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The effects of the site-directed removal of N-glycosylation sites from beta-1,4-N-acetylgalactosaminyltransferase on its function.
- Source :
-
The Biochemical journal [Biochem J] 1995 Nov 15; Vol. 312 ( Pt 1), pp. 273-80. - Publication Year :
- 1995
-
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.
- Subjects :
- Animals
Base Sequence
Blotting, Southern
CHO Cells
Cricetinae
G(M2) Ganglioside biosynthesis
Glycosylation
Humans
Immunohistochemistry
Kinetics
Mice
Molecular Sequence Data
N-Acetylgalactosaminyltransferases chemistry
N-Acetylgalactosaminyltransferases genetics
Point Mutation
Protein Biosynthesis
Sequence Analysis
Transcription, Genetic
Transfection
Tumor Cells, Cultured
Polypeptide N-acetylgalactosaminyltransferase
Mutagenesis, Site-Directed
N-Acetylgalactosaminyltransferases metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0264-6021
- Volume :
- 312 ( Pt 1)
- Database :
- MEDLINE
- Journal :
- The Biochemical journal
- Publication Type :
- Academic Journal
- Accession number :
- 7492324
- Full Text :
- https://doi.org/10.1042/bj3120273