Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.4.99.10 (sialyltransferase)
 1,547 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Iodine incorporation into thyroglobulin is known to occur within the lumen of the thyroid follicle. Since incorporation of sialic acid, which occupies a terminal position in the oligosaccharide chains, is also a later event in thyroglobulin synthesis, the possibility that sialic acid might be incorporated after thyroglobulin secretion was investigated. In one experimental approach normal rat thyroid hemilobes were incubated with radioactive precursors. Thyroglobulin, analyzed by equilibrium centrifugation in RbCl, had a median density which varied according to the moiety labeled in the following increasing order: leucine smaller than galactose smaller than sialic acid smaller than iodine. The molecules having the highest density were labeled only with iodine. In the second approach, thyroid hemilobes were taken from rats treated with cycloheximide for 16 hours to stop protein synthesis and allow nascent molecules to be secreted, and incorporation of precursors into thyroglobulin was analyzed by sucrose gradient centrifugation. Leucine incorporation was 6% of control but the amino acid was found in the NH2-terminal position. N-Acetylmannosamine (sialic acid precursor) and galactose incorporation were also completely inhibited whereas iodine incorporation was 10% of control. Incorporation was not restored by thyrotropin treatment, and the sialyltransferase and iodination systems were reduced only to 50 to 70% of control. The results indicate that sialic acid is incorporated only in nascent thyroglobulin and not in thyroglobulin molecules already secreted into the follicular lumen. A large fraction of the iodine incorporation also seems to occur in newly synthesized thyroglobulin.
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PMID:The site of sialic acid incorporation into thyroglobulin in the thyroid gland. 111 19

Sequence information obtained by NH2-terminal sequence analysis of two molecular weight forms (45 and 48 kDa) of the porcine Gal beta 1,3GalNAc alpha 2,3-sialyltransferase was used to clone a full-length cDNA of the enzyme. The cDNA sequence revealed an open reading frame coding for 343 amino acids and a putative domain structure consisting of a short NH2-terminal cytoplasmic domain, a signal-anchor sequence, and a large COOH-terminal catalytic domain. This domain structure was confirmed by construction of a recombinant sialyltransferase in which the cytoplasmic domain and signal-anchor sequence of the enzyme was replaced with the cDNA of insulin signal sequence. Expression of the resulting construct in COS-1 cells produced an active sialyltransferase which was secreted into the medium in soluble form. Comparison of the cDNA sequence of the sialyltransferase with GenBank produced no significant homologies except with the previously described Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase. Although the cDNA sequences of these two enzymes were largely nonhomologous, there was a 45-amino acid sequence which exhibited 65% identity. This observation suggests that the two sialyltransferases were derived, in part, from a common gene.
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PMID:Cloning and expression of the Gal beta 1, 3GalNAc alpha 2,3-sialyltransferase. 138 14

We have isolated, by immunological screening of a lambda gt11 expression library, a cDNA clone that represents the complete coding sequence for bovine alpha 1----3-galactosyltransferase. The coding sequence predicts a membrane-bound protein with three distinct structural features: a large, potentially glycosylated COOH-terminal domain (346 amino acids), a single transmembrane domain (16 amino acids), and a short NH2-terminal domain (6 amino acids). Thus, the domain structure for this transferase is similar to that deduced for beta 1----4-galactosyltransferase (Shaper, N. L., Hollis, G. F., Douglas, J. G., Kirsch, I. R., and Shaper, J. H. (1988) J. Biol. Chem. 263, 10420-10428) and alpha 2----6-sialyltransferase (Weinstein, J., Lee, E. V., McEntee, K., Lai, P.-H., and Paulson, J. C. (1987) J. Biol. Chem. 262, 17735-17743). S1 analysis demonstrates that two sets of mRNAs, which are heterogeneous at their 5' ends, are transcribed. Because both sets initiate upstream of the translational start site, only one protein is encoded by this gene. alpha 1----3-Galactosyltransferase is widely expressed in different mammalian species, with the notable exception of man and Old World monkeys (Galili, U., Shohet, S. B., Kobrin, E., Stults, C.L.M., and Macher, B. A. (1988) J. Biol. Chem. 263, 17755-17762). By Northern blot analysis we were indeed unable to detect transcripts for this enzyme in various human and Old World monkey cell lines; transcripts were readily detected in other mammalian species. However, by Southern blot analysis, homologous sequences for alpha 1----3-galactosyltransferase were identified in human genomic DNA. This suggests that the gene, although present in the human genome, is normally not expressed. These observations have potential medical implications. Because many humans have high levels of circulating antibodies directed against the enzymatic product of alpha 1----3-galactosyltransferase (Gal alpha 1----3Gal beta 1----4GlcN Ac) (Galili, U., Clark, M. R., Shohet, S. B., Buehler, J., and Macher, B. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1369-1373), it has been suggested that activation of this normally silent gene may play a role in autoimmune disease in man (Etienne-Decerf, J., Malaise, M., Mahieu, P., and Winand, R. (1987) Acta Endocrinol. 115, 67-74).
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PMID:Bovine alpha 1----3-galactosyltransferase: isolation and characterization of a cDNA clone. Identification of homologous sequences in human genomic DNA. 250 16

The beta-galactoside alpha 2,6 sialyltransferase, an integral membrane protein localized to the trans-region of the Golgi apparatus, has been converted into a catalytically active secreted protein by the replacement of the NH2-terminal signal-anchor domain with the cleavable signal peptide of human gamma-interferon. Pulse-chase analysis of the wild type and recombinant proteins expressed in stably transfected Chinese hamster ovary cells showed that the wild type sialyltransferase (47 kDa) remained cell-associated. In contrast, the signal peptide-sialyltransferase fusion protein yielded an enzymatically active 41-kDa polypeptide which was secreted with a half-time of 2-3 h, consistent with cleavage of the signal peptide. The data indicate that the catalytic domain does not contain sufficient information for retention in the Golgi apparatus and that retention signals are likely to be found in the NH2-terminal 57 amino acids of the wild type enzyme.
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PMID:Conversion of a Golgi apparatus sialyltransferase to a secretory protein by replacement of the NH2-terminal signal anchor with a signal peptide. 280 34

This report describes the primary structure of a rat liver beta-galactoside alpha 2,6-sialyltransferase (EC 2.4.99.1), a Golgi apparatus enzyme involved in the terminal sialylation of N-linked carbohydrate groups of glycoproteins. The complete amino acid sequence was deduced from the nucleotide sequence of cDNA clones of the enzyme. The primary structure suggests that the topology of the enzyme in the Golgi apparatus consists of a short NH2-terminal cytoplasmic domain, a 17-residue hydrophobic sequence which serves as the membrane anchor and signal sequence, and a large lumenal, catalytic domain. NH2-terminal sequence analysis of a truncated form of the enzyme, obtained by purification from tissue homogenates, reveals that it is missing a 63-residue NH2-terminal peptide which includes the membrane binding domain. These and supporting results show that soluble forms of the sialyltransferase can be generated by proteolytic cleavage between the NH2-terminal signal-anchor and the catalytic domain.
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PMID:Primary structure of beta-galactoside alpha 2,6-sialyltransferase. Conversion of membrane-bound enzyme to soluble forms by cleavage of the NH2-terminal signal anchor. 312 4

Sialyltransferases are a family of 10-12 enzymes that catalyze the transfer of sialic acid to carbohydrate groups of glycoproteins and glycolipids. Three sialyltransferase cDNAs have been cloned, revealing a highly conserved sialylmotif in the catalytic domain of these enzymes. Using a polymerase chain reaction-based approach, we cloned a 150-base pair fragment of a new sialymotif from human placenta mRNA, which was then used as a probe to clone the complete coding sequence of the corresponding gene from a cDNA library. Like the other members of the sialyltransferase gene family cloned to date, the new cDNA coded for a protein predicted to have an NH2-terminal signal-anchor sequence and had the sialylmotif located in the center of the molecule. Comparison with the three other cloned sialyltransferases revealed extensive sequence homology that was not recognized earlier. Expression of a soluble recombinant form of the protein in COS-1 cells produced an active sialyltransferase, which used oligosaccharide, glycoprotein, and glycolipid acceptor substrates with terminal galactose in the Gal beta 1,3GalNAc and Gal beta 1, 4GlcNAc sequences but not the Gal beta 1,3GlcNAc sequence. The sialylated products were sensitive to digestion with the Newcastle disease virus sialidase, which is specific for sialic acid-galactose linkages in the alpha 2,3 linkage. The results suggest that this new member of the sialyltransferase gene family is the enzyme previously described as a glycolipid sialyltransferase activity (SAT-3), which forms the terminal sequences NeuAc alpha-2,3Gal beta 1,3GalNAc-R and NeuAc alpha 2,3Gal beta 1, 4GlcNAc-R.
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PMID:Cloning of a novel alpha 2,3-sialyltransferase that sialylates glycoprotein and glycolipid carbohydrate groups. 828 6

cDNA clones encoding GalNAc alpha 2,6-sialyltransferase (EC 2.4.99.3) have been isolated from chick embryo cDNA libraries using sequence information obtained from the conserved amino acid sequence of the previously cloned enzymes. The cDNA sequence included an open reading frame coding for 566 amino acids, and the deduced amino acid sequence showed 12% identity with that of Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase from chick embryo. The primary structure of this enzyme suggested a putative domain structure, like that in other glycosyltransferases, consisting of a short NH2-terminal cytoplasmic domain, a signal-membrane anchor domain, a proteolytically sensitive stem region, and a large COOH-terminal active domain. The identity of this enzyme was confirmed by the construction of a recombinant sialyltransferase in which the NH2-terminal part (232 amino acid residues) was replaced with the immunoglobulin signal sequence. The expression of this recombinant in COS-7 cells resulted in secretion of a catalytically active and soluble form of the enzyme into the medium. The expressed enzyme exhibited activity toward only asialomucin and (asialo)fetuin, no significant activity being detected toward the other glycoprotein and glycolipid substrates tested. 14C-Sialylated glycols obtained from asialomucin re-sialylated with this enzyme were identical to NeuAc alpha 2,6-GalNAc-ol and GlcNAc beta 1,3(NeuAc alpha 2,6) GalNAc-ol. Synthetic GalNAc-SerNAc also served as an acceptor for alpha 2,6-sialylation. These results clearly showed that the expressed enzyme is GalNAc alpha 2,6-sialyltransferase.
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PMID:Molecular cloning and expression of GalNAc alpha 2,6-sialyltransferase. 828 7

DNA clones encoding beta-galactoside alpha 2,3-sialyltransferase have been isolated from mouse brain cDNA libraries using sequence information obtained from the conserved amino acid sequence of the previously cloned enzymes. The cDNA sequence revealed an open reading frame coding for 337 amino acids, and the deduced amino acid sequence showed 80% identity with that of porcine submaxillary gland Gal beta 1,3GalNAc alpha 2,3-sialyltransferase. The primary structure of this enzyme suggested a putative domain structure, like that in other glycosyltransferases, consisting of a short NH2-terminal cytoplasmic domain, a signal-membrane anchor domain, a proteolytically sensitive stem region, and a large COOH-terminal active domain. The identity of this enzyme was confirmed by construction of a recombinant sialyltransferase in which the NH2-terminal part including the cytoplasmic tail, signal-anchor domain and stem region was replaced with an immuno-globulin signal sequence. The expression of this recombinant in COS-7 cells resulted in secretion of a catalytically active and soluble form of the enzyme into the medium. This enzyme exhibited the transferase activity toward only the disaccharide moiety of Gal beta 1,3GalNAc of glycoproteins and glycolipids, no significant activity being detected for the other substrates tested.
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PMID:Molecular cloning and expression of Gal beta 1,3GalNAc alpha 2,3-sialyltransferase from mouse brain. 837 77

To examine the role of the NH2-terminal region of the 402-residue-long beta-1,4-galactosyltransferase (beta-1,4-GT), a series of mutants and chimeric cDNA were constructed by polymerase chain reaction and transiently expressed in COS-7 cells, the enzyme activities were measured, and the protein was localized in the cells by subcellular fractionation or indirect immunofluorescence microscopy. We showed earlier that the deletion of the amino-terminal cytoplasmic tail and transmembrane domain from GT abolishes the stable expression of this protein in mammalian cells (Masibay, A.S., Boeggeman, E., and Qasba, P.K. (1992) Mol. Biol. Rep. 16, 99-104). Further deletion analyses of the amino-terminal region show that the first 21 amino acids of beta-1,4-GT are not essential for the stable production of the protein and are consistently localized in the Golgi apparatus. In addition, analysis of hybrid constructs showed that residues 1-25 of alpha-1,3-galactosyltransferase can functionally replace the beta-1,4-GT amino-terminal domain (residues 1-43). This fusion protein also showed Golgi localization. On the other hand, the alpha-2,6-sialyltransferase/beta-1,4-GT fusion protein (alpha-2,6-ST/beta-1,4-GT) needed additional COOH-terminal sequences flanking the transmembrane domain of the alpha-2,6-ST for stability and Golgi localization. Substitution of Arg-24, Leu-25, Leu-26, and His-33 of the beta-1,4-GT transmembrane by Ile (pLFM) or substitution of Tyr by Ile at positions 40 and 41 coupled with the insertion of 4 Ile residues at position 43 (pLB) released the mutant proteins from the Golgi and was detected on the cell surface. Our results show that (a) the transmembrane domains of beta-1,4-GT, alpha-1,3-galactosyltransferase, and alpha-2,6-ST, along with its stem region, all play a role in Golgi targeting and participate in a common mechanism that allows the protein to be processed properly and not be degraded in vivo; (b) increasing the length of the transmembrane domain overrides the Golgi retention signal and directs the enzyme to the plasma membrane; and (c) the length of the hydrophobic region of the transmembrane domain of beta-1,4-GT is an important parameter but is not sufficient by itself for Golgi retention.
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PMID:Mutational analysis of the Golgi retention signal of bovine beta-1,4-galactosyltransferase. 838 8

Our goal was to engineer a Golgi glycosyltransferase epitope-tagged on its cytoplasmically exposed, short, N-terminal domain that gave normal subcellular localization. Partial replacement of the cytoplasmic tail of human alpha-2,6-sialyltransferase (SialylT) with the negatively charged myc or FLAG epitope resulted in almost complete mislocalization of the chimera expressed in Vero cells. A granular cytoplasmic staining pattern was seen by immunofluorescence. Spacing the negatively charged residues progressively outward from the negative N-terminus resulted in increasingly more normal localization of myc or FLAG-tagged protein to a juxtanuclear Golgi-like distribution. Substitution of a neutrally charged VSV-G sequence for these tags resulted in normal localization of the chimera to the juxtanuclear Golgi region. Insertion of the myc epitope within the N-terminal domain of the short form of bovine beta-1,4-galactosyltransferase (GalT) gave a chimeric protein that mislocalized in BHK cells. No signal was detected with a monoclonal anti-epitope antibody indicating that the myc epitope was masked. Placement of myc or FLAG epitopes at the NH2-terminus of human N-acetylglucosaminyltransferase I (GlcNAc-T) resulted in chimeric proteins that in Vero cells displayed little Golgi localization. We conclude that positioning of negative charge, in particular, close to the membrane, typically produces a failure of type II Golgi glycosyltransferases to exit the ER/CGN, presumably due to quality control mechanisms. These proteins may be successfully epitope-tagged on their N-terminal domain either using a neutral or positively charged sequence or spacing any negatively charged sequence out from the membrane.
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PMID:Modification of the cytoplasmic domain affects the subcellular localization of Golgi glycosyl-transferases. 888 78


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