Sultan A S, Miyoshi E, Ihara Y, Nishikawa A, Tsukada Y, Taniguchi N
Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan.
J Biol Chem. 1997 Jan 31;272(5):2866-72. doi: 10.1074/jbc.272.5.2866.
The bisecting N-acetylglucosamine residue is formed by UDP-N-acetylglucosamine:beta-D-mannoside-beta-1, 4-N-acetylglucosaminyltransferase III (GnT-III), a key branching enzyme for N-glycans. We found that forskolin, an adenylyl cyclase activator, markedly enhanced GnT-III at the transcriptional level in various hepatoma cells and hepatocytes, resulting in an increase of bisecting GlcNAc residues in various glycoproteins, as judged from the lectin binding to erythroagglutinating phytohemagglutinin (E-PHA). In whole cell lysates, the E-PHA binding was increased, and leukoagglutinating phytohemagglutinin (L-PHA) binding was decreased at 12 h after forskolin treatment, by time, both GnT-III activity and mRNA had reached the maximum levels. In contrast, the binding capacity as to E-PHA, determined by fluorescence-activated cell sorting on the cell surface, was decreased, suggesting that bisecting GlcNAc structures in certain glycoproteins changed the expression levels of glycoproteins and decreased their sorting on the cell surface. Fractionated organelles of M31 cells showed that the binding capacity as to E-PHA was mainly localized in Golgi membranes and lysosomes. This was also supported by a fluorescence microscopy. In order to determine whether or not the bisecting GlcNAc residue acts as a sorting signal for glycoproteins, N-oligosaccharide structures of lysosomal-associated membrane glycoprotein 1 and beta-glucuronidase, gamma-glutamyltranspeptidase, and secretory glycoproteins such as ceruloplasmin and alpha-fetoprotein were measured by E-PHA and L-PHA blotting after immunoprecipitation. The expression levels of lysosomal membrane glycoprotein 1 and gamma-glutamyltranspeptidase on the cell surface were decreased at 12 h after forskolin treatment, indicating that the bisecting GlcNAc structure may act as a negative sorting signal for the cell surface glycoproteins and may alter the characteristics of hepatoma cells. This is the first report on glycoprotein sorting related to a specific structure of oligosaccharides, bisecting GlcNAc.
平分型N-乙酰葡糖胺残基由UDP-N-乙酰葡糖胺：β-D-甘露糖苷-β-1,4-N-乙酰葡糖胺基转移酶III（GnT-III）形成，它是N-聚糖的关键分支酶。我们发现，腺苷酸环化酶激活剂福斯可林在转录水平上显著增强了各种肝癌细胞和肝细胞中的GnT-III，导致各种糖蛋白中平分型GlcNAc残基增加，这从凝集素与红细胞凝集植物血凝素（E-PHA）的结合情况判断得出。在全细胞裂解物中，福斯可林处理12小时后，E-PHA结合增加，而白细胞凝集植物血凝素（L-PHA）结合减少，此时GnT-III活性和mRNA均达到最高水平。相反，通过细胞表面荧光激活细胞分选测定的对E-PHA的结合能力降低，这表明某些糖蛋白中的平分型GlcNAc结构改变了糖蛋白的表达水平并降低了它们在细胞表面的分选。M31细胞的分级细胞器显示，对E-PHA的结合能力主要定位于高尔基体膜和溶酶体中。荧光显微镜检查也支持了这一点。为了确定平分型GlcNAc残基是否作为糖蛋白的分选信号，在免疫沉淀后通过E-PHA和L-PHA印迹法测量了溶酶体相关膜糖蛋白1、β-葡萄糖醛酸酶、γ-谷氨酰转肽酶以及分泌性糖蛋白如铜蓝蛋白和甲胎蛋白的N-寡糖结构。福斯可林处理12小时后，溶酶体膜糖蛋白1和γ-谷氨酰转肽酶在细胞表面的表达水平降低，表明平分型GlcNAc结构可能作为细胞表面糖蛋白的负分选信号，并可能改变肝癌细胞的特性。这是关于与寡糖特定结构（平分型GlcNAc）相关的糖蛋白分选的首次报道。
