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Effects of site-directed removal of N-glycosylation sites in human erythropoietin on its production and biological properties
- Source :
- Journal of Biological Chemistry. 266:20434-20439
- Publication Year :
- 1991
- Publisher :
- Elsevier BV, 1991.
-
Abstract
- Erythropoietin (Epo) has three N-linked sugar chains. Codons for asparagine at N-glycosylation sites in genomic human Epo DNA were replaced with those for glutamine. The wild-type Epo gene and seven mutants that lacked N-glycosylation sites in every possible combination were introduced into baby hamster-kidney cells. To study the role of the N-linked sugars in Epo biosynthesis, Epo protein expressed transiently was measured by an enzyme-linked immunoassay. The elimination of all three N-glycosylation sites decreased Epo production to 10% of that of the wild-type Epo. Wild-type and mutant Epos produced by stably transfected cells were partially purified to investigate their properties. Removal of N-glycosylation sites changed affinity of Epo to the receptor. The in vitro activity of Epo that lost all N-glycosylation sites was comparable with that of the wild-type Epo, while the in vivo activity severely decreased. These results indicate that N-linked sugars of Epo have two major functions; N-linked sugars are important for 1) proper biosynthesis and/or secretion and 2) expression of the in vivo activity probably by enhancing survival in the circulation. N-Linked sugars of Epo affect binding affinity of the ligand to the receptor but do not play a key role in expression of the in vitro activity.
Details
- ISSN :
- 00219258
- Volume :
- 266
- Database :
- OpenAIRE
- Journal :
- Journal of Biological Chemistry
- Accession number :
- edsair.doi...........940fef2c0a6c65c309a05a75c426e238