography was employed, using a fluorescence-labeled acceptor substrate GlcNAcbeta1,2-Manalpha1-Ser-pyridylaminoethylsuccinamyl (PAES). Consistent with the above data, GnT-IX generated a new product which was identified as GlcNAcbeta1,2-(GlcNAcbeta1,6-)Manalpha1-Ser-PAES by mass spectrometry and (1)H NMR. Furthermore, incorporation of an additional GlcNAc residue into a synthetic mannosyl peptide Ac-Ala-Ala-Pro-Thr(Man)-Pro-Val-Ala-Ala-Pro-NH(2) by GnT-IX was only observed in the presence of POMGnT1. Collectively, these results strongly suggest that GnT-IX may be a novel beta1,6-N-acetylglucosaminyltransferase that is responsible for the formation of the 2,6-branched structure in the brain O-mannosyl glycan.

Brain↗
PubMed2003

Expression of alpha1,6-fucosyltransferase (FUT8) in papillary carcinoma of the thyroid: its linkage to biological aggressiveness and anaplastic transformation.

Previous studies have demonstrated that terminal fucosylation is associated with the biological aggressiveness of carcinomas, but the significance of core fucosylation (alpha1,6-fucosylation) through alpha1,6-fucosyltransferase (FUT8) has not been studied in depth. Herein, we investigated the expression of alpha1,6-fucosyltransferase (FUT8) in 133 cases of thyroid carcinomas using an immunohistochemical approach. The expression of FUT8 was quite low in normal follicules. A high expression of FUT8 was observed in 33.3% of papillary carcinoma and the incidence was directly linked to tumor size and lymph node metastasis. In contrast, this phenomenon was less frequently observed in follicular carcinoma and anaplastic (undifferentiated) carcinoma. These results suggest that FUT8 expression may be a key factor in the progression of thyroid papillary carcinomas, but not follicular carcinomas, and decreases in FUT8 expression might be linked to anaplastic transformation.

Carcinoma↗
PubMed2003

Cerebroside sulfotransferase deficiency ameliorates L-selectin-dependent monocyte infiltration in the kidney after ureteral obstruction.

Mononuclear cells infiltrating the interstitium are involved in renal tubulointerstitial injury. The unilateral ureteral obstruction (UUO) is an established experimental model of renal interstitial inflammation. In our previous study, we postulated that L-selectin on monocytes is involved in their infiltration into the interstitium by UUO and that a sulfated glycolipid, sulfatide, is the physiological L-selectin ligand in the kidney. Here we tested the above hypothesis using sulfatide- and L-selectin-deficient mice. Sulfatide-deficient mice were generated by gene targeting of the cerebroside sulfotransferase (Cst) gene. Although the L-selectin-IgG chimera protein specifically bound to sulfatide fraction in acidic lipids from wild-type kidney, it did not show such binding in fractions of Cst(-/-) mice kidney, indicating that sulfatide is the major L-selectin-binding glycolipid in the kidney. The distribution of L-selectin ligand in wild-type mice changed after UUO; sulfatide was relocated from the distal tubules to the peritubular capillaries where monocytes infiltrate, suggesting that sulfatide relocated to the endothelium after UUO interacted with L-selectin on monocytes. In contrast, L-selectin ligand was not detected in Cst(-/-) mice irrespective of UUO treatment. Compared with wild-type mice, Cst(-/-) mice showed a considerable reduction in the number of monocytes/macrophages that infiltrated the interstitium after UUO. The number of monocytes/macrophages was also reduced to a similar extent in L-selectin(-/-) mice. Our results suggest that sulfatide is a major L-selectin-binding molecule in the kidney and that the interaction between L-selectin and sulfatide plays a critical role in monocyte infiltration into the kidney interstitium.

Animals↗
PubMed2003

Beta1,4-N-Acetylglucosaminyltransferase III down-regulates neurite outgrowth induced by costimulation of epidermal growth factor and integrins through the Ras/ERK signaling pathway in PC12 cells.

A rat pheochromocytoma cell line (PC12), when transfected with beta1,4-N-acetylglucosaminyltransferase III (GnT-III), which catalyzes the formation of a bisecting GlcNAc structure in N-glycans, resulted in the suppression of neurite outgrowth induced by costimulation of epidermal growth factor (EGF) and integrins. The neurite outgrowth was restored by the overexpression of a constitutively activated mitogen- or extracellular signal-regulated kinase kinase-1 (MEK-1). Consistent with this, the EGF receptor (EGFR)-mediated ERK activation was blocked in GnT-III transfectants. Conversely, the overexpression of dominant negative MEK-1 or treatment with PD98059, a specific inhibitor of MEK-1, inhibited neurite outgrowth in controls transfected with mock. Furthermore GnT-III activity is required for these inhibitions, because the overexpression of a dominant negative GnT-III mutant (D321A) failed to reduce neurite outgrowth and EGFR-mediated ERK activation. Lectin blot analysis confirmed that EGFR from wild-type GnT-III transfectants had been modified by bisecting GlcNAc in its N-glycan structures. This modification led to a significant decrease in EGF binding and EGFR autophosphorylation. Collectively, the results constitute a comprehensive body of evidence to show clearly that the overexpression of GnT-III prevents neurite outgrowth induced by costimulation of EGF and integrins through the Ras/MAPK activation pathway and indicates that GnT-III may be an important regulator for cell differentiation in neural tissues.

Animals↗
PubMed2003

Addition of beta1-6 GlcNAc branching to the oligosaccharide attached to Asn 772 in the serine protease domain of matriptase plays a pivotal role in its stability and resistance against trypsin.

beta1-6 GlcNAc branching, a product of N-acetylglucosaminyltransferase V (GnT-V), is a key structure that is associated with malignant transformations and cancer metastasis. Although a number of reports concerning tumor metastasis-related glycoproteins that contain beta1-6 GlcNAc branching have appeared, the precise function of beta1-6 GlcNAc branching on glycoproteins remains to be elucidated. We previously reported on the importance of beta1-6 GlcNAc branching on matriptase in terms of proteolytic degradation in tumor metastasis. We report here that matriptase purified from GnT-V transfectant (beta1-6 GlcNAc matriptase) binds strongly to L4-PHA, which preferentially recognizes beta1-6 GlcNAc branches of tri- or tetraantennary sugar chains, indicating that the isolated matriptase contains beta1-6 GlcNAc branching. The beta1-6 GlcNAc matriptase was resistant to autodegradation, as well as trypsin digestion, compared with matriptase purified from mock-transfected cells. Furthermore, N-glycosidase-F treatment of beta1-6 GlcNAc matriptase greatly reduced its resistance to degradation. An analysis of matriptase mutants that do not contain potential N-glycosylation sites clearly shows that the beta1-6 GlcNAc branching on N-glycans attached to Asn 772 in the serine protease domain plays a major role in trypsin resistance. This is the first example of a demonstration of a direct relationship between beta1-6 GlcNAc branching and a biological function at the protein level.

Amino Acid Sequence↗
PubMed2003

Relationship between elevated FX expression and increased production of GDP-L-fucose, a common donor substrate for fucosylation in human hepatocellular carcinoma and hepatoma cell lines.

The levels of fucosylated glycoproteins in various cancers and inflammatory processes have been a subject of intense study. The level of fucosyltransferases and intracellular GDP-L-fucose, a sugar nucleotide and a common donor substrate for all fucosyltransferases, may regulate the level of fucosylated glycoproteins. This study reports on the determination of GDP-L-fucose levels in human hepatocellular carcinoma (HCC) and surrounding tissues, using a recently established high-throughput assay system. Levels of GDP-L-fucose in HCC tissues were significantly increased compared with adjacent nontumor tissues or normal livers. The mean +/- SD for GDP-L-fucose level was 3.6 +/- 0.2 micro mol/mg in control liver, 4.6 +/- 0.9 micro mol/mg in adjacent noninvolved liver tissues (chronic hepatitis, 4.4 +/- 0.7 micro mol/mg; liver cirrhosis, 4.8 +/- 0.9 micro mol/mg), and 7.1 +/- 2.5 micro mol/mg in HCC tissues. The level of GDP-L-fucose in HCC decreased in proportion with tumor size (r = -0.675, P = 0.0002). When expression of the series of genes responsible for GDP-L-fucose synthesis was investigated, the gene expression of FX was found to be increased in 70% (7 of 10) of the HCC tissues examined compared with that in their surrounding tissues. The levels of GDP-L-fucose were positively correlated with the expression of FX mRNA (r = 0.599, P = 0.0074). The levels of FX gene expression in some human hepatoma and hepatocyte cell lines were determined. FX mRNA production was strongly increased in HepG2 and Chang liver, moderately increased in Hep3B and HLF, and, in HLE, was similar to that of a normal human liver tissue. To investigate the effect of GDP-L-fucose on core fucosylation, FX cDNA was transfected into Hep3B cells, which express a relatively low level of GDP-L-fucose:N-acetyl-beta-D-glucosaminide alpha1-6 fucosyltransferase (alpha1-6 FucT) and FX mRNA. Transfection of this gene caused an increase in GDP-L-fucose levels as well as the extent of fucosylation on glycoproteins, including alpha-fetoprotein, as judged by reactivity to lectins. Collectively, the results herein suggest that the high level of fucosylation in HCC is dependent on a high expression of FX followed by increases in GDP-L-fucose, as well as an enhancement in alpha1-6 FucT expression. Thus, an elevation in GDP-L-fucose levels and the up-regulation of FX expression represent potential markers for HCC.

Carbohydrate Sequence↗
PubMed2003

Molecular cloning and characterization of human GnT-IX, a novel beta1,6-N-acetylglucosaminyltransferase that is specifically expressed in the brain.

A novel beta1,6-N-acetylglucosaminyltransferase (beta1, 6GnT) cDNA was identified by a BLAST search using the amino acid sequence of human GnT-V as a query. The full-length sequence was determined by a combination of 5'-rapid amplification of cDNA end analysis and a further data base search. The open reading frame encodes a 792 amino acid protein with a type II membrane protein structure typical of glycosyltransferases. The entire sequence identity to human GnT-V is 42%. When pyridylaminated (PA) agalacto biantennary N-linked oligosaccharide was used as an acceptor substrate, the recombinant enzyme generated a novel product other than the expected GnT-V product, (GlcNAcbeta1,2-Manalpha1,3-)[GlcNAcbeta1,2-(GlcNAcbeta1,6-)Manalpha1,6-]Manbeta1,4-GlcNAcbeta1,4-GlcNAc-PA. This new product was identified as [GlcNAcbeta1,2-(GlcNAcbeta1,6-)Manalpha1,3-][Glc-NAcbeta1,2-(GlcNAcbeta1,6-)Manalpha1,6-]Manbeta1,4-GlcNAcbeta1,4-GlcNAc-PA by mass spectrometry and 1H NMR. Namely, the new GnT (designated as GnT-IX) has beta1,6GnT activity not only to the alpha1,6-linked mannose arm but also to the alpha1,3-linked mannose arm of N-glycan, forming a unique structure that has not been reported to date. Northern blot analysis showed that the GnT-IX gene is exclusively expressed in the brain, whereas the GnT-V gene is expressed ubiquitously. These results suggest that GnT-IX is responsible for the synthesis of a unique oligosaccharide structure in the brain.

Amino Acid Sequence↗
PubMed2003

Caveolin-1 regulates the functional localization of N-acetylglucosaminyltransferase III within the golgi apparatus.

In an investigation of the mechanism underlying the functional sublocalization of glycosyltransferases within the Golgi apparatus, caveolin-1 was identified as a possible cellular factor. Caveolin-1 appears to regulate the localization of N-acetylglucosaminyltransferase III (GnT-III) in the intra-Golgi subcompartment. Structural analyses of total cellular N-glycans indicated that the overexpression of GnT-III in human hepatoma cells, in which caveolin-1 is not expressed, failed to reduce branch formation, whereas expression of caveolin-1 led to a dramatic decrease in the extent of branching with no enhancement in GnT-III activity. Because the addition of a bisecting GlcNAc by GnT-III to the core beta-Man in N-glycans prevents the action of GnT-IV and GnT-V, both of which are involved in branch formation, this result suggests that caveolin-1 facilitates the prior action of GnT-III, relative to the other GnTs, on the nascent sugar chains in the Golgi apparatus and that GnT-III is redistributed in the earlier Golgi subcompartment by caveolin-1. Indeed, when caveolin-1 was expressed in human hepatoma cells, it was found to be co-localized with GnT-III, as evidenced by the fractionation of Triton X-100-insoluble cellular membranes by density gradient ultracentrifugation. Caveolin-1 may modify the biosynthetic pathway of sugar chains via the regulation of the intra-Golgi subcompartment localization of this key glycosyltransferase.

Caveolin 1↗