Effects of site-directed removal of N-glycosylation sites in human erythropoietin on its production and biological properties.
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.
Yamaguchi
K
K
Research Institute of Life Science, Snow Brand Milk Products Co., Ltd., Tochigi, Japan.
Akai
K
K
Kawanishi
G
G
Ueda
M
M
Masuda
S
S
Sasaki
R
R
D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011956:Receptors, Cell Surface / Q000378:metabolism; D017467:Receptors, Erythropoietin; D014162:Transfection
D011956:Receptors, Cell Surface; D017467:Receptors, Erythropoietin; D004921:Erythropoietin