Taeko Miyagi

Evidence for mitochondrial localization of a novel human sialidase (NEU4).

Based on the human cDNA sequence predicted to represent the NEU4 sialidase gene in public databases, a cDNA covering the entire coding sequence was isolated from human brain and expressed in mammalian cells. The cDNA encodes two isoforms: one possessing an N-terminal 12-amino-acid sequence that is predicted to be a mitochondrial targeting sequence, and the other lacking these amino acids. Expression of the isoforms is tissue specific, as assessed by reverse transcription-PCR. Brain, muscle and kidney contained both isoforms; liver showed the highest expression, and the short form was predominant in this organ. In transiently transfected COS-1 cells, enzyme activity was markedly increased with gangliosides as well as with glycoproteins and oligosaccharides as substrates compared with the control levels. This differs from findings with other human sialidases. Although the isoforms were not distinguishable with regard to substrate specificity, they exhibited differential subcellular localizations. Immunofluorescence microscopy and biochemical fractionation demonstrated that an exogenously expressed haemagglutinin-tagged long form of NEU4 was concentrated in mitochondria in several human culture cell types, whereas the short form was present in intracellular membranes, indicating that the sequence comprising the N-terminal 12 amino acid residues acts as a targeting signal for mitochondria. Co-localization of the long form to mitochondria was further supported by efficient targeting of the N-terminal region fused to enhanced green fluorescent protein, and by the targeting failure of a mutant with an amino acid substitution in this region. NEU4 is possibly involved in regulation of apoptosis by modulation of ganglioside G(D3), which accumulates in mitochondria during apoptosis and is the best substrate for the sialidase.

Plasma membrane production of ceramide from ganglioside GM3 in human fibroblasts

Ceramide is a key lipid molecule necessary to regulate some cellular processes, including apoptosis and cell differentiation. In this context, its production has been shown to occur via sphingomyelin hydrolysis or sphingosine acylation. Here, we show that in human fibroblasts, plasma membrane ceramide is also produced from ganglioside GM3 by detachment of sugar units. Membrane‐bound glycosylhydrolases have a role in this process. In fact, the production of ceramide from GM3 has been observed even under experimental conditions able to block endocytosis or lysosomal activity, and the overexpression of the plasma membrane ganglioside sialidase Neu3 corresponded to a higher production of ceramide in the plasma membrane. The increased activity of Neu3 was paralleled by an increase of GM3 synthase mRNA and GM3 synthase activity. Neu3‐overexpressing fibroblasts were characterized by a reduced proliferation rate and higher basal number of apoptotic cells in comparison with wild‐type cells. A similar behavior was observed when normal fibroblasts were treated with exogenous C2‐ceramide.—Valaperta, R., Chigorno, V., Basso, L., Prinetti, A., Bresciani, R., Preti, A., Miyagi, T., and Sonnino, S. Plasma membrane production of ceramide from ganglioside GM3 in human fibroblasts. FASEB J. 20, E450–E461 (2006)

Down-regulation of sialidase NEU4 may contribute to invasive properties of human colon cancers

In mammalian cells, four types of sialidase have been described and found to behave in different ways during carcinogenesis. We previously demonstrated that a human sialidase associated with plasma membranes (NEU3) is up‐regulated in human colon cancer and is involved in suppression of apoptosis. Here we document altered expression of another human sialidase, the recently identified NEU4, and evidence of its influence on the malignant phenotype in colon cancers. Human colon mucosa was relatively rich in NEU4, which has been observed to possess short and long isoforms, but hardly contained the latter form. In clear contrast to the NEU3 case, the levels of mRNA for this sialidase were found by quantitative RT‐PCR to be markedly decreased in colon cancers. In cultured human colon cancer cells, the enzyme was up‐regulated in the early stage of apoptosis induced by either the death ligand TRAIL or serum‐depletion, and transfection of NEU4 resulted in acceleration of apoptosis and in decreased invasion and motility. The siRNA‐mediated NEU4 targeting, on the other hand, caused a significant inhibition of apoptosis and promotion of invasion and motility. Lectin blot analyses revealed that desialylated forms of nearly 100 kDa glycoproteins were prominently increased with PNA in NEU4 transfectants, whereas only slight changes in glycolipids were detected as assessed by thin layer chromatography. These results suggest that NEU4 plays important roles for maintenance of normal mucosa mostly through desialylation of glycoproteins and that down‐regulation may contribute to invasive properties of colon cancers. (Cancer Sci 2007; 98: 299–307)

Role of Neu4L sialidase and its substrate ganglioside GD3 in neuronal apoptosis induced by catechol metabolites

Mammalian sialidases are key enzymes in the degradation of glycoconjugates. Neu4L sialidase is localized to mitochondria and specifically expressed in brain. To elucidate the pathophysiological roles of Neu4L in the nervous system, we investigated the possible involvement of Neu4L in the apoptotic neurodegeneration under the existence of catechol metabolites generated by tyrosinase. We demonstrated that: (i) the expression level of Neu4L was dramatically decreased prior to apoptosis; (ii) the apoptotic phenotype was characterized by cytochrome c release into cytosol concomitant with the trafficking of ganglioside GD3 to mitochondria; and (iii) the inhibitor of glucosylceramide synthase partially recovered cell viability. Neu4L and its substrate GD3 may act as key molecules in the mitochondrial apoptotic pathway in neuronal cells.

Evidence for sialidase hydrolyzing gangliosides GM2 and GM1 in rat liver plasma membrane

Rat liver plasma membrane removed sialic acid from mixed bovine brain gangliosides more efficiently than from sialyllactose and orosomucoid with an optimal pH of 4.5. When individual gangliosides, each labeled with [14C]sialic acid or [3H]sphingosine, were tested, not only GDla and GM3 but also GM2 and GM1, both of which had been considered to resist mammalian sialidases, were desialylated. The products of GM2 and GM1 hydrolysis were identified as asialo‐GM2 and asialo‐GM1, respectively, by thin‐layer chromatography.

Effect of phosphoglucose isomerase and glucose 6-phosphate on UDP-N-acetylglucosamine inhibition of L-glutamine-D-fructose 6-phosphate aminotransferase.

1. n1. l-Glutamine:d-fructose 6-phosphate aminotransferase (EC 2.6.1.16) was purified about 30-fold from rat liver extracts with a recovery of about 70%. Fractionation involved (NH4)2SO4 precipitation and DEAE-Sephadex column chromatography. Further purification (240-fold) was possible by use of hydroxylapatite chromatography. n n2. n2. The sensitivity of aminotransferase to UDP-N-acetylglucosamine inhibition was markedly reduced when the (NH4)2SO4 precipitate was purified by DEAE-Sephadex column chromatography. The original high sensitivity, however, was restored by addition of phosphoglucose isomerase (d-glucose 6-phosphate ketol-isomerase, EC 5.3.1.9) or glucose 6-phosphate. n n3. n3. Glucose 6-phosphate enhanced the UDP-N-acetylglucosamine inhibition of aminotransferase purified by DEAE-Sephadex chromatography. The kinetics of UDP-N-acetylglucosamine inhibition with respect to fructose 6-phosphate were of the competitive type in the purified preparation but of the mixed type in the ammonium sulfate precipitate. The former preparation, however, exhibited the mixed-type kinetics when fructose 6-phosphate/glucose 6-phosphate ratio was maintained at 0.32, the equilibrium of phosphoglucose isomerase reaction. n n4. n4. It. was concluded that the higher sensitivity to UDP-N-acetylglucosamine observed in crude preparations of aminotransferase is due to phosphoglucose isomerase which converts fructose 6-phosphate added as substrate to glucose 6-phosphate. n n5. n5. Comparison of the rat liver and Yoshida sarcoma aminotransferases purified by DEAE-Sephadex chromatography confirmed the previous observation that the tumor enzyme is more sensitive to feedback inhibition than is the liver enzyme.

Tumor‐promoting Phorbol Ester Induces Alterations of Sialidase and Sialyltransferase Activities of JB6 Cells

Sialidase and sialyltransferase activities were studied in JB6 mouse epidermal cells before and after exposure to phorbol ester, 12‐O‐tetradecanoyl phorbol‐13‐acetate (TPA), which irreversibly induces anchorage‐independent growth and tumorigenicity. JB6 cells exhibited sialidase activities toward 4‐methylumbelliferyl‐α‐d‐N‐acetylneuraminic acid (4MU‐NeuAc) and gangliosides at pH 4.5 in the particulate fraction but apparently not in the cytosol at pH 4.5 or 6.0. In JB6 cells exposed to TPA and in the anchorage‐independent transformants, the sialidase activity toward 4MU‐NeuAc was decreased and the activity toward gangliosides was increased compared with those in untreated JB6 cells. Immunological analysis with antisera against membrane‐associated sialidases I and II revealed that plasma membrane‐associated sialidase I was increased and lysosomal membrane‐associated sialidase II was decreased under these conditions. TPA treatment also affected the sialyltransferase activities of JB6 cells: an elevation of the transfer activities toward asialo‐orosomucoid and asialo‐porcine submaxillary mucin but a reduction of GM3 and GD3 synthase activities were observed on exposure to TPA and in cells transformed by TPA to retain anchorage‐independency. These results suggest that an increase in sialic acid bound to glycoproteins and a decrease in that bound to glycolipids may occur in JB6 cells exposed to TPA and in the anchorage‐independent transformants.

Comparative Study of the Levels of Sialyltransferases Responsible for the Formation of Sugar Chains in Glycoproteins and Gangliosides in Rat Liver and Hepatomas

Sialyltransferases responsible for the formation of sugar chains in glycoproteins were studied in rat hepatoma in comparison with rat liver. Hepatoma induced by feeding Wistar rats with 3′‐ methyl‐4‐dimethyIaminoazobenzene (MeDAB) was more active than Wistar liver in sialylating asialo‐orosomucoid, and this was due to an increased activity of GaI(β1→4)GlcNAc (α→6) sialyltransferase, the major sialyltransferase in these tissues, Gal(β1→3,4)GlcNAc (α→3) sialyltransferase and the sialyltransferases acting on asialo‐bovine submaxillary mucin were, however, decreased in the hepatoma. A similar pattern of sialyltransferase alterations was observed in regenerating liver and other tumors such as AH‐109A hepatoma and Sato lung cancer, both of which had been inoculated into Donryu rats. In contrast to these sialyltransferases, the activities of the sialyltransferases responsible for the formation of gangliosides were markedly different even between Wistar and Donryu livers. When compared with Wistar liver, MeDAB induced hepatoma was higher in lactosylceramide‐ and lower in GM3‐sialyltransferase activity, but these two activities were both lower in AH‐109A compared with Donryu liver.

Neoplastic alterations of glucosamine 6-phosphate synthase in rat liver

Abstract Glucosamine 6-phosphate (GlcN-6-P) synthase catalyzes the formation of GlcN-6-P from fructose 6-phosphate and glutamine and is the rate-limiting enzyme for the biosynthesis of UDP-N-acetylglucosamine. In the rat, the enzyme is rich in proliferating and fetal tissues as well as those engaged in glycoprotein export and exists in multi-molecular forms. The enzyme purified from adult liver has a pI at 5.0 and differs in many respects from the major enzyme of adult brain. The latter form, with a pI of 4.1, is also found in early embryos. Sequential alterations in enzyme species thus occurs in the liver with the progress of differentiation. Upon the hepatocarcinogenesis, GlcN-6-P synthase undergoes profound alterations. The activity is especially high in fast growing ascites hepatomas, which possess as the major component the brain-type enzyme. In a Morris hepatoma (5123D), however, the liver form is replaced partially by a form with a pI of 4.3. Partial hepatectomy induces profound changes in GlcN-6-P synthase of the remnant liver. Following the operation, the transient and sequential appearance of the three forms with respective isoelectric points of 4.3, 4.8, and 4.5 is noted. Comparison of these alterations with those induced by laparotomy and by injection of the mixture of triiodothyronine, amino acids, glucagon and heparin enables us to postulate that the sequential appearance and disappearance of the 4.3 and 4.5 forms are associated with the proliferative cycle of regenerating liver. The 4.8 form is induced in response to surgical stress. The interrelation between GlcN-6-P synthase and tissue proliferation is discussed in the light of these results.

Comparison of the properties of glucosaminephosphate isomerase (glutamine-forming) from rat liver and a hepatoma.

Abstract Properties of glucosaminephosphate isomerase (glutamine-forming (EC 5.3.1.19)) partially purified from a rat ascites hepatoma have been compared with properties of rat liver enzyme. Hepatoma glucosaminephosphate isomerase is more unstable, more sensitive to inhibition by UDP -N- acetylglucosamine and more tightly bound to DEAE-Sephadex gel than the liver enzyme. The two enzymes can also be distinguished immunologically. Isoelectric focusing studies have demonstrated that the major form of hepatoma glucosaminephosphate isomerase has a pI at 4.5 and is distinctly different from the major liver form, with a pI of 5.0. The tumor in addition contains a minor component having a pI at 4.1. A small amount of the major tumor form has also been found in liver. The possible significance of the difference in the glucosaminephosphate isomerase forms from liver and hepatoma is discussed.