Reference - Detail
| RRC ID | 38805 |
|---|---|
| Author | Haraguchi M, Yamashiro S, Furukawa K, Takamiya K, Shiku H, Furukawa K. |
| Title | The effects of the site-directed removal of N-glycosylation sites from beta-1,4-N-acetylgalactosaminyltransferase on its function. |
| Journal | Biochem J |
| Abstract |
The amino acid sequence deduced from the cloned human cDNA of beta-1,4-N-acetylgalactosaminyltransferase (GalNAc-T; EC 2.4.1.92) 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 that all potential N-glycosylation sites could be used. Although cell lines transfected with mutant cDNAs showed equivalent levels of GalNAc beta 1-->4(NeuAc alpha 2-->3)Gal beta 1-->4Glc-Cer (GM2) 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 Km 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. |
| Volume | 312 ( Pt 1)(Pt 1) |
| Pages | 273-80 |
| Published | 1995-11-15 |
| DOI | 10.1042/bj3120273 |
| PMID | 7492324 |
| PMC | PMC1136255 |
| MeSH | Animals Base Sequence Blotting, Southern CHO Cells Cricetinae G(M2) Ganglioside / biosynthesis Glycosylation Humans Immunohistochemistry Kinetics Mice Molecular Sequence Data Mutagenesis, Site-Directed* N-Acetylgalactosaminyltransferases / chemistry N-Acetylgalactosaminyltransferases / genetics N-Acetylgalactosaminyltransferases / metabolism* Point Mutation Protein Biosynthesis Sequence Analysis Transcription, Genetic Transfection Tumor Cells, Cultured |
| IF | 4.097 |
| Times Cited | 51 |
| WOS Category | BIOCHEMISTRY & MOLECULAR BIOLOGY |
| Altmetric score |
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| The most frequently cited source | Patent(IFI CLAIMS) |
| Total number of mentions | 1 |
| Altmetric score changes over past 6months | 0.0 |
| Resource | |
| Human and Animal Cells | CHO-K1(RCB0285) |