Structural requirements for additional N-linked carbohydrate on recombinant human erythropoietin.
N-Linked glycosylation is a post-translational event whereby carbohydrates are added to secreted proteins at the consensus sequence Asn-Xaa-Ser/Thr, where Xaa is any amino acid except proline. Some consensus sequences in secreted proteins are not glycosylated, indicating that consensus sequences are necessary but not sufficient for glycosylation. In order to understand the structural rules for N-linked glycosylation, we introduced N-linked consensus sequences by site-directed mutagenesis into the polypeptide chain of the recombinant human erythropoietin molecule. Some regions of the polypeptide chain supported N-linked glycosylation more effectively than others. N-Linked glycosylation was inhibited by an adjacent proline suggesting that sequence context of a consensus sequence could affect glycosylation. One N-linked consensus sequence (Asn123-Thr125) introduced into a position close to the existing O-glycosylation site (Ser126) had an additional O-linked carbohydrate chain and not an additional N-linked carbohydrate chain suggesting that structural requirements in this region favored O-glycosylation over N-glycosylation. The presence of a consensus sequence on the protein surface of the folded molecule did not appear to be a prerequisite for oligosaccharide addition. However, it was noted that recombinant human erythropoietin analogs that were hyperglycosylated at sites that were normally buried had altered protein structures. This suggests that carbohydrate addition precedes polypeptide folding.
Elliott
Steve
S
Amgen, Thousand Oaks, California 91320, USA. selliott@amgen.com
Chang
David
D
Delorme
Evelyne
E
Eris
Tamer
T
Lorenzini
Tony
T
D002241:Carbohydrates / Q000737:chemistry; D004921:Erythropoietin / Q000737:chemistry* / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D016296:Mutagenesis; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship
D002241:Carbohydrates; D011994:Recombinant Proteins; D004921:Erythropoietin