1997 Fiscal Year Final Research Report Summary
Studies on the role of N-glycan by analysis of Golgi alpha-mannosidase II gene
| Project/Area Number |
08680666
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Structural biochemistry
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| Research Institution | University of Occupational and Environmental Health (1997)
University Occupational and Environmental Health, School of Nursing and Medical Technology (1996) |
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Principal Investigator |
MISAGO Masahiro UOEH,School of Health Sciences, Associate Prof., 産業保健学部, 助教授 (30157474)
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| Co-Investigator(Kenkyū-buntansha) |
ETO Sumiya UOEH,1st.Dep.of Int.Medicine, Professor, 医学部, 教授 (90010347)
TSUKADA Junichi UOEH,1st.Dep.of Int.Medicine, Assistant Prof., 医学部, 助手 (20227367)
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| Project Period (FY) |
1996 – 1997
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| Keywords | Asn-linked glycan / N-glycan / alpha-mannosidase II / alpha-mannosidase II^x / Promoter |
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| Research Abstract |
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 cells
The 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 gene
We 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.
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