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的底物专一性分析为了分析人α-甘露糖苷酶II^x的底物专一性，我们以酵母为宿主制备了重组人α-甘露糖苷酶。该酶在合成底物‘甘露糖苷’和α-MII上具有活性，其活性不受苦马豆素的抑制。该酶被认为是与N-葡聚糖加工有关的酶之一。在以吡二胺寡糖为底物的实验中，我们发现α-MII具有不同于α-MII的另一种底物特异性。2.α-MII和α-MII蛋白在细胞中的表达分析用抗α-MII和α-MII^x的抗血清进行荧光原位杂交鉴定，证明这两种蛋白均存在于Medium-Golgi.3.人α-MII^x基因启动子区域的分析我们鉴定了包含5‘-侧翼序列的约40kBP的粘粒克隆。该克隆至少包含8个外显子，编码396个氨基酸残基，共1139个氨基酸残基。没有观察到典型的TATA或CAAT盒，但在转录起始点附近发现了一个(G+C)富集区和6个Sp1共同序列。-12~+11序列参与了启动子的功能。在该位点的上游区域发现了增强子活性。

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.