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 sialyltransferase gene family is comprised of 16 cloned enzymes. All members contain two conserved protein domains, termed the S- and L-sialylmotifs, that participate in substrate binding. Of only six invariant amino acids, two are cysteines, with one found in each sialylmotif. Although the recombinant soluble form of ST6Gal I has six cysteines, quantitative analysis indicated the presence of only one disulfide linkage, and thiol reducing agents dithiothreitol and beta-mercaptoethanol inactivated the enzyme. Analysis of site-directed mutants showed that alanine or serine mutants of invariant Cys(181) or Cys(332) exhibit no detectable activity, either by direct assay or by staining of the transfected cells with Sambucus nigra agglutinin, which recognizes the product NeuAcalpha2,6Galbeta1,4GlcNAc on glycoproteins. In contrast, alanine mutations of charged residues adjacent to either cysteine showed little or no effect on enzyme activity. Immunofluorescence microscopy showed that although the wild type sialyltransferase is properly localized in the Golgi apparatus, the inactive cysteine mutants are retained in the endoplasmic reticulum. The results suggest that the invariant cysteine residues in the L- and S-sialylmotifs participate in the formation of an intradisulfide linkage that is essential for proper conformation and activity of ST6Gal I.
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PMID:Conserved cysteines in the sialyltransferase sialylmotifs form an essential disulfide bond. 1127 97

Sialic acids play important roles in various biological functions. In the brain, evidence suggests that sialylation of glycoproteins and glycolipids affects neural plasticity. While the 18 sialyltransferase isoenzymes (STs) identified to date synthesize individual sialyl-oligosaccharide structures, they each exhibit activity toward more than one substrate and can overlap in their specificity. Therefore, the distribution of STs is a secondary factor in the study of specific sialylation. Here, seven STs; ST3Gal I-IV, ST8Sia IV, ST6Gal I and ST6GalNAc II, the expressions of which were identified in the adult hippocampus by RT-PCR, showed diverse localization patterns in the hippocampus on in situ hybridization, suggesting that the individual cells expressed relevant STS: Furthermore, to assay activity-related changes in ST expression, we used amygdaloid-kindling among models of neural plasticity. Differential expression of the STs participating in the kindling, notably, up-regulation of ST3Gal IV and ST6GalNAc II mRNAs, and down-regulation of ST3Gal I and ST8Sia IV mRNAs, were observed in the hippocampus following kindled seizures. These results indicate that ST expressions are regulated by physiological activity and may play a role in neural plasticity.
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PMID:Differential expression of mRNAs for sialyltransferase isoenzymes induced in the hippocampus of mouse following kindled seizures. 1138 69

Sialyltransferase activity has traditionally been studied by determining the rate at which the enzyme transfers a labeled donor sugar to an acceptor substrate. These types of assays can be difficult to quantitate, and the separation of untransfered donor sugar from the sialylated acceptor is time-consuming. The biosensor-based method described here is both rapid and semi-automated. The NeuAc-alpha2-6Gal-R-specific lectin Sambucus nigra agglutinin (SNA) immobilized to the carboxymethyl dextran surface of a BIAcore sensor chip was used to detect and measure the formation of the NeuAc-alpha2-6Gal-R moieties. The sialyltransferase assays were carried out using modified protocols based on the method described in Rearick, J.I., Sadler, J.E., Paulson, J.C., and Hill, R.L. (1979) Enzymatic characterization of betaD-galactoside alpha2-3 sialyltransferase from porcine submaxillary gland. J. Biol. Chem., 254, 4444-4451. The complete assay mixture was simply diluted before injection into the instrument. All injections were performed automatically using the robotics of the BIAcore instrument. Using this technique it is possible to detect product from 0.4 microU of commercial Galbeta1-4GlcNAc alpha2,6-sialyltransferase (EC 2.4.99.1) (ST6Gal I). One unit of sialyltransferase is defined as the quantity that will transfer 1 micromol of N-acetylneuraminic acid from cytidine monophosphate (CMP)-N-acetylneuraminic acid to asialofetuin per min at pH 6.5 and 37 degrees C. The method described here requires as little as 10 microl total assay volume, thus reducing the consumption of reagents. In addition, the sample is completely recoverable from the sensor chip surface, which allows for downstream analysis of the reaction product if desired. This method eliminates the need for labeled donor and acceptor molecules and does not require the separation of the substrates from the product before analysis. Although some kinetic properties of the enzyme can be estimated using this method, further development and validation is required. The method is most useful in determining qualitative estimates of ST6Gal I activity in tissue extracts and in characterizing the production of enzymes in cultured cell systems. The use of a microtiter plate assay format enables the rapid screening of multiple fractions for sialyltransferase activity.
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PMID:A rapid, semi-automated method for detection of Galbeta1-4GlcNAc alpha2,6-sialyltransferase (EC 2.4.99.1) activity using the lectin Sambucus nigra agglutinin. 1144 35

Natural human interferon-gamma (hIFN-gamma) contains mainly biantennary complex-type sugar chains. We previously remodeled the branch structures of N-glycans on hIFN-gamma in Chinese hamster ovary (CHO) cells by overexpressing UDP-N-acetylglucosamine: alpha1,6-D-mannoside beta1,6-N-acetylglucosaminyltransferase (GnT-V). Normal CHO cells primarily produced hIFN-gamma having biantennary sugar chains, whereas a CHO clone, designated IM4/Vh, transfected with GnT-V, primarily produced hIFN-gamma having GlcNAcbeta1-6 branched triantennary sugar chains when sialylation was incomplete and an increase in poly-N-acetyllactosamine (Galbeta1-4GlcNAcbeta1-3)n was observed. In the present study, we introduced mouse Galbeta1-3/4GlcNAc-R alpha2,3-sialyltransferase (ST3Gal IV) and/or rat Galbeta1-4GlcNAc-R alpha2,6-sialyltransferase (ST6Gal I) cDNAs into the IM4/Vh cells to increase the extent of sialylation and to examine the effect of sialyltransferase (ST) type on the linkage of sialic acid. Furthermore, we speculated that sialylation extent might affect the level of poly-N-acetyllactosamine. We isolated four clones expressing different levels of alpha2,3-ST and/or alpha2,6-ST. The extent of sialylation of hIFN-gamma from the IM4/Vh clone was 61.2%, which increased to about 80% in every ST transfectant. The increase occurred regardless of the type of overexpressed ST, and the proportion of alpha2,3- and alpha2,6-sialic acid corresponded to the activity ratio of alpha2,3-ST to alpha2,6-ST. Furthermore, the proportion of N-glycans containing poly-N-acetyllactosamine was significantly reduced (less than 10%) in the ST transfectants compared with the parental IM4/Vh clone (22.9%). These results indicated that genetic engineering of STs is highly effective for regulating the terminal structures of sugar chains on recombinant proteins in CHO cells.
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PMID:Genetic engineering of CHO cells producing human interferon-gamma by transfection of sialyltransferases. 1151 14

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

Despite numerous reports suggesting that beta(1) integrin receptors undergo differential glycosylation, the potential role of N-linked carbohydrates in modulating integrin function has been largely ignored. In the present study, we find that beta(1) integrins are differentially glycosylated during phorbol ester (PMA)-stimulated differentiation of myeloid cells along the monocyte/macrophage lineage. PMA treatment of two myeloid cell lines, U937 and THP-1, induces a down-regulation in expression of the ST6Gal I sialyltransferase. Correspondingly, the beta(1) integrin subunit becomes hyposialylated, suggesting that the beta(1) integrin is a substrate for this enzyme. The expression of hyposialylated beta(1) integrin isoforms is temporally correlated with enhanced binding of myeloid cells to fibronectin, and, importantly, fibronectin binding is inhibited when the Golgi disrupter, brefeldin A, is used to block the expression of the hyposialylated form. Consistent with the observation that cells with hyposialylated integrins are more adhesive to fibronectin, we demonstrate that the enzymatic removal of sialic acid residues from purified alpha(5)beta(1) integrins stimulates fibronectin binding by these integrins. These data support the hypothesis that unsialylated beta(1) integrins are more adhesive to fibronectin, although desialylation of alpha(5) subunits could also contribute to increased fibronectin binding. Collectively our results suggest a novel mechanism for regulation of the beta(1) integrin family of cell adhesion receptors.
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PMID:Hyposialylation of integrins stimulates the activity of myeloid fibronectin receptors. 1209 85

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

The synthesis of the common and well-documented Siaalpha 2,6 to Galbeta 1,4GlcNAc structure (Sia6LacNAc) is principally mediated by the sialyltransferase ST6Gal I, which is particularly highly expressed in liver, lactating mammary gland, intestinal epithelia of newborn animals, and B cells. Multiple independent promoters govern the expression of Siat1, the ST6Gal I gene. In liver, elevation of hepatic and serum ST6Gal is part of the acute phase reaction, the hepatic response to systemic trauma, and is governed by the inducible, liver-specific promoter-regulatory region, P1. A constitutive and nontissue-specific promoter, P3, mediates low-level, basal hepatic Siat1 transcription. We generated a mouse specifically unable to use the transcriptional initiation site uniquely used in P1-mediated ST6Gal I expression. These animals, Siat1deltaP1, are viable and display reduced ST6Gal I mRNA in liver with concomitantly reduced sialyltransferase activities in liver and in serum. Siat1deltaP1 animals are unable to elevate hepatic Siat1 mRNA as part of the inflammatory response induced by turpentine. Surprisingly, serum glycoprotein components exhibit normal extent of sialylation, with no noticeable difference in binding to SNA, the alpha2,6-sialyl-specific lectin. Siat1deltaP1 animals also exhibit an outwardly normal B cell response. On intraperitoneal challenge with the pathogen Salmonella typhimurium, a significantly greater accumulation of neutrophils within the peritoneal space was observed in Siat1deltaP1 animals compared to wild-type mice. Siat1deltaP1 mice also exhibit a greater bacterial burden in liver and spleen, accompanied by more pronounced spleno-/hepatomegaly and greater leukocyte infiltration into affected organs than their wild-type counterparts.
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PMID:Biologic contribution of P1 promoter-mediated expression of ST6Gal I sialyltransferase. 1267

The aspartyl protease BACE1 cleaves the amyloid precursor protein and the sialyltransferase ST6Gal I and is important in the pathogenesis of Alzheimer's disease. The normal function of BACE1 and additional physiological substrates have not been identified. Here we show that BACE1 acts on the P-selectin glycoprotein ligand 1 (PSGL-1), which mediates leukocyte adhesion in inflammatory reactions. In human monocytic U937 and human embryonic kidney 293 cells expressing endogenous or transfected BACE1, PSGL-1 was cleaved by BACE1 to generate a soluble ectodomain and a C-terminal transmembrane fragment. No evidence of the cleavage fragment was seen in primary cells derived from mice deficient in BACE1. By using deletion constructs and enzymatic deglycosylation of the C-terminal PSGL-1 fragments, the cleavage site in PSGL-1 was mapped to the juxtamembrane region within the ectodomain. In an in vitro assay BACE1 catalyzed the formation of the PSGL-1 products seen in vivo. The cleavage occurred at a Leu-Ser peptide bond as identified by mass spectrometry using a synthetic peptide. We conclude that PSGL-1 is an additional substrate for BACE1.
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PMID:The cell adhesion protein P-selectin glycoprotein ligand-1 is a substrate for the aspartyl protease BACE1. 1450 29


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