Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.4.99.6 (sialyltransferase)
1,546 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The postnatal sialylation of individual neural cell adhesion molecule (N-CAM) polypeptides by a developmentally regulated sialyltransferase in Golgi-enriched fractions isolated from rat brain is described. The 120-kilodalton polypeptide of N-CAM was found to be sialylated at each developmental age examined. This was in contrast to the 140- and 180-kilodalton N-CAM polypeptides which were only sialylated until postnatal day 10 and from postnatal day 12, respectively. Immunoblotting procedures demonstrated that all N-CAM polypeptides were expressed in the Golgi fractions at each developmental stage examined. The heavily sialylated "embryonic" form of N-CAM was found to be reexpressed at postnatal days 10 and 12, a time coincident with extensive fibre outgrowth. The "embryonic" form of N-CAM incorporated similar amounts of [14C]sialic acid into its constituent polypeptides reflecting the difference in sialic acid to protein ratio, as this form of N-CAM was virtually undetectable in the immunoblots of postnatal material.
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PMID:Differentiation-dependent sialylation of individual neural cell adhesion molecule polypeptides during postnatal development. 333 47

The neural cell adhesion molecule N-CAM is believed to be intimately involved in the structuring of the central nervous system. During post-natal development the molecule exists in 2 forms--a sialic acid-rich form which is preferentially expressed during cell acquisition and fibre outgrowth and a sialic acid-poor form which appears at times coincident with synaptogenesis. The developmental changes between these 2 forms have been demonstrated to be impaired by chronic low-level lead exposure and this is consistent with the reduced synaptic elaboration associated with this action. Here is described the effect of lead on the Golgi-associated sialyltransferase which regulates N-CAM sialylation state. Lead chloride was found to markedly stimulate sialyltransferase with an ED50 of 5 X 10(-7) M in adult Golgi fractions. This was not observed in fractions derived from 12-day old animals. At the concentration of 5 X 10(-5) M lead was found to have a differential effect on the developmental expression of this enzyme. During the early phases of development (days 4-16) sialyltransferase activity was inhibited. However, in coincidence with periods of N-CAM desialylation (days 16-30), it was significantly stimulated. These findings are related to the perturbations of N-CAM function during chronic low-level lead exposure.
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PMID:Lead stimulates Golgi sialyltransferase at times coincident with the embryonic to adult conversion of the neural cell adhesion molecule (N-CAM). 337 25

Cytosol- and Golgi-enriched fractions were obtained from whole rat brain homogenates by density gradient centrifugation. Using a 4-methylumbelliferyl neuraminic acid substrate a soluble neural sialidase has been identified and characterised. The enzyme had optimal activity at pH 6.0 and a Km of 0.44 +/- 0.18 mM. The specific activity increased during postnatal development and this was in parallel with the described temporal changes in total brain neuraminic acid turnover. The potential of this enzyme to influence the intracellular processing of sialoglycoconjugates was also investigated. Cytosol fractions were incapable of releasing [14C]NeuNAC [( 14C]N-acetylneuramic acid) transferred to the glycoproteins of isolated Golgi membranes by their associated sialyltransferase. Further preincubation of Golgi membranes with soluble sialidase had no effect on their intrinsic sialyltransferase activity. These results demonstrate that no epigenetic regulation of processed sialoglycoconjugates occurs intracellularly and these finding are related to post-translational control of neural cell adhesion molecule (N-CAM) sialylation state.
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PMID:Soluble rat brain sialidase does not influence intracellular glycosylation of Golgi sialyltransferase or its constitutive glycoproteins. 338 77

Golgi-enriched fractions have been isolated from rat brain of increasing postnatal age and defined by electron microscopy and distribution of marker enzymes. The expression of sialyltransferase activity associated with these fractions has been demonstrated to developmentally decrease and this appeared to be, in part, dependent on endogenous competitive inhibition. The developmental regulation of this activity paralleled the sialylation state of the neural cell adhesion molecule (D2-CAM/N-CAM) and could be demonstrated to be capable of endogenously sialylating this protein in the isolated Golgi fractions. In 12-day-old animals the majority of the transferred [14C]sialic acid was found to be associated with the high-molecular-weight [greater than 200 kilodaltons (kd)] form of D2-CAM/N-CAM, indicative of the protein having been heavily sialylated. Sialylation of the individual D2-CAM/N-CAM polypeptides was also demonstrated in both 12-day and adult animals and transfer was evident only in the 180-kd and 115-kd components and not in the 140-kd component. In contrast, Golgi-enriched fractions prepared from adult animals showed little capability of heavily sialylating D2-CAM/N-CAM to any significant extent.
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PMID:Postnatal D2-CAM/N-CAM sialylation state is controlled by a developmentally regulated Golgi sialyltransferase. 355 63

Polysialic acid, or PSA, is a term used to refer to linear homopolymers of alpha(2,8)-sialic acid residues displayed at the surface of some mammalian cells. PSA is typically linked to the neural cell adhesion molecule N-CAM, where it can modulate the homotypic adhesive properties of this polypeptide. PSA expression is developmentally regulated, presumably through mechanisms involving regulated expression of sialyltransferases involved in PSA biosynthesis. Several different sialytransferase sequences have been implicated in PSA expression, although the precise roles of these enzymes in this context remain unclear. One such sequence, termed STX, maintains approximately 59% amino acid sequence identity with another sialyltransferase (PST-1, from hamster; PST, human) that is known to participate in PSA expression. While a murine STX fusion protein can catalyze the synthesis of a single alpha(2,8)-sialic acid linkage in vitro, the ability of STX to participate in PSA expression in vivo has not been demonstrated. We show here that STX transcripts are present in a PSA-positive, N-CAM-positive human small cell carcinoma line (NCI-H69/F3), but are absent in a variant of this line (NCI-H69/E2) selected to be PSA-negative and N-CAM-positive. To functionally confirm this correlation, we have cloned a human cDNA encoding the human STX sequence, and show, by transfection studies, that human STX can restore PSA expression when expressed in the PSA-negative, N-CAM-positive small cell carcinoma variant. We furthermore show that STX can confer PSA expression when expressed in a PSA-negative, N-CAM-positive murine cell line (NIH-3T3 cells), or when expressed in PSA-negative, N-CAM-negative COS-7 cells. These observations imply that STX, like PST-1/PST, can determine PSA expression in vivo. When considered together with the correlation between STX expression and PSA expression in vivo in the brain, these results suggest a regulatory role for STX in PSA expression in the developing central nervous system and small cell lung carcinoma.
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PMID:A human STX cDNA confers polysialic acid expression in mammalian cells. 755 89

We previously showed that mouse ST8Sia II (STX) exhibits polysialic acid (PSA) synthase activity in vivo as well as in vitro (Kojima, N., Yoshida, Y., and Tsuji, S. (1995) FEBS Lett. 373, 119-122, 1995). In this paper, we reported that the neural cell adhesion molecule (NCAM) was specifically polysialylated by a single enzyme, ST8Sia II. PSA-expressing Neuro2a cells (N2a-STX) were established by stable transfection of the mouse ST8Sia II gene. Only the 140- and 180-kDa isoforms of NCAM in N2a-STX cells were specifically polysialylated in vivo, although other membrane proteins of N2a-STX were polysialylated in vitro. A recombinant soluble mouse ST8Sia II synthesized PSA on a recombinant soluble NCAM fused with the Fc region of human IgG1 (NCAM-Fc) as well as fetuin. However, NCAM-Fc served as a 1500-fold better acceptor for ST8Sia II than fetuin. Treatment of NCAM-Fc with Charonia lampas alpha-fucosidase, which is able to cleave alpha1,6-linked fucose, clearly reduced the polysialylation of NCAM-Fc by ST8Sia II. PSA was not synthesized on the N-glycanase-treated NCAM-Fc polypeptide or the free N-glycans of NCAM-Fc. When fetuin and its glycopeptide and N-glycans of fetuin were used as substrates for ST8Sia II, PSA was found to be synthesized on native fetuin and its glycopeptide but not on free N-glycans. These results strongly suggested that core alpha1, 6-fucose on N-glycans as well as the antennary structures of N-glycans and the polypeptide regions are required for the polysialylation by ST8Sia II. Furthermore, oligo and single alpha2, 8-sialylated glycoproteins were no longer polysialylated by mouse ST8Sia II. Therefore, the single enzyme, ST8Sia II, directly transferred all alpha2,8-sialic acid residues on the alpha2,3-linked sialic acids of N-glycans of specific NCAM isoforms to yield PSA-NCAM. Polysialylation did not require any initiator alpha2, 8-sialyltransferase but did depend on the carbohydrate and protein structures of NCAM.
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PMID:Characterization of mouse ST8Sia II (STX) as a neural cell adhesion molecule-specific polysialic acid synthase. Requirement of core alpha1,6-linked fucose and a polypeptide chain for polysialylation. 870 35

A method for the assay of CMP-NeuAc:(NeuAc alpha 2-->8)n (colominic acid) sialyltransferase activity was developed. Using a 1-day-old rat brain membrane fraction as an enzyme preparation optimal activity was obtained at pH 6.5, 0.3% Triton X-100, and 5 mM MnCl2. However, no absolute cation requirement was found as EDTA only partially inhibited the activity. Within a concentration range of 0.3-3 mg colominic acid (which consists of a mixture of oligomers of alpha 2-->8-linked sialic acid) per 50 microliters a V of 0.61 nmol per mg protein h-1 was estimated while a half-maximal reaction velocity was obtained at a concentration of 1.75 mg per 50 microliters. High performance anion-exchange chromatography of the radioactive products formed in the reaction showed that sialic acid oligomers ranging in size from a degree of polymerization (DP) of 2 up to at least DP 9 could serve as acceptor substrates. Comparison of the acceptor properties of DP 3 and DP 6 showed that the larger oligomer was acted upon with a 10-fold higher efficiency. Periodate oxidation of the products followed by reduction and hydrolysis yielded the C7 analogue of NeuAc as the only radioactive product, indicating that under the conditions of the assay only a single sialic acid residue was introduced into the acceptor molecules. Using the assay it appeared that in rat brain the activity of this sialyltransferase decreased six-fold during postnatal development to the adult stage. The assay method was also applied to lysates of several neuroblastoma and small cell lung tumour cell lines, which differ in the expression of polysialic acid as well as of the neural cell adhesion molecule NCAM, a major carrier of this polymer. Activity of the sialyltransferase appeared to be correlated with the expression of polysialic acid present on NCAM. These results indicate that this sialyltransferase might function in the process of poly-sialylation.
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PMID:CMP-NeuAc:(NeuAc alpha 2-->8)n (colominic acid) sialyltransferase activity in rat brain and in tumour cells that express polysialic acid on neural cell adhesion molecules. 874 61

The up- and downregulation of polysialic acid-neural cell adhesion molecule (PSA-NCAM) expression on motorneurons during development is associated respectively with target innervation and synaptogenesis, and is regulated at the level of PSA enzymatic biosynthesis involving specific polysialyltransferase activity. The purpose of this study has been to describe the cellular mechanisms by which that regulation might occur. It has been found that developmental regulation of PSA synthesis by ciliary ganglion motorneurons is not reflected in the levels of polysialyltransferase-1 (PST) or sialyltransferase-X (STX) mRNA. On the other hand, PSA synthesis in both the ciliary ganglion and the developing tectum appears to be coupled to the concentration of calcium in intracellular compartments. This study documents a calcium dependence of polysialyltransferase activity in a cell-free assay over the range of 0.1-1 mM, and a rapid sensitivity of new PSA synthesis, as measured in a pulse-chase analysis of tissue explants, to calcium ionophore perturbation of intracellular calcium levels. Moreover, the relevant calcium pool appears to be within a specific intracellular compartment that is sensitive to thapsigargin and does not directly reflect the level of cytosolic calcium. Perturbation of other major second messenger systems, such as cAMP and protein kinase-dependent pathways, did not affect polysialylation in the pulse chase analysis. These results suggest that the shuttling of calcium to different pools within the cell can result in the rapid regulation of PSA synthesis in developing tissues.
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PMID:Regulation of neural cell adhesion molecule polysialylation: evidence for nontranscriptional control and sensitivity to an intracellular pool of calcium. 949 Jul 30

Brain dysmorphogenesis and persistent psychomotor disturbances are hallmarks of developmental methylmercury (MeHg) exposure, but the molecular mechanisms underlying these effects are poorly understood. Targets of developmental MeHg exposure include neural cell adhesion molecules (NCAMs), sialoglycoconjugate molecules whose proper temporal and spatial expression is important at all stages of neurodevelopment and especially during synaptic structuring. To investigate the effects of MeHg on the temporal expression of NCAM during development, rat pups were dosed with 7.0 mg/kg MeHgCl (s.c.) on alternate days from postnatal days (PNDs) 3-13 and killed on PNDs 15, 30 and 60. Brain MeHg concentrations were determined in a subset of litters injected with CH(3)203Hg. Expression of NCAM180 protein and of NCAM180 polysialylation was examined in whole cerebellum homogenates, cerebellar synaptosomes and isolated cerebellar growth cones by Western blotting and immunocytochemical staining. NCAM sialyltransferase activity was assayed in preparations of purified Golgi apparatus from the cerebelli of rats treated in vivo, or following in vitro incubation with 0, 1, 2.5, or 7.5 microM MeHg for 2 h. At PND15, no change in NCAM180 protein expression was observed in any cerebellar preparations, but decreased polysialylation of NCAM180 was observed in cerebellar whole homogenates, synaptosomes and isolated growth cones. At PND30, both NCAM180 protein expression and NCAM180 polysialylation were elevated in whole homogenate preparations but not in synaptosomes. NCAM180 expression in MeHg-treated rats was similar to controls at PND60, 47 days after the last methylmercury administration. In vivo studies of cerebellar Golgi sialyltransferase activity revealed significant reductions in PND15 MeHg-treated rats as compared to controls, but no changes in sialyltransferase activity in PND30 and PND60 animals. In vitro experiments revealed decreasing sensitivity of cerebellar sialyltransferases to MeHg as the developmental age of the rat increased. Toxic perturbation of the developmentally-regulated expression of polysialylated NCAM during brain formation may disturb the stereotypic formation of neuronal contacts and could contribute to the behavioral and morphological disturbances observed following MeHg poisoning.
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PMID:Developmental methylmercury administration alters cerebellar PSA-NCAM expression and Golgi sialyltransferase activity. 1053 93

The pre-existence of alpha2-->8-linked disialic acid (di-Sia) and oligosialic acid (oligo-Sia) structures with up to 7 Sia residues was shown to occur on a large number of brain glycoproteins, including neural cell adhesion molecules (N-CAMs), by two highly sensitive chemical methods (Sato, C., Inoue, S., Matsuda, T., and Kitajima, K. (1998) Anal. Biochem. 261, 191-197; Sato, C., Inoue, S., Matsuda, T., and Kitajima, K. (1999) Anal. Biochem. 266, 102-109). This unexpected finding was also confirmed using a newly developed antibody prepared using a copolymer of alpha2-->8-linked N-acetylneuraminyl p-vinylbenzylamide and acrylamide as an immunogen and known antibodies whose immunospecificities were determined to be di- and oligo-Sia residues with defined chain lengths. The major significance of the new finding that di- and oligo-Sia chains exist on a large number of brain glycoproteins is 2-fold. First, it reveals a surprising diversity in the number and M(r) of proteins distinct from N-CAM that are covalently modified by these short sialyl glycotopes. Second, it suggests that synthesis of di- and/or oligo-Sia units may be catalyzed by alpha2-->8-sialyltransferase(s) that are distinct from the known polysialyltransferases, STX and PST, which are partially responsible for polysialylation of N-CAM.
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PMID:Frequent occurrence of pre-existing alpha 2-->8-linked disialic and oligosialic acids with chain lengths up to 7 Sia residues in mammalian brain glycoproteins. Prevalence revealed by highly sensitive chemical methods and anti-di-, oligo-, and poly-Sia antibodies specific for defined chain lengths. 1080 78


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