van den Eijnden D H, Koenderman A H, Schiphorst W E
Department of Medical Chemistry, Vrije Universiteit, Amsterdam, The Netherlands.
J Biol Chem. 1988 Sep 5;263(25):12461-71.
An N-acetylglucosaminyltransferase has been partially purified from Novikoff tumor cell ascites fluid by affinity chromatography on concanavalin A-Sepharose. The enzyme was obtained in a highly concentrated form after lyophilization. The enzyme appeared to be highly specific for acceptor oligosaccharides and glycoproteins carrying a terminal Gal beta 1----4GlcNAc beta 1----R unit. Characterization of products formed by the enzyme in vitro by methylation analysis and 1H NMR spectroscopy revealed that the enzyme catalyzed the formation of a GlcNAc beta 1----3Gal beta 1----4GlcNAc beta-R sequence. The enzyme therefore could be described as an UDP-GlcNAc:Gal beta 1----4GlcNAc beta-R beta 1----3-N-acetylglucosaminyltransferase. Acceptor specificity studies with oligosaccharides that form part of N-glycans revealed that the presence of a Gal beta 1----4GlcNAc beta 1----2(Gal beta 1----4GlcNAc beta 1----6)Man pentasaccharide in the acceptor structure is a requirement for optimal activity. Studies on the branch specificity of the enzyme showed that the branches of this pentasaccharide structure, when contained in tri- and tetraantennary oligosaccharides, are highly preferred over other branches for attachment of the 1st and 2nd mol of GlcNAc into the acceptor molecule. The enzyme also showed activity toward oligosaccharides related to blood group I- and i-active polylactosaminoglycans. In addition the enzyme together with calf thymus UDP-Gal:GlcNAc beta-R beta 1----4-galactosyltransferase was capable of catalyzing the synthesis of a series of oligomers of N-acetyllactosamine. Competition studies revealed that all acceptors were acted upon by a single enzyme species. It is concluded that the N-acetylglucosaminyltransferase functions in both the initiation and the elongation of polylactosaminoglycan chains of N-glycoproteins and possibly other glycoconjugates.
一种N - 乙酰葡糖胺基转移酶已通过伴刀豆球蛋白A - 琼脂糖亲和层析从诺维科夫肿瘤细胞腹水液中部分纯化。该酶经冻干后以高浓缩形式获得。该酶似乎对带有末端Galβ1----4GlcNAcβ1----R单元的受体寡糖和糖蛋白具有高度特异性。通过甲基化分析和1H NMR光谱对该酶体外形成的产物进行表征，结果表明该酶催化形成了GlcNAcβ1----3Galβ1----4GlcNAcβ - R序列。因此，该酶可被描述为UDP - GlcNAc:Galβ1----4GlcNAcβ - Rβ1----3 - N - 乙酰葡糖胺基转移酶。对构成N - 聚糖一部分的寡糖进行的受体特异性研究表明，受体结构中存在Galβ1----4GlcNAcβ1----2(Galβ1----4GlcNAcβ1----6)Man五糖是实现最佳活性的必要条件。对该酶的分支特异性研究表明，当该五糖结构的分支存在于三触角和四触角寡糖中时，与其他分支相比，它们在将第1和第2摩尔GlcNAc连接到受体分子中时具有更高的优先性。该酶对与血型I - 和i - 活性多乳糖胺聚糖相关的寡糖也表现出活性。此外，该酶与小牛胸腺UDP - Gal:GlcNAcβ - Rβ1----4 - 半乳糖基转移酶一起能够催化一系列N - 乙酰乳糖胺寡聚物的合成。竞争研究表明，所有受体都由单一酶作用。结论是，N - 乙酰葡糖胺基转移酶在N - 糖蛋白以及可能的其他糖缀合物的多乳糖胺聚糖链的起始和延伸过程中发挥作用。
