Relationship between sugar chain structure and biological activity of recombinant human erythropoietin produced in Chinese hamster ovary cells.
Two forms of erythropoietin, EPO-bi and EPO-tetra, with different biological activities were isolated from the culture medium of a recombinant Chinese hamster ovary cell line, B8-300, into which the human erythropoietin gene had been introduced. EPO-bi, an unusual form, showed only one-seventh the in vivo activity and 3 times higher in vitro activity of the previously described recombinant human EPO (standard EPO). In contrast, EPO-tetra showed both in vivo and in vitro activities comparable to those of the standard EPO. EPO-bi, EPO-tetra, and the standard EPO had the same amino acid composition and immunoreactivity. However, structural analyses of their N-linked sugar chains revealed that EPO-bi contains the biantennary complex type as the major sugar chain, while EPO-tetra and the standard EPO contain the tetraantennary complex type as the major sugar chain. From examination of various preparations of recombinant human EPO, we found a positive correlation between the in vivo activity of EPO and the ratio of tetraantennary to biantennary oligosaccharides. These results suggest that higher branching of the N-linked sugar chains is essential for effective expression of in vivo biological activity of EPO.
Takeuchi
M
M
Pharmaceutical Laboratory, Kirin Brewery, Gunma, Japan.
Inoue
N
N
Strickland
T W
TW
Kubota
M
M
Wada
M
M
Shimizu
R
R
Hoshi
S
S
Kozutsumi
H
H
Takasaki
S
S
Kobata
A
A
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004921:Erythropoietin / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D005260:Female; D005796:Genes; D006801:Humans; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000302:isolation & purification*; D010053:Ovary; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D014162:Transfection
D009844:Oligosaccharides; D011994:Recombinant Proteins; D004921:Erythropoietin