Increase of sialyltransferase activity in the serum and liver of inflamed rates

Induction of inflammation by turpentine injection caused 1.5–2-fold increase of both sialy- and galactosyltransferase activity in liver homogenates. The effect was apparent after 12 h turpentine treatment. Serum sialytransferase activity started to increase in the inflamed rats after 18 h, reaching a maximum of 4-fold at 48 h. In contrast, galactosyltransferase activity in serum showed no significant increase. The coordinated and temporal increase of sialytransferase activity in liver and serum suggest involvement of a specific mechanism for the preferential release of this enzyme into serum.     

Developmental Changes in Ganglioside Composition and Synthesis in Embryonic Rat Brain

Developmental changes in ganglioside composition and biosynthesis was studied in rat brain between embryonic day (E) 14 and birth. In E14 brains, GM3 and GD3 were predominant. At E16, "b" series gangliosides, such as GD1b, GT1b, and GQ1b, increased in content. After E18, "a" series gangliosides such as GM1, GD1a, and GT1a increased in content, and the content of GM3 and GD3 markedly decreased. Because of these changes in composition, we determined the activities, in homogenates of embryonic brains, of two key enzymes of ganglioside synthesis: sialyltransferase for the synthesis of GD3 from GM3 and N-acetylgalactosaminyltransferase for GM2 synthesis from GM3. The sialyltransferase activity (GM3----GD3) was constant between E14 and E18 but decreased rapidly from E18 to birth. In contrast, the N-acetylgalactosaminyltransferase activity (GM3----GM2) increased between E14 and E18 but was constant from E18 to birth. These changes in ganglioside composition and enzymatic activities indicate that during development there is a shift from synthesis of the simplest gangliosides of the "a" and "b" pathways to synthesis of the more complex gangliosides.     

Effect of Desipramine on a Glycoprotein Sialyltransferase Activity in C6 Cultured Glioma Cells

The tricyclic antidepressant desipramine, when added to culture medium, gave rise in C6 rat glioma cells to a decrease of the activity of the enzyme asialofetuin sialyltransferase. The inhibition was dose and time dependent and was observed in both multiplying cells and cells blocked with 2 mM thymidine or depletion of amino acids. This inhibition was rather specific to the sialyltransferase, as under the conditions where this enzyme was inhibited up to 70%, other enzymes such as dolichol phosphate mannose synthetase, glutamine synthetase, and glycerol phosphate dehydrogenase remained unaffected. This inhibition was not reversed after removal of desipramine from the medium and was not observed by direct addition of desipramine to the sialyltransferase incubation assay. Under the same conditions, W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], which is known to be a potent calmodulin antagonist and an inhibitor of calmodulin-dependent kinases, gave the same concentration-dependent inhibition profile of sialyltransferase as desipramine, whereas H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine], which is an inhibitor of protein kinase C and cyclic nucleotide-dependent kinases, had no effect. So, it is suggested that desipramine inhibits the sialyltransferase activity in C6 glioma cells through a calmodulin-dependent system.     

Sialyl-transferase mitochondriale

A sialyl transferase activity is found in purified mitochondria. It is not due to residual contamination and this enzymatic system is located in the outer mitochondrial membrane. This proves mitochondrial autonomy in regard to glycoconjugate sialylation.     

Alcohol exposure alters the expression pattern of neural cell adhesion molecules during brain development

Neural cell adhesion molecules (NCAMs) play critical roles during development of the nervous system. The aim of this study is to investigate the possible effect of ethanol exposure on the pattern of expression and sialylation of NCAM isoforms during postnatal rat brain development because alterations in NCAM content and distribution have been associated with defects in cell migration, synapse formation, and memory consolidation, and deficits in these processes have been observed after in utero alcohol exposure. The expression of NCAM isoforms in the developing cerebral cortex of pups from control and alcohol-fed mothers was assessed by western blotting, ribonuclease protection assay, and immunocytochemistry. The highly sialylated form of NCAM [polysialic acid (PSA)-NCAM] is mainly expressed during the neonatal period and then is down-regulated in parallel with the appearance of NCAM 180 and NCAM 140. Ethanol exposure increases PSA-NCAM levels during the neonatal period, delays the loss of PSA-NCAM, decreases the amount of NCAM 180 and NCAM 140 isoforms, and reduces sialyltransferase activity during postnatal brain development. Neuraminidase treatment of ethanol-exposed neonatal brains leads to more intense band degradation products, suggesting a higher content of NCAM polypeptides carrying PSA in these samples. However, NCAM mRNA levels are not changed by ethanol. Immunocytochemical analysis demonstrates that ethanol triggers an increase in PSA-NCAM immunolabeling in the cytoplasm of astroglial cells, accompanied by a decrease in immunogold particles over the plasma membrane. These findings indicate that ethanol exposure during brain development alters the pattern of NCAM expression and suggest that modification of NCAM could affect neuronal-glial interactions that might contribute to the brain defects observed after in utero alcohol exposure.     

Modified GM3 gangliosides produced by metabolic oligosaccharide engineering

Metabolic oligosaccharide engineering is powerful approach to altering the structure of cellular sialosides. This method relies on culturing cells with N-acetylmannosamine (ManNAc) analogs that are metabolized to their sialic acid counterparts and added to glycoproteins and glycolipids. Here we employed two cell lines that are deficient in ManNAc biosynthesis and examined their relative abilities to metabolize a panel of ManNAc analogs to sialosides. In addition to measuring global sialoside production, we also examined biosynthesis of the sialic acid-containing glycolipid, GM3. We discovered that the two cell lines differ in their ability to discriminate among the variant forms of ManNAc. Further, our data suggest that modified forms of sialic acid may be preferentially incorporated into certain sialosides and excluded from others. Taken together, our results demonstrate that global analysis of sialoside production can obscure sialoside-specific differences. These findings have implications for downstream applications of metabolic oligosaccharide engineering, including imaging and proteomics.     

Efficient synthesis of 6′-sialyllactose, 6,6′-disialyllactose, and 6′-KDO-lactose by metabolically engineered E. coli expressing a multifunctional sialyltransferase from the Photobacterium sp. JT-ISH-224

We have previously reported the efficient conversion of lactose into 3′-sialyllactose by high cell density cultures of a genetically engineered Escherichia coli strain expressing the Neisseria meningitidis gene for α-(2→3)-sialyltransferase [Fierfort, N.; Samain, E. J. Biotechnol. 2008, 134, 261-265.]. First attempts to use a similar strategy to produce 6′-sialyllactose with a strain expressing α-(2→6)-sialyltransferase from the Photobacterium sp. JT-ISH-224 led to the production of a trisaccharide that was identified as KDO-lactose (2-keto-3-deoxy-manno-octonyllactose). This result showed that α-(2→6)-sialyltransferase was able to use CMP-KDO as sugar donor and preferentially used CMP-KDO over CMP-Neu5Ac. By reducing the expression level of the sialyltransferase gene and increasing that of the neuABC genes, we have been able to favour the formation of 6′-sialyllactose and to prevent the formation of KDO-lactose. However, in this case, a third lactose derivative, which was identified as 6,6′-disialyllactose, was also produced. Formation of 6,6′-disialyllactose was mainly observed under conditions of lactose shortage. On the other hand, when the culture was continuously fed with an excess of lactose, 6′-sialyllactose was almost the only product detected and its final concentration was higher than 30 g/L of culture medium.     

Enhancement of sialyltransferase-catalyzed transfer of sialic acid onto glycoprotein oligosaccharides using silkworm hemolymph and its 30K protein

Silkworm hemolymph (SH) has been reported to inhibit apoptosis in both insect and human cells, and increase the high-sialylation structure of recombinant glycoprotein in insect cells. This indicates that SH might increase glycosyltransferase activity. Therefore, this study examined the effect of SH on the activity of sialyltransferase, which catalyzes the sialylation of the glycoprotein. When 10 μg/mL of SH was added to the reaction mixture, almost complete sialylation was observed even under the reaction conditions where sialyltransferase-catalyzed sialylation rarely occurs. The effect of deproteinized SH (dSH) and the 30K protein, which is a major plasma protein in SH, was examined to determine which component in SH enhances sialylation. The 30K protein promoted sialylation, while the dSH did not. This suggests that SH and its 30K protein can be used as an additive to a medium for efficient glycosylation when mammalian cells are being cultured for the production of valuable biopharmaceuticals, many of which are glycoproteins.     

Sialidase NEU4 hydrolyzes polysialic acids of neural cell adhesion molecules and negatively regulates neurite formation by hippocampal neurons

Modulation of levels of polysialic acid (polySia), a sialic acid polymer, predominantly associated with the neural cell adhesion molecule (NCAM), influences neural functions, including synaptic plasticity, neurite growth, and cell migration. Biosynthesis of polySia depends on two polysialyltransferases ST8SiaII and ST8SiaIV in vertebrate. However, the enzyme involved in degradation of polySia in its physiological turnover remains uncertain. In the present study, we identified and characterized a murine sialidase NEU4 that catalytically degrades polySia. Murine NEU4, dominantly expressed in the brain, was found to efficiently hydrolyze oligoSia and polySia chains as substrates in sialidase in vitro assays, and also NCAM-Fc chimera as well as endogenous NCAM in tissue homogenates of postnatal mouse brain as assessed by immunoblotting with anti-polySia antibodies. Degradation of polySia by NEU4 was also evident in neuroblastoma Neuro2a cells that were co-transfected with Neu4 and ST8SiaIV genes. Furthermore, in mouse embryonic hippocampal primary neurons, the endogenously expressed NEU4 was found to decrease during the neuronal differentiation. Interestingly, GFP- or FLAG-tagged NEU4 was partially co-localized with polySia in neurites and significantly suppressed their outgrowth, whereas silencing of NEU4 showed the acceleration together with an increase in polySia expression. These results suggest that NEU4 is involved in regulation of neuronal function by polySia degradation in mammals.     

N-Terminal 112 amino acid residues are not required for the sialyltransferase activity of Photobacterium damsela α2,6-sialyltransferase

Photobacterium damsela α2,6-sialyltransferase was cloned as N- and C- His-tagged fusion proteins with different lengths (16–497 aa or 113–497 aa). Expression and activity assays indicated that the N-terminal 112 amino acid residues of the protein were not required for its α2,6-sialyltransferase activity. Among four truncated forms tested, N-His-tagged Δ15Pd2,6ST(N) containing 16–497 amino acid residues had the highest expression level. Similar to the Δ15Pd2,6ST(N), the shorter Δ112Pd2,6ST(N) was active in a wide pH range of 7.5–10.0. A divalent metal ion was not required for the sialyltransferase activity, and the addition of EDTA and dithiothreitol did not affect the activity significantly. Mingchi Sun and Yanhong Li contributed equally to this work.