Watanabe Tae, Ihara Hideyuki, Miyoshi Eiji, Honke Koichi, Taniguchi Naoyuki, Taguchi Tomohiko
Department of Biochemistry, Osaka University Graduate School of Medicine 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Glycobiology. 2006 May;16(5):431-9. doi: 10.1093/glycob/cwj079. Epub 2006 Jan 20.
Malignant transformation is often accompanied by an aberrant glycosylation profile of the cell surface-in particular, the production of GlcNAcbeta1-6Manalpha1 branches in N-linked glycoproteins. To identify the target glycoproteins, we show a method using recombinant chicken N-acetylglucosaminyltransferase VI (GnT VI) and radiolabeled uridine (5'-)diphosphate-GlcNAc. The assay exploits the fact that GnT VI has a strict requirement for the GlcNAcbeta1-6Manalpha1 structure for activity, when a pyridylaminated free N-glycan is used as the acceptor substrate. Human asialo-agalacto alpha1-acid glycoprotein (AGP), which is known to contain GlcNAcbeta1-6Manalpha1 branches in its N-linked glycan chains, was radiolabeled when reacted with GnT VI, whereas human asialo-agalacto transferrin and bovine fetuin, neither of which contains a GlcNAcbeta1-6Manalpha1 structure were not, thus corroborating the specificity of the assay. Several proteins from human serum after pretreatment with sialidase and beta-galactosidase could be detected using the assay. One was identified as AGP from its mobility on SDS-PAGE, demonstrating the potential of this assay even with crude materials. Furthermore, this method could detect a protein that was also positively stained with leukoagglutinating phytohemagglutinin (L(4)-PHA) using glycoproteins prepared from WiDr human colon cancer cells. This method should provide a useful complement to the current method, which relies on the specificity of L(4)-PHA.
恶性转化通常伴随着细胞表面异常的糖基化谱——特别是在N-连接糖蛋白中产生β1,6分支的N-乙酰葡糖胺（GlcNAc）。为了鉴定目标糖蛋白，我们展示了一种使用重组鸡N-乙酰葡糖胺转移酶VI（GnT VI）和放射性标记的尿苷（5'-）二磷酸-GlcNAc的方法。该检测利用了这样一个事实，即当使用吡啶基化的游离N-聚糖作为受体底物时，GnT VI对β1,6分支的N-乙酰葡糖胺（GlcNAc）结构具有严格的活性要求。人去唾液酸-去半乳糖α1-酸性糖蛋白（AGP），已知其N-连接聚糖链中含有β1,6分支的N-乙酰葡糖胺（GlcNAc）结构，与GnT VI反应时会被放射性标记，而人去唾液酸-去半乳糖转铁蛋白和牛胎球蛋白，两者均不含有β1,6分支的N-乙酰葡糖胺（GlcNAc）结构，则不会被放射性标记，从而证实了该检测的特异性。用唾液酸酶和β-半乳糖苷酶预处理后的人血清中的几种蛋白质可以用该检测方法检测到。其中一种通过SDS-PAGE上的迁移率被鉴定为AGP，这表明即使使用粗原料，该检测方法也具有潜力。此外，使用从WiDr人结肠癌细胞制备的糖蛋白，该方法可以检测到一种也能用白细胞凝集植物血凝素（L(4)-PHA）阳性染色的蛋白质。该方法应该为目前依赖L(4)-PHA特异性的方法提供有用的补充。
