Mucha Jan, Svoboda Barbara, Kappel Sonja, Strasser Richard, Bencur Peter, Fröhwein Ulrike, Schachter Harry, Mach Lukas, Glössl Josef
Zentrum für Angewandte Genetik, Universität für Bodenkultur Wien, Muthgasse 18, A-1190 Wien, Austria.
Glycoconj J. 2002 Mar;19(3):187-95. doi: 10.1023/A:1024201824354.
UDP-GlcNAc:alpha6-D-mannoside beta1,2-N-acetylglucosaminyltransferase II (GnT II; EC 2.4.1.143) is a medial-Golgi resident enzyme that catalyses an essential step in the biosynthetic pathway leading from high mannose to complex N-linked oligosaccharides. Screening a cDNA library from Xenopus laevis ovary with a human GnT II DNA probe resulted in the isolation of two cDNA clones encoding two closely related GnT II isoenzymes, GnT II-A and GnT II-B. Analysis of the corresponding genomic DNAs revealed that the open reading frame of both X. laevis GnT II genes resides within a single exon. The GnT II-A gene was found to be transcriptionally active in all X. laevis tissues tested. In contrast, expression of the GnT II-B gene was detected only in a limited number of tissues. Both GnT II-A and GnT II-B exhibit a type II transmembrane protein topology with a putative N-terminal cytoplasmic tail of 9 amino acids followed by a transmembrane domain of 18 residues, and a C-terminal luminal domain of 405 residues. The two proteins differ at 28 amino acid positions within their luminal regions. Heterologous expression of soluble forms of the enzymes in insect cells showed that GnT II-A and GnT II-B are both catalytically active and exhibit similar specific activities. Both recombinant proteins are modified with N-linked oligosaccharides. N-terminal deletion studies demonstrated that the first 49 amino acid residues are not essential for proper folding and enzymatic activity of X. laevis GnT II.
UDP - N - 乙酰葡糖胺：α6 - D - 甘露糖苷β1,2 - N - 乙酰葡糖胺基转移酶II（GnT II；EC 2.4.1.143）是一种定位于高尔基体中部的酶，它催化从高甘露糖型到复杂N - 连接寡糖的生物合成途径中的关键步骤。用人GnT II DNA探针筛选非洲爪蟾卵巢的cDNA文库，分离出了两个编码两种密切相关的GnT II同工酶GnT II - A和GnT II - B的cDNA克隆。对相应基因组DNA的分析表明，两种非洲爪蟾GnT II基因的开放阅读框都位于单个外显子内。发现GnT II - A基因在所有检测的非洲爪蟾组织中都具有转录活性。相比之下，仅在有限数量的组织中检测到GnT II - B基因的表达。GnT II - A和GnT II - B都表现出II型跨膜蛋白拓扑结构，具有一个推定的由9个氨基酸组成的N端胞质尾巴，接着是一个由18个残基组成的跨膜结构域，以及一个由405个残基组成的C端腔结构域。这两种蛋白质在其腔区域内的28个氨基酸位置上存在差异。在昆虫细胞中对这些酶的可溶性形式进行异源表达表明，GnT II - A和GnT II - B都具有催化活性且表现出相似的比活性。两种重组蛋白都用N - 连接寡糖进行了修饰。N端缺失研究表明，前49个氨基酸残基对于非洲爪蟾GnT II的正确折叠和酶活性不是必需的。
