title: Role of glycosylation on the secretion and biological activity of erythropoietin.
abstract: The erythropoietin (EPO) molecule contains four carbohydrate chains. Three contain N-linkages to asparagines at positions 24, 38, and 83, and one contains an O-linkage to a serine at position 126. We constructed human EPO variants that eliminated the three N-glycosylation sites by replacing the asparagines with glutamines singly or in combination. The O-linked carbohydrate chain was removed by replacing the serine with glutamine, valine, histidine, or alanine. A variant with a double mutation (Gln38,83) and another with a triple mutation (Gln24,38,83) were secreted poorly from COS1 and CHO cells even though RNA encoding these variants was present. All other variants with mutations in N-linked glycosylation sites were secreted normally. Removal of any of the N-glycosylation sites reduced the in vivo but not the in vitro biological activity of the EPO molecule. All the mutations at Ser126, the O-glycosylation site, were secreted normally. In vitro activity was also unaffected except for Ala126 which had a 50-fold decrease. The Val126 variant was tested in vivo, and its specific activity was only slightly less than that of the native EPO, which indicates that the O-linked carbohydrate is not essential for activity.
authors.affiliation: Amgen Inc., Amgen Center, Thousand Oaks, California 91320.
authors.forename: E
authors.initials: E
authors.lastname: Delorme
authors.forename: T
authors.initials: T
authors.lastname: Lorenzini
authors.forename: J
authors.initials: J
authors.lastname: Giffin
authors.forename: F
authors.initials: F
authors.lastname: Martin
authors.forename: F
authors.initials: F
authors.lastname: Jacobsen
authors.forename: T
authors.initials: T
authors.lastname: Boone
authors.forename: S
authors.initials: S
authors.lastname: Elliott
mesh_terms: D000818:Animals; D001216:Asparagine / Q000737:chemistry; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002241:Carbohydrates / Q000737:chemistry*; D002460:Cell Line; D006224:Cricetinae; D004921:Erythropoietin / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D005973:Glutamine / Q000737:chemistry; D006031:Glycosylation; D015151:Immunoblotting; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D002241:Carbohydrates; D005973:Glutamine; D004921:Erythropoietin; D001216:Asparagine