1994 Fiscal Year Final Research Report Summary
Peptide : N-glycanase-catalyzed De-N-glycosylation of Glycoproteins in Mammalian Cells
| Project/Area Number |
05808053
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Structural biochemistry
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| Research Institution | THE UNIVERSITY OF TOKTO |
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Principal Investigator |
KITAJIMA Ken THE UNIVERSITY OF TOKTO,GRADUATE SCHOOL OF SCIENCE,SENIOR RESEARCH FELLOW, 大学院・理学系研究科, 助手 (80192558)
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| Project Period (FY) |
1993 – 1994
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| Keywords | PNGase (Peptide : N-glycanase) / Post-translational Modification Of Proteins / Mammalian PNGase / L-929 PNGase / De-N-glycosylation Of Glycoproteins / Biological Function Of PNGase / Carbohydrate-Binding Activity / PNGase Family |
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| Research Abstract |
Recently, we have proposed the hypothesis that peptide : N-glycanase (PNGase) -catalyzed de-N-glycosylation of glycoproteins is a post-translational remodification process to modulate physicochemical and physiological properties of certain functional proteins. In order to validate our hypothesis, we have anticipated the general occurrence of PNGase and PNGase-catalyzed reaction in a wider variety of living cells, and we have initiated to examine the distribution of PNGase among mammalian organs and tissues. In this reserch project, we have carried out the following studies on distribution and properties of mammalian PNGases.
1. PNGase was purified to homogeneity from mouse fibroblast L-929 cells (designated as L-929 PNGase) , and its unique properties, which were different from those of plant and bacterial PNGases, were revealed : (1) requirement of-SH group (s) ; (2) neutral and alkaline pH optima ; (3) inhibition by free N-linked glycan chains.
2. L-929 PNGase was shown to have carbohy
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drate-binding activity, suggesting that L-929 PNGase may play dual roles as a deglycosylating enzyme and as a carbohydrate-binding protein.
3. The simple assay method for PNGase activity using ^<14>C-labeled asialo-fetuin glycopeptide I as a substrate was developed, which was based on the paper chromatographical and paper electrophoretical analyzes of the deglycosylated peptide product.
4. A wide occurrence of PNGase in mouse organs was demonstrated. PNGase activities were detected in both soluble and membranous fractions, although the levels of the activities were different from organ to organ.
5. Soluble PNGases were partially purified from pig spleen. At least four PNGases were identified, which differed in physicoshemical properties (hydrophobicity and molecular weight) from each other, suggesting soluble PNGases may possibly constitute a "PNGase family" . These soluble enzymes shared some enzymatic properties (see above) with L-929 PNGase, and these soluble and neutral PNGases may be involved in basic biological processes in certain intracellular non-lysosomal events. Less
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