Wilson disease is a genetic disorder characterized by the accumulation of copper in the body by defective biliary copper excretion. Wilson disease gene product (ATP7B) functions in copper ...incorporation to ceruloplasmin (Cp) and biliary copper excretion. However, copper metabolism in hepatocytes has been still unclear. Niemann-Pick disease type C (NPC) is a lipid storage disorder and the most commonly mutated gene is NPC1 and its gene product NPC1 is a late endosome protein and regulates intracellular vesicle traffic. In the present study, we induced NPC phenotype and examined the localization of ATP7B and secretion of holo-Cp, a copper-binding mature form of Cp. The vesicle traffic was modulated using U18666A, which induces NPC phenotype, and knock down of NPC1 by RNA interference. ATP7B colocalized with the late endosome markers, but not with the trans-Golgi network markers. U18666A and NPC1 knock down decreased holo-Cp secretion to culture medium, but did not affect the secretion of other secretory proteins. Copper accumulated in the cells after the treatment with U18666A. These findings suggest that ATP7B localizes in the late endosomes and that copper in the late endosomes is transported to the secretory compartment via NPC1-dependent pathway and incorporated into apo-Cp to form holo-Cp.
A novel sulfotransferase gene (designated GP3ST) was identified on human chromosome 2q37.3 based on its similarity to the cerebroside 3′-sulfotransferase (CST) cDNA (Honke, K., Tsuda, M., Hirahara, ...Y., Ishii, A., Makita, A., and Wada, Y. (1997) J. Biol. Chem. 272, 4864–4868). A full-length cDNA was obtained by reverse transcription-polymerase chain reaction and 5′- and 3′-rapid amplification of cDNA ends analyses of human colon mRNA. The isolated cDNA clone predicts that the protein is a type II transmembrane protein composed of 398 amino acid residues. The amino acid sequence indicates 33% identity to the human CST sequence. A recombinant protein that is expressed in COS-1 cells showed no CST activity, but did show sulfotransferase activities toward oligosaccharides containing nonreducing β-galactosides such as N-acetyllactosamine, lactose, lacto-N-tetraose (Lc4), lacto-N-neotetraose (nLc4), and Galβ1–3GalNAcα-benzyl (O-glycan core 1 oligosaccharide). To characterize the cloned sulfotransferase, a sulfotransferase assay method was developed that uses pyridylaminated (PA) Lc4 and nLc4 as enzyme substrates. The enzyme product using PA-Lc4 as an acceptor was identified as HSO3-3Galβ1–3GlcNAcβ1–3Galβ1–4Glc-PA by two-dimensional 1H NMR. Kinetics studies suggested that GP3ST is able to act on both type 1 (Galβ1–3GlcNAc-R) and type 2 (Galβ1–4GlcNAc-R) chains with a similar efficiency. In situ hybridization demonstrated that the GP3ST gene is expressed in epithelial cells lining the lower to middle layer of the crypts in colonic mucosa, hepatocytes surrounding the central vein of the liver, extravillous cytotrophoblasts in the basal plate and septum of the placenta, renal tubules of the kidney, and neuronal cells of the cerebral cortex. The results of this study indicate the existence of a novel β-Gal-3′-sulfotransferase gene family.
Sexual dimorphism in LEC rat liver Kuhara, Makihiko; Wang, Jingshu; Jolina Flores, Maria ...
Biomedical research (Tokyo),
03/2011, Volume:
32, Issue:
2
Journal Article
Peer reviewed
We examined age-related changes in the protein expression of carbonic anhydrase III (CAIII) in livers of Long-Evans with a cinnamon-like color (LEC) rats using an agouti color (LEA) rats as controls. ...The levels of the protein of CAIII in the liver of LEC male rats increased before 20 weeks of age, at the stage of acute hepatitis, and were decreased at 54 weeks of age, while those of CAIII in the liver of LEA male rats were highly expressed at all ages. In the normal LEA rats, CAIII showed sexual dimorphism. The level of CAIII in LEA male rat liver relative to female was four times higher. On the other hand, young LEC rat (at 4-12 weeks) showed a higher protein level of CAIII than LEA rats, and then decreased during development of hepatitis. CAIII mRNA also decreased in the LEC rat liver during hepatocarcinogenesis. The level of CAIII in the tumor region was lower than that in the tumor-free region. Immunohistochemical analysis showed that glutathione S-transferase P (GST-P) was positive and CAIII was negative in the precancerous region. The expression of CAIII was suppressed in cancerous lesions in hepatoma-bearing LEC rat liver compared to uninvolved surrounding tissues. These results indicated that suppression of CAIII accompanied hepatocarcinogenesis and it is a secondary consequence of the high copper levels in the liver.
The down-regulation of the alpha-Gal epitope (Galalpha1,3Galbeta-R) in swine tissues would be highly desirable, in terms of preventing hyperacute rejection in pig-to-human xenotransplantation. In an ...earlier study, we reported that the introduction of the beta1,4-N-acetylglucosaminyltransferase (GnT) III gene into swine endothelial cells resulted in a substantial reduction in the expression of the alpha-Gal epitope. In this study, we report on the mechanism for this down-regulation of the alpha-Gal epitope by means of structural and kinetic analyses. The structural analyses revealed that the amount of N-linked oligosaccharides bearing the alpha-Gal epitopes in the GnT-III-transfected cells was less than 10% that in parental cells, due to the alteration of the terminal structures as well as a decrease in branch formation. In addition, it appeared that the addition of a bisecting GlcNAc, which is catalyzed by GnT-III, leads to a more efficient sialylation rather than alpha-galactosylation. In vitro kinetic analyses showed that the bisecting GlcNAc has an inhibitory effect on alpha-galactosylation, but does not significantly affect the sialylation. These results suggest that the bisecting GlcNAc in the core is capable of modifying the biosynthesis of the terminal structures via its differential effects on the capping glycosyltransferase reactions. The findings may contribute to the development of a novel strategy to eliminate carbohydrate xenoantigens.
A reliable marker of chemoradiosensitivity that would enable appropriate and individualized treatment of thoracic squamous cell esophageal cancer has long been sought. We investigated whether ...regenerating gene (REG) Iα is such a marker. We assessed expression of REG Iα in untreated endoscopic biopsy specimens and examined the correlation between REG Iα expression and the clinical responses to definitive chemoradiotherapy and prognosis. We also examined the relationship between REG Iα expression in the resected tumor and the prognosis of patients who received esophagectomy for thoracic squamous cell esophageal cancer. Among the 42 patients treated with definitive chemoradiotherapy, 8 of the 23 REG I-positive patients (35%) showed complete responses to chemoradiotherapy, while only one of the 19 REG I-negative patients did so. The survival rate among the REG I-positive patients was significantly better than among the REG I-negative patients. For the 76 patients treated surgically, there was no significant difference in the survival rates among the REG I-positive and REG I-negative patients. REG Iα expression in squamous cell esophageal carcinoma may be a reliable marker of chemoradiosensitivity. We anticipate that it will enable us to provide more appropriate and individualized treatment to patients of advanced esophageal squamous cell carcinoma. PUBLICATION ABSTRACT
The α-Gal epitope (Gal-α1-3Gal-β1-4-GlcNAc-R), which is biosynthesized by the UDP-Gal:α1-3-galactosyltransferase (α1,3GT), is highly associated with hyperacute rejection in swine to human ...xenotransplantation. A variety of strategies have been pursued to reduce or eliminate this epitope from swine tissues. Since swine ES cells are not available at present, the targeted knock out of the α1,3GT is restricted. Other strategies, such as enzyme competition of the α1,3GT with other glycosyltransferases and/or control of sugar processing by the glycosyltransferases, provide a new insight into the downregulation of the α-Gal epitope. This review will focus on this type of strategy, which involves a gene transfection of variety of glycosyltransferases as competitors against α1,3GT.
The down-regulation of the α-Gal epitope (Galα1,3Galβ-R) in swine tissues would be highly desirable, in terms of preventing hyperacute rejection in pig-to-human xenotransplantation. In an earlier ...study, we reported that the introduction of the β1,4-N-acetylglucosaminyltransferase (GnT) III gene into swine endothelial cells resulted in a substantial reduction in the expression of the α-Gal epitope. In this study, we report on the mechanism for this down-regulation of the α-Gal epitope by means of structural and kinetic analyses. The structural analyses revealed that the amount of N-linked oligosaccharides bearing the α-Gal epitopes in the GnT-III-transfected cells was less than 10% that in parental cells, due to the alteration of the terminal structures as well as a decrease in branch formation. In addition, it appeared that the addition of a bisecting GlcNAc, which is catalyzed by GnT-III, leads to a more efficient sialylation rather than α-galactosylation. In vitro kinetic analyses showed that the bisecting GlcNAc has an inhibitory effect on α-galactosylation, but does not significantly affect the sialylation. These results suggest that the bisecting GlcNAc in the core is capable of modifying the biosynthesis of the terminal structures via its differential effects on the capping glycosyltransferase reactions. The findings may contribute to the development of a novel strategy to eliminate carbohydrate xenoantigens.
The effect of the various glycosyltransferases on glycosphingolipids was examined, using transfected swine endothelial cell (SEC) lines. The reactivity of parental SEC to normal human serum (NHS) and ...Griffonia simplicifolia IB(4) (GSIB4) lectin, which binds to the Gal alpha1-3 Gal beta 1-4 GlcNAc-R (alpha-galactosyl epitope), was reduced by approximately 20% by the treatment with D-PDMP (D-threo-1-phenyl-2-decan- oylamino-3-morpholino-1-propanol), suggesting that glycosphingolipids contained by SEC have a considerable amount of the alpha-galactosyl epitope. The overexpression of two different types of glycosyltransferase, N-acetylglucosaminyl transferase III (GnT-III), as well as alpha2, 6-sialyltransferase (ST6Gal I), alpha2,3-sialyltransferase (ST3Gal III), and alpha1,2-fucosyltransferase (alpha1,2FT), suppresses the total antigenicity of SEC significantly. However, the reduction in reactivities toward NHS and GSIB4 lectin in the case of GnT-III transfectants was milder than those in other transfectants. Western blot analysis indicated that the glycoproteins in all transfectants had diminished reactivity to NHS and GSIB4 lectin to approximately the same extent. Therefore, the neutral glycosphingolipids of these transfectants were separated by thin layer chromatography, followed by immunostaining with NHS and GSIB4 lectin. The levels of the alpha-galactosyl epitope in glycosphingolipids were not decreased in the GnT-III transfectants but were in the ST6Gal I, ST3Gal III, and alpha1,2FT transfectants. These data indicate that ST6Gal I, ST3Gal III, and alpha1,2FT reduced the alpha-galactosyl epitope in both glycoproteins and glycosphingolipids, while GnT-III reduced them only in glycoproteins.
The kinetic basis of the donor substrate specificity of β1,4-N-acetylgiucosaminyltrans-ferase HI (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 Vmax values, whereas the Km 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 giycosyl donors. Thus, GnT-HI 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.
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