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- Title
Stabilization of Human Recombinant Erythropoietin through Interactions with the Highly Branched N-Glycans1.
- Authors
Toyoda, Teruko; Itai, Tomokazu; Arakawa, Tsutomu; Aoki, Kenneth H.; Yamaguchi, Haruki
- Abstract
Human erythropoietin (EPO) produced in Chinese hamster ovary cells (CHO-EPO) is a hydrophobic protein stabilized by the highly branched complex-type N-glycans. To characterize the stabilizing effect of the N-glycans, the properties of enzymatically N-glycan-modified CHO-EPO species were compared spectrophotometrically. CD and fluorescence spectra following the protein unfolding induced by guanidine hydrochloride or pH revealed that the inner regions including the galactose residues of the N-glycans stabilize the protein conformation. The decrease in the conformational stability caused by enzymatic trimming of the N-glycans was associated with the exposure of the hydrophobic protein surface areas accessible to 1-anilino-8-naphthalenesulfonic acid (ANS) binding. Further, the ANS binding and heat denaturation of Escherichia coli-expressed EPO (nonglycosylated EPO) were depressed in dilute solutions (1 mM or so) of free N-glycans of the complex type. These results, together with the finding that the N-glycans of CHO-EPO make little contact with the aromatic amino acid residues exposed on the protein surface, indicate that the inner regions including the galactose residues of the intramolecular N-glycans stabilize the protein conformation by clinging to the hydrophobic protein surface areas mainly made up of nonaromatic hydrocarbon groups
- Subjects
ERYTHROPOIETIN; GLYCANS; FLUORESCENCE; NAPHTHALENESULFONIC acids; ESCHERICHIA coli
- Publication
Journal of Biochemistry, 2000, Vol 128, Issue 5, p731
- ISSN
0021-924X
- Publication type
Article
- DOI
10.1093/oxfordjournals.jbchem.a022809