Stabilization of human recombinant erythropoietin through interactions with the highly branched N-glycans.
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.
Toyoda
T
T
Course of Applied Biochemistry, Graduate School of Agriculture and Biological Science, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan.
Itai
T
T
Arakawa
T
T
Aoki
K H
KH
Yamaguchi
H
H
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D006224:Cricetinae; D004921:Erythropoietin / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000378:metabolism*; D011489:Protein Denaturation; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D011134:Polysaccharides; D011994:Recombinant Proteins; D004921:Erythropoietin