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
|
Resource |
Human and Animal Cells |
CHO-K1(RCB0285) |