The importance of N- and O-linked oligosaccharides for the biosynthesis and in vitro and in vivo biologic activities of erythropoietin.
Erythropoietin (EPO) plays a critical role in stimulating the proliferation and differentiation of erythroid precursor cells. EPO is heavily glycosylated with three asparagine (N)-linked tetraantennary oligosaccharides that may contain N-acetyl-lactosamine repeats and a single serine (O)-linked oligosaccharide. EPO expressed in Chinese hamster ovary cells exhibits biologic properties and amino acid and carbohydrate composition similar to natural urinary EPO. The importance of the complex N-linked and the O-linked carbohydrate was studied by expressing EPO in cells that are deficient in UDP-galactose/UDP-N-acetylgalactosamine 4-epimerase activity. In these cells, the ability to add galactose and N-acetylgalactosamine to glycoproteins can be controlled by the addition of these sugars to the culture medium. The results demonstrate that a block in O-linked glycosylation and/or the ability to process N-linked carbohydrate to completion does not alter EPO secretion. EPO produced without O-linked carbohydrate exhibits normal in vitro and in vivo biologic activity and in vivo clearance. However, EPO produced with incompletely processed N-linked oligosaccharides exhibits normal in vitro activity but is at least 500-fold less effective in stimulating erythropoiesis in vivo. Studies on the survival of bioactive EPO remaining in the circulation demonstrated that EPO with incomplete N-linked oligosaccharides exhibits a sevenfold increased rate of clearance. However, this increased clearance may not fully account for the 500-fold loss of in vivo activity. These results suggest a potentially important unique requirement for appropriate complex N-linked oligosaccharides for the intrinsic biologic activity of EPO in vivo.
Wasley
L C
LC
Genetics Institute, Cambridge, MA 02140.
Timony
G
G
Murtha
P
P
Stoudemire
J
J
Dorner
A J
AJ
Caro
J
J
Krieger
M
M
Kaufman
R J
RJ
D000116:Acetylgalactosamine / Q000378:metabolism; D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D015672:Erythroid Precursor Cells / Q000166:cytology / Q000187:drug effects; D004921:Erythropoietin / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D005690:Galactose / Q000378:metabolism; D015870:Gene Expression; D006031:Glycosylation; D006207:Half-Life; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D013329:Structure-Activity Relationship; D014162:Transfection
D009844:Oligosaccharides; D004921:Erythropoietin; D004247:DNA; D000116:Acetylgalactosamine; D005690:Galactose