Stabilization of Human Recombinant Erythropoietin through Interactions with the Highly Branched N-Glycans

• Published in: Journal of biochemistry (Tokyo) Vol. 128; no. 5; pp. 731 - 737
• Main Authors: Toyoda, Teruko, Itai, Tomokazu, Arakawa, Tsutomu, Aoki, Kenneth H., Yamaguchi, Haruki
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.11.2000
• Subjects: Animals; Carbohydrate Conformation; Carbohydrate Sequence; Cricetinae; erythropoietin; Erythropoietin - chemistry; Erythropoietin - metabolism; glycoprotein; Humans; Hydrogen-Ion Concentration; Molecular Sequence Data; N-glycan; N-glycan function; N-glycan-protein interaction; Polysaccharides - metabolism; Protein Denaturation; Recombinant Proteins; Spectrometry, Fluorescence; Structure-Activity Relationship
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a022809

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