Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.4.99.6 (sialyltransferase)
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A Gal beta 1 to 4GlcNAc alpha 2 to 6 sialyltransferse and a Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferase have been purified 23,000- and 860,000-fold to homogeneity from Triton CF-54 extracts of rat liver membranes. The two enzymes were concentrated by affinity chromatography on CDP-hexanolamine-agarose and resolved by NaCl gradient elution from the same adsorbent. Final purification of the Gal beta 1 to 4GlcNAc alpha 2 to 6 sialytransferase, the most abundant enzyme, was achieved by specific elution from CDP-agarose with CDP. The Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferase was also purified further by CDP elution from CDP-agarose, but final purification required affinity chromatography on an adsorbent prepared by coupling asialoprothrombin to cyanogen bromide-activated agarose. Asialoprothrombin contains the terminal sequence Gal beta 1 to 3GlcNAc on N-linked oligosaccharides and is the best acceptor substrate of the enzyme (Km congruent to 6 microM). The Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferase was found to bind to asialoprothrombin-agarose in the presence of CDP and could be eluted with a solution containing 0.2 M lactose and no CDP. Sodium dodecyl sulfate-gel electrophoresis of the Gal beta 1 to 4GlcNAc alpha 2 to 6 and Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferases revealed a single major protein band for each enzyme with apparent molecular weights of 40,500 and 44,000, respectively. Rabbit antibodies raised to the Gal beta 1 to 4GlcNAc alpha 2 to 6 sialyltransferase inhibit its enzymatic activity greater than 99% but caused little or no inhibition of Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialytransferase. Moreover, the Gal beta 1 to 4GlcNAc alpha 2 to 6 sialyltransferase quantitatively bound to a column containing antibody adsorbed to Protein A-agarose, while the Gal beta 1 to 3(4) GlcNAc alpha 2 to 3 sialyltransferase did not bind. This demonstrated that the two sialyltransferases are antigenically unrelated and formed the basis for removal of contaminating Gal beta 1 to 4GlcNAc alpha 2 to 6 sialyltransferase from solutions of the Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferase. Enzymatic characterization of the two sialyltransferases suggests that their major biological roles are in the terminal glycosylation of N-linked oligosaccharides of glycoproteins. (Weinstein, J., de Souza-e-Silva, U., and Paulson J. C. (1982) J. Biol. Chem. 257, 13845-13853. The alpha 2 to 6 sialyltransferase efficiently forms the NeuAc alpha 2 to 6Gal beta 1 to 4GlcNAc sequence, and the alpha 2 to 3 sialyltransferase forms the NeuAc alpha 2 to 3Gal beta 1 to 3GlcNAc and NeuAc alpha 2 to 3Ga; beta 1 to 4GlcNAc sequences.
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PMID:Purification of a Gal beta 1 to 4GlcNAc alpha 2 to 6 sialyltransferase and a Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferase to homogeneity from rat liver. 714 79

In the framework of a project aimed at the elucidation of the nature of the functional importance of the N-glycosylation of the alpha-subunit of the glycoprotein hormones human lutropin and human chorionic gonadotropin, the structural element alpha-Neu p5Ac-(2-->6)-beta-D-GalpNac-(1-->4)- beta-D-GlcpNAc-(1-->2)-alpha-D-Manp, which is part of the carbohydrate chains of human lutropin, has been prepared by chemical and chemo-enzymatic synthesis in the form of its propyl glycoside. Condensation of 4-O- acetyl-3,6-di-O-benzyl-2-deoxy-2-phthalimido-alpha/beta-D-glucopyranosyl trichloroacetimidate with allyl 3,4,6-tri-O-benzyl-alpha-D-mannopyranoside gave after deacetylation allyl (3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl) -(1-->2)-3,4,6-tri-O-benzyl-alpha-D-mannopyranoside. Ethyl 3-O-benzyl-2-deoxy-2-phthalimido-l-thio-beta-D-glucopyranoside was converted into the galacto-derivative ethyl 4,6-di-O-acetyl-3-O-benzyl-2-deoxy-2-phthalimido-1-thio-beta-D -galactopyranoside via an oxidation-reduction route, as well as via SN2-type substitution with acetate. The use of this galacto thioglycoside, after its conversion into the corresponding bromide, as GaIN donor for condensation with the mentioned disaccharide derivative yielded after deacetylation allyl (3-O-benzyl-2-deoxy-2-phthalimido-beta-D-galactopyranosyl)-(1-->4) -(3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1-->2) -3,4,6-tri-O-benzyl-alpha-D-mannopyranoside. Methylsulfenyl bromide-silver triflate promoted sialylation of this trisaccharide derivative with O-ethyl S-[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D -glycero-alpha-D-galacto-non-2-ulopyranosyl)onate] dithiocarbonate and subsequent deprotection resulted into the aimed tetrasaccharide structural element. Alternatively, this compound was prepared via a block synthesis, which, however, was not superior to the linear strategy. Finally, a stereose lective sialylation of synthetically prepared beta-D-GalpNAc-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O) CH2CH2CH3 with CMP-Neu5Ac and rat liver alpha-2,6-sialyltransferase was accomplished affording the same tetrasaccharide structural element.
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PMID:Chemical and chemo-enzymatic synthesis of the alpha-Neu p5Ac-(2-->6)- beta-D-GalpNAc-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp element that is part of N-linked carbohydrate chains of human lutropin. 920 38