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Abstract
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.
Highlights
Secreted proteins are often glycosylated during transit through the secretory apparatus in eukaryotic cells
In order to identify the rules that define a N-linked glycosylation site, we introduced N-linked glycosylation consensus sequences into defined regions of the recombinant human erythropoietin (rHuEPO) gene by using in vitro mutagenesis. 62 different rHuEPO glycosylation analogs were constructed with consensus sequences introduced into 47 different positions
If a Pro residue was present at the Xxx position, it was substituted with another amino acid because a Pro at the Xxx position inhibits N-linked glycosylation[17]
Summary
Secreted proteins are often glycosylated during transit through the secretory apparatus in eukaryotic cells These carbohydrates can be attached to the hydroxyl group on a Serine or Threonine (Olinked glycosylation), or the amine of an asparagine via an N-glycosidic bond (N-linked glycosylation). While at least 12– 14 amino acids must be synthesized and have entered the luminal surface of the endoplasmic reticulum for carbohydrate addition, the synthesis of the protein need not be completed for glycosylation to take place[19;20]. This suggests that the structures for carbohydrate addition are recognized in partially folded molecules. Distance from the C-terminus may affect glycosylation [23]
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