Structural requirements for addition of O-linked carbohydrate to recombinant erythropoietin.
To define the structural requirements for addition of O-linked glycosylation in vivo, recombinant erythropoietin (rEPO) variants were constructed. Thirty-three independent Ser or Thr substitutions were constructed and examined to see which were subject to O-linked carbohydrate addition. Variants with Thr mutations at positions 123 and 125, but not elsewhere, contained additional carbohydrate, which suggests that several positions around the existing O-linked glycosylation site (Ser126), but not elsewhere, contain the necessary information for O-linked carbohydrate addition. Two forms of the Thr125 variant were identified. One form was glycosylated only at residue 125, and a second form was glycosylated at both Thr125 and Ser126, the normal O-glycosylation site. We have also found that glycosylation is less efficient when rEPO is improperly folded and that prolines at -1 and +1 relative to the O-glycosylation site enhance glycosylation.
Elliott
S
S
Amgen Center, Thousand Oaks, California 91320, USA.
Bartley
T
T
Delorme
E
E
Derby
P
P
Hunt
R
R
Lorenzini
T
T
Parker
V
V
Rohde
M F
MF
Stoney
K
K
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D006224:Cricetinae; D004220:Disulfides; D004921:Erythropoietin / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D014644:Genetic Variation; D006031:Glycosylation; D006801:Humans; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009439:Neuraminidase; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D012694:Serine; D013912:Threonine; D014162:Transfection
D004220:Disulfides; D009844:Oligosaccharides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D004921:Erythropoietin; D013912:Threonine; D012694:Serine; D009439:Neuraminidase