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)

In this study, we report the first isolation and characterization of a bovine sialyltransferase gene. Bovine cDNAs prepared from different tissues contain an open-reading frame encoding a 405 amino acid sequence showing 83%, 75%, and 60% identity with human, murine, and chicken ST6Gal I (beta-galactoside alpha2,6-sialyltransferase) sequences, respectively. When transfected into COS-7 cells, a recombinant enzyme was obtained which catalyzed the in vitro alpha2, 6-sialylation of LacNAc (NeuAcalpha2-6Galbeta1-4GlcNAc) and LacdiNAc (NeuAcalpha2-6GalNAcbeta1-4GlcNAc) acceptor substrates. The K (m) values were 2.8 and 6.9 mM, respectively. Different relative efficiencies (Vmax/Km) for the two precursors (36 for LacNAc and 4.3 for LacdiNAc) were observed. Bovine ST6Gal I gene consists of four 5'-untranslated exons E(-2) to E(1), and five coding exons from E(2) to E(6). This later carries a 3'-untranslated region of 2. 7 kb. Gene sequence spans at least 80 kb of genomic DNA. Two processed pseudogenes have been identified. They are 94.3 and 95.6% similar to the bovine cDNA, respectively. Three families of mRNA isoforms were isolated. They differed by their 5'-untranslated regions and could be generated by three tissue-specific promoters. Family 1 is made up of exons E(-2) and E(1) to E(6), family 2 of exons E(-1) to E(6), and family 3 of exons E(1) to E(6). Tissular distribution of transcript families appears noticeably different than those described in human and rat.
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PMID:Molecular cloning, expression and exon/intron organization of the bovine beta-galactoside alpha2,6-sialyltransferase gene. 1046 Aug 27

The ST6Gal I is a sialyltransferase that modifies N-linked oligosaccharides of glycoproteins. Previous results suggested a role for luminal stem and active domain sequences in the efficiency of ST6Gal I Golgi retention. Characterization of a series of STtyr isoform deletion mutants demonstrated that the stem is sensitive to proteases and that preventing cleavage in this region leads to increased cell surface expression. A mutant lacking amino acids 32-104 (STDelta4) is not active or cleaved and secreted like the wild type STtyr, but does exhibit increased cell surface expression. It is probable that the STDelta4 mutant lacks the stem region and some amino acids of the active domain because the STDelta5 mutant lacking amino acids 86-104 is also not active but is cleaved and secreted. In contrast, deletion of stem amino acids between residues 32 and 86 in the STDelta1, STDelta2, and STDelta3 mutants does not inactive these enzyme forms, eliminate their cleavage and secretion, or increase their cell surface expression. Surprisingly, cleavage occurs even though the previously identified Asn63-Ser 64 cleavage site is missing. Further evaluation demonstrated that a cleavage site between Lys 40 and Glu 41 is used in COS cells. Mutagenesis of Lys 40 significantly decreased, but did not eliminate cleavage, suggesting that there are additional secondary sites of cleavage in the ST6Gal I stem.
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PMID:The relationship between ST6Gal I Golgi retention and its cleavage-secretion. 1056 65

A cDNA clone encoding a human Galbeta1-3GalNAc alpha2, 6-sialyltransferase (designated hST6GalNAc II) was identified employing the PCR with degenerated primers to the sialylmotifs, followed by BLAST analysis of databanks. This sialyltransferase sequence is similar to that of previously cloned ST6GalNAc II (chicken and mouse) and shows the sialylmotifs that are present in all eukaryotic members of the sialyltransferase gene family. The predicted amino acid sequence encodes a putative type II transmembrane protein as found for other eukaryotic sialyltransferases and shows significant similarity to chicken (56. 8% identity) and mouse (74.6% identity) enzymes. Expression of a secreted form of hST6GalNAc II in COS-7 cells showed that the gene product had Galbeta1-3GalNAc (sialyl to GalNAc) alpha2, 6-sialyltransferase activity. In vitro analysis of substrate specificity revealed that the enzyme required a peptide aglycone fraction to be active and used both Galbeta1-3GalNAc and Neu5Acalpha2-3Galbeta1-3GalNAc as acceptor substrates. Northern analysis revealed a restricted expression pattern of two hST6GalNAc II transcripts, a 2.0 kb mRNA found mainly in skeletal muscle, heart and kidney and a 1.8 kb mRNA found in placenta, lung and leukocytes. No transcriptional expression was detected in brain, thymus or spleen. Transcriptional expression of the ST6GalNAc II gene was followed in various human cell lines and found to be expressed in almost all cell types with notable exceptions for several myeloid and lymphoid cell lines.
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PMID:Molecular cloning and functional expression of human ST6GalNAc II. Molecular expression in various human cultured cells. 1074

The ST6Gal I is a sialyltransferase that functions in the late Golgi to modify the N-linked oligosaccharides of glycoproteins. The ST6Gal I is expressed as two isoforms with a single amino acid difference in their catalytic domains. The STcys isoform is stably retained in the cell and is predominantly found in the Golgi, whereas the STtyr isoform is only transiently localized in the Golgi and is cleaved and secreted from a post-Golgi compartment. These two ST6Gal I isoforms were used to explore the role of the bilayer thickness mechanism and oligomerization in Golgi localization. Analysis of STcys and STtyr proteins with longer transmembrane regions suggested that the bilayer thickness mechanism is not the predominant mechanism used for ST6Gal I Golgi localization. In contrast, the formation and quantity of Triton X-100-insoluble oligomers was correlated with the stable or transient localization of the ST6Gal I isoforms in the Golgi. Nearly 100% of the STcys and only 13% of the STtyr were found as Triton-insoluble oligomers when Golgi membranes of COS-1 cells expressing these proteins were solubilized at pH 6.3, the pH of the late Golgi. In contrast, both proteins were found in the soluble fraction when these membranes were solubilized at pH 8.0. Analysis of other mutants suggested that a conformational change in the catalytic domain rather than increased disulfide bond-based cross-linking is the basis for the increased ability of STcys protein to form oligomers and the stable localization of STcys protein in the Golgi.
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PMID:Formation of insoluble oligomers correlates with ST6Gal I stable localization in the golgi. 1078 4

On the basis of the detection of expressed sequence tag ('EST') similar to the rat N-acetylgalactosamine alpha2,6-sialyltransferase (ST6GalNAc) III cDNA, we have identified a novel member of the human ST6GalNAc family. We have isolated a cDNA clone containing an open reading frame that codes for a type II membrane protein of 302 amino acids with a seven-amino-acid cytoplasmic domain, an 18-amino-acid transmembrane domain and the smallest described catalytic domain of 277 amino acids. This predicted sialyltransferase sequence is similar to the rat ST6GalNAc III (46.6%), but was found to be even more similar to the recently reported mouse ST6GalNAc IV (88.1%) on the basis of amino acid sequence identity. Northern-blot analysis showed that the newly identified gene is expressed constitutively in various adult human tissues as a 2.2kb transcript, but was also found to be expressed at lower levels in brain, heart and skeletal muscle as a 2.5kb transcript. Expression of the hST6GalNAc IV gene was investigated by reverse transcription PCR in various human cancer cells, and was found to be present in the majority of cell types with the exception of the carcinoma cell line T47D and pro-monocyte THP cells. The transient expression in COS-7 cells of the full-length cDNA led to the production of an active enzyme sharing the acceptor specificity of the ST6GalNAc family towards Neu5Ac alpha 2-3Gal beta 1-3GalNAc alpha-O-R (where 'R' denotes H, benzyl, or a peptidic chain). Detailed analysis in vitro of substrate specificity revealed that the enzyme required the trisaccharide Neu5Ac alpha 2-3Gal beta1-3GalNAc found on O-glycans and arylglycosides. In addition, we have clarified the genomic organization of ST6GalNAc IV gene.
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PMID:Cloning, expression and gene organization of a human Neu5Ac alpha 2-3Gal beta 1-3GalNAc alpha 2,6-sialyltransferase: hST6GalNAcIV. 1106 56

The CMP-Neu5Ac:Galbeta1-3GalNAc alpha2,3-sialyltransferase (ST3Gal I, EC 2.4.99.4) is a Golgi membrane-bound type II glycoprotein that catalyses the transfer of sialic acid residues to Galbeta1-3GalNAc disaccharide structures found on O-glycans and glycolipids. In order to gain further insight into the structure/function of this sialyltransferase, we studied protein expression, N-glycan processing and enzymatic activity upon transient expression in the COS-7 cell line of various constructs deleted in the N-terminal portion of the protein sequence. The expressed soluble polypeptides were detected within the cell and in the cell culture media using a specific hST3Gal I monoclonal antibody. The soluble forms of the protein consisting of amino acids 26-340 (hST3-Delta25) and 57-340 (hST3-Delta56) were efficiently secreted and active. In contrast, further deletion of the N-terminal region leading to hST3-Delta76 and hST3-Delta105 gave also rise to various polypeptides that were not active within the transfected cells and not secreted in the cell culture media. The kinetic parameters of the active secreted forms were determined and shown to be in close agreement with those of the recombinant enzyme already described (H. Kitagawa, J.C. Paulson, J. Biol. Chem. 269 (1994)). In addition, the present study demonstrates that the recombinant hST3Gal I polypeptides transiently expressed in COS-7 cells are glycosylated with complex and high mannose type glycans on each of the five potential N-glycosylation sites.
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PMID:Delineation of the minimal catalytic domain of human Galbeta1-3GalNAc alpha2,3-sialyltransferase (hST3Gal I). 1169 Jun 53

The deposition of amyloid beta-peptide (A beta) in the brain is closely associated with the development of Alzheimer's disease. A beta is generated from the amyloid precursor protein (APP) by sequential action of beta-secretase (BACE1) and gamma-secretase. Although BACE1 is distributed among various other tissues, its physiological substrates other than APP have yet to be identified. ST6Gal I is a sialyltransferase that produces a sialyl alpha 2,6galactose residue, and the enzyme is secreted out of the cell after proteolytic cleavage. We report here that BACE1 is involved in the proteolytic cleavage of ST6Gal I, on the basis of the following observations. ST6Gal I was colocalized with BACE1 in the Golgi apparatus by immunofluorescence microscopy, suggesting that BACE1 acts on ST6Gal I within the same intracellular compartment. When BACE1 was overexpressed with ST6Gal I in COS cells, the secretion of ST6Gal I markedly increased. When APP(SW) (Swedish familial Alzheimer's disease mutation), a preferable substrate for BACE1, was coexpressed with ST6Gal I in COS cells, the secretion of ST6Gal I significantly decreased, suggesting that that the beta-cleavage of overexpressed APP(SW) competes with ST6Gal I processing. In addition, BACE1-Fc (Fc, the hinge and constant region of IgG) chimera cleaved protein A-ST6Gal I fusion protein in vitro. Thus, we conclude that BACE1 is responsible for the cleavage and secretion of ST6Gal I.
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PMID:Alzheimer's beta-secretase, beta-site amyloid precursor protein-cleaving enzyme, is responsible for cleavage secretion of a Golgi-resident sialyltransferase. 1169 69

BACE1 is a membrane-bound aspartic protease that cleaves the amyloid precursor protein (APP) at the beta-secretase site, a critical step in the Alzheimer disease pathogenesis. We previously found that BACE1 also cleaved a membrane-bound sialyltransferase, ST6Gal I. By BACE1 overexpression in COS cells, the secretion of ST6Gal I markedly increased, and the amino terminus of the secreted ST6Gal I started at Glu(41). Here we report that BACE1-Fc chimera protein cleaved the A-ST6Gal I fusion protein, or ST6Gal I-derived peptide, between Leu(37) and Gln(38), suggesting that an initial cleavage product by BACE1 was three amino acids longer than the secreted ST6Gal I. The three amino acids, Gln(38)-Ala(39)-Lys(40), were found to be truncated by exopeptidase activity, which was detected in detergent extracts of Golgi-derived membrane fraction. These results suggest that ST6Gal I is cleaved initially between Leu(37) and Gln(38) by BACE1, and then the three-amino acid sequence at the NH(2) terminus is removed by exopeptidase(s) before secretion from the cells.
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PMID:Characterization of alpha 2,6-sialyltransferase cleavage by Alzheimer's beta -secretase (BACE1). 1247 67

BLAST analysis of the human and mouse genome sequence databases using the sequence of the human CMP-sialic acid:beta-galactoside alpha-2,6-sialyltransferase cDNA (hST6Gal I, EC2.4.99.1) as a probe allowed us to identify a putative sialyltransferase gene on chromosome 2. The sequence of the corresponding cDNA was also found as an expressed sequence tag of human brain. This gene contained a 1590 bp open reading frame divided in five exons and the deduced amino-acid sequence didn't correspond to any sialyltransferase already known in other species. Multiple sequence alignment and subsequent phylogenic analysis showed that this new enzyme belonged to the ST6Gal subfamily and shared 48% identity with hST6Gal-I. Consequently, we named this new sialyltransferase ST6Gal II. A construction in pFlag vector transfected in COS-7 cells gave raise to a soluble active form of ST6Gal II. Enzymatic assays indicate that the best acceptor substrate of ST6Gal II was the free disaccharide Galbeta1-4GlcNAc structure whereas ST6Gal I preferred Galbeta1-4GlcNAc-R disaccharide sequence linked to a protein. The alpha2,6-linkage was confirmed by the increase of Sambucus nigra agglutinin-lectin binding to the cell surface of CHO transfected with the cDNA encoding ST6Gal II and by specific sialidases treatment. In addition, the ST6Gal II gene showed a very tissue specific pattern of expression because it was found essentially in brain whereas ST6Gal I gene is ubiquitously expressed.
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PMID:Identification and functional expression of a second human beta-galactoside alpha2,6-sialyltransferase, ST6Gal II. 1260 28

Molecular events that lead to the development of autonomously functioning thyroid nodules (AFTNs) are somatic mutations of the thyrotropin receptor (TSHR) in approximately 60% of the nodules and less frequently, somatic mutations in the Gsalpha protein. However, AFTNs without known mutations indicate that other causes remain to be identified. Moreover, the impact of constitutively activating TSHR mutations on the signal transduction network of the thyroid epithelial cell is unknown. We therefore investigated gene expression in 15 AFTNs and their surrounding tissue using Affymetrix GeneChips. Most prominently, data analysis revealed a changed pattern of gene expression in the TGF-beta signaling cascade and 25 differentially regulated genes in AFTNs, including thyroid peroxidase, type I iodothyronine deiodinase and sialyltransferase (SIAT) 1. Strikingly coexpression of SIAT 1 and TSHR in COS-7 cells increased TSH binding and cell surface expression of the TSHR. Moreover, differences in gene expression patterns for AFTNs with and without TSHR mutations indicate specific alterations of signal transduction in AFTNs without TSHR mutations. These results suggest that AFTNs with TSHR mutations harbor further mechanisms of forward stimulation. Furthermore, they give important leads to elucidate the molecular etiology of AFTNs without TSHR mutations.
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PMID:Gene expression analysis reveals evidence for inactivation of the TGF-beta signaling cascade in autonomously functioning thyroid nodules. 1473 14


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