Studies on the role of N-glycan by analysis of Golgi alpha-mannosidase II gene

1.人α -甘露糖苷酶II^x （α - mii ^x）底物特异性分析为了分析人α -甘露糖苷酶II^x的底物特异性，我们以酵母为宿主制备了重组α - mii ^x。该酶对合成底物甘露诺吡喃苷和α - mii均有活性，且不受苦马豆素的抑制。该酶被认为是与n -聚糖加工有关的酶之一。在以吡啶二氨基低聚糖为底物的实验中，表明α - mii ^x具有不同于α - mii的另一种底物特异性。我们现在正在研究这一点。细胞中α - mii和α - mii ^x蛋白的表达分析α - mii和α - mii ^x cdna的表达载体在COS-1细胞中共表达。用抗α - mii和α - mii ^x血清荧光原位杂交鉴定了蛋白的细胞位点。这两种蛋白均已被证明存在于中位高尔基蛋白中。人类α - mii ^x基因启动子区分析我们鉴定了包含5'-侧翼序列的约40-kbp的cosmid克隆。该克隆包含至少8个外显子，编码α - mii ^x基因1139个氨基酸残基中的396个氨基酸残基。没有观察到典型的TATA或CAAT盒子，但在转录起始位点附近发现了一个（G+C）丰富区域和6个Sp1一致序列。从-12到+11的序列参与启动子功能。在该位点的上游区域发现了增强子活动。

1.Analysis of substrate specificity of human alpha-mannosidase II^x (alpha-MII^x)In order to analyze the substrate specificity of human alpha-mannosidase II^x, we made recombinant alpha-MII^x using yeast as host. The enzyme possessed the activity on synthesized substrate 'mannnopyranoside' as well as alpha-MII and its activity was not inhibited with swainsonine. The enzyme was thought to be one of enzymes associated with processing of N-glycan. In experiments using pyridilamino-oligosaccharides as substrates, it was suggested that alpha-MII^x had another substrate specificity different from alpha-MII.We are now examining on this point.2.Analysis of expression of alpha-MII and alpha-MII^x proteins in cellsThe expression vectors including alpha-MII and alpha-MII^x cDNAs were co-expressed in COS-1 cells. Cellular Loci of the proteins were indentified with fluorescent in-situ hybridization using antisera against alpha-MII and alpha-MII^x. Either protein has proven to be exist in median-Golgi.3.Analysis of the promoter region of human alpha-MII^x geneWe characterized the cosmid clone of about 40-kbp that include the 5'-flanking sequence. This clone contains at least eight exons which encode 396 amino acid residues of a total of 1139 amino acid residues of alpha-MII^x gene. No canonical TATA or CAAT boxes were observed, but a (G+C) -rich region and six Sp1 consensus sequence was found in close proximity to the transcription-initiation site. The sequence from -12 to +11 was involved in the promoter function. Enhancer activities were found in the region upstream of this site.