Glycosylation at specific sites of erythropoietin is essential for biosynthesis, secretion, and biological function.
The glycoprotein hormone erythropoietin (Ep), the primary regulator of erythropoiesis, is synthesized by the kidney and secreted as the mature protein with three N-linked and one O-linked oligosaccharide chains. To investigate the role(s) of each carbohydrate moiety in the biosynthesis and function of Ep, we have used oligonucleotide-directed mutagenesis of a cDNA for human Ep to alter the amino acids at each of the carbohydrate attachment sites. Each mutated cDNA construct was expressed in stably transfected sublines of a kidney cell line, baby hamster kidney. We show, by preventing attachment of N-linked carbohydrate at asparagines 38 or 83, or preventing O-linked glycosylation at serine 126, that glycosylation of each of these specific sites is critical for proper biosynthesis and secretion of Ep. Fractionation of cellular extracts demonstrated that the mutant proteins lacking glycosylation at each of these three sites, (38, 83, and 126) were associated mainly with membrane components or were degraded rapidly. Less than 10% of these three mutant proteins were processed properly and secreted from the cells. The Ep protein lacking N-linked glycosylation at asparagine 24 is synthesized and secreted as efficiently as native Ep. The carbohydrates at positions 24 and 38 may be involved in the biological activity of Ep, since the absence of either of the oligosaccharide side chains at these positions reduced the hormone's biological activity.
Dubé
S
S
Division of Hematology, University of Washington, Seattle 98195.
Fisher
J W
JW
Powell
J S
JS
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004921:Erythropoietin / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D005796:Genes; D006023:Glycoproteins / Q000235:genetics*; D006031:Glycosylation; D006801:Humans; D009154:Mutation; D011499:Protein Processing, Post-Translational; D014162:Transfection
D006023:Glycoproteins; D004921:Erythropoietin; D004247:DNA