1995 Fiscal Year Final Research Report Summary
Analvsis of localization of a glycosyltransferase and transfort of glycolipids by gene manipulation
| Project/Area Number |
06670146
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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 |
General medical chemistry
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| Research Institution | Nagasaki University |
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Principal Investigator |
FURUKAWA Keiko Nagasaki University, School of Medicine, Assistent Professor, 医学部, 助手 (50260732)
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| Co-Investigator(Kenkyū-buntansha) |
FURUKAWA Koichi Nagasaki University, School of Medicine, Assistent Professor, 医学部, 助教授 (80211530)
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| Project Period (FY) |
1994 – 1995
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| Keywords | glycosyltransferase / N-glycosylation / localization / gene modification / ganglioside |
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| Research Abstract |
The amino acid sequence deduced from the cloned human cDNA of beta-1,4-N-acetyl-galactosaminyltransferase (GalNAc-T ; EC2.4.1.92) gene predicted three potential sites for N-linked glycosylation. Although many glycosyltransferases isolated contain from 2 to 6 N-glycosylation sites, their significance has not been adequately demonstrated. To clarify the roles of N-glycosylation in GalNAc-T function, we generated a series of mutant cDNAs, in which some or all of the glycosylation recognition sites were eliminated by polymerase chain reaction( PCR)-mediated site directed mutagenesis. Using transcription/translation in vitro, we confirmed the all potential N-glycosylation sites could be used. Although cell lines transfected with mutant cDNAs showed equivalent levels of GalNAcbeta1*4 (NeuAcalpha2*3) Galbeta1*4GlcCer (G_<M2>) to that of the wild-type, the extracts from mutant cDNA transfectants demonstrated lower enzyme activity than in the wild-type. The decrease in enzyme activity was more evident as the number of deglycosylated sites increased, with about 90% decrease in a totally deglycosylated mutant. The enzyme kinetics analysis revealed no significant change of K_m among wild-type and mutant cDNA products. The intracellular localization of GalNAc-T expressed in transfectants with wild-type or mutant cDNAs also showed a similar perinuclear pattern (Golgi pattern). These results suggest that N-linked carbohydrates on GalNAc-T are required for regulating the stability of the enzyme structure.
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Research Products
(8 results)
