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

通过分析高尔基体α-甘露糖苷酶II基因研究N-聚糖的作用

• 批准号: 08680666
• 负责人: MISAGO Masahiro
• 金额: $ 1.6万
• 依托单位: University of Occupational and Environmental Health (1997)University Occupational and Environmental Health, School of Nursing and Medical Technology (1996)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08680666/
• 关键词: Asn-linked glycan; N-glycan; alpha-mannosidase II; alpha-mannosidase II^x; Promoter; アルファマンノシダーゼII^x

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.

1.人α-甘露糖苷酶II^x（α-MII ^x）的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，我们使用酵母作为宿主制备重组α-MII ^x。该酶对合成的底物“甘露吡喃糖苷”和α-MII具有活性，且其活性不受苦马豆素的抑制。该酶被认为是与N-聚糖加工相关的酶之一。在以吡啶氨基寡糖为底物的实验中，发现α-MII ^x具有不同于α-MII的另一种底物特异性，我们正在对此进行研究。2. α-MII和α-MII ^x蛋白在细胞中的表达分析使用抗α-MII和α-MII ^x的抗血清，通过荧光原位杂交鉴定蛋白质的细胞基因座。3.人α-MII^x基因启动子区的分析我们克隆了一个大小约为40-kbp的粘粒，其中包括5 ′-侧翼序列。该克隆包含至少8个外显子，其编码α-MII ^x基因的总共1139个氨基酸残基中的396个氨基酸残基。没有典型的TATA或CAAT盒观察到，但（G+C）丰富的区域和六个Sp1的共识序列被发现在接近转录起始位点。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性。

项目成果

Ogawa R,et al.: "Structure and transcriptional reulation of human alpha-mannosidase II^x (alpha-mannosidase II isotype) gene." Eur J Biochem. 242. 446-453 (1996)

Okawa R,et al.：“人类 α-甘露糖苷酶 II^x（α-甘露糖苷酶 II 同型）基因的结构和转录调控。”

Misago M, et al.: "Molecular cloning and expression of cDNAs encoding human α-mannosidase II and a previously unrecognized α-mannosidase II^X isozyme." Proc Natl Acad Sci USA. 92. 11766-11770 (1995)

Misago M 等人：“编码人 α-甘露糖苷酶 II 和先前未识别的 α-甘露糖苷酶 II^X 同工酶的 cDNA 的分子克隆和表达。”Proc Natl Acad Sci USA。 92. 11766-11770 (1995)

Ogawa, R.: "Structure and transcriptional regulation of human α-mannosidase IIx (α-mannosidase II isotype)gene." Eur.J.Biochem.242. 446-453 (1996)

小川，R.：“人 α-甘露糖苷酶 IIx（α-甘露糖苷酶 II 同型）基因的结构和转录调控。Eur.J.Biochem.242（1996）。

Ogawa R, et al: "Structure and transcriptional regulation of human α-mannosidase II^X(α-mannosidase II isotype)gene." Eur J Biochem. 242. 446-453 (1996)

Okawa R 等人：“人 α-甘露糖苷酶 II^X（α-甘露糖苷酶 II 同种型）基因的结构和转录调控。”Eur J Biochem 242. 446-453 (1996)

Misago M,et al.: "Molecular cloning and expression of cDNAs encoding human alpha-mannosidase II and a previously unrecognized alpha-mannosidase II^x isozyme." Proc Natl Acad Sci USA. 92. 11766-11770 (1995)

Misago M 等人：“编码人 α-甘露糖苷酶 II 和以前未被识别的 α-甘露糖苷酶 II^x 同工酶的 cDNA 的分子克隆和表达。”