Stabilization of human recombinant erythropoietin through interactions with the highly branched N-glycans

Human erythropoietin (EPO) produced in. Chinese hamster ovary cells (CHO-EP O) is a hydrophobic protein stabilized by the highly branched complex-type N-glycans, To characterize the stabilizing effect of the N-glycans, the pro perties of enzymatically N-glycan-modified CHO-EPO species were compared sp ectrophotometrically. CD and fluorescence spectra following the protein unf olding induced by guanidine hydrochloride or pH revealed that the inner reg ions including the galactose residues of the N-glycans stabilize the protei n conformation. The decrease in the conformational stability caused by enzy matic trimming of the N-glycans was associated with the exposure of the hyd rophobic protein surface areas accessible to 1-anilino-8-naphthalenesulfoni c acid (ANS) binding. Further,the ANS binding and heat denaturation of Esch erichia 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 conta ct with the aromatic amino acid residues exposed on the protein surface, in dicate that the inner regions including the galactose residues of the intra molecular N-glycans stabilize the protein conformation by clinging to the h ydrophobic protein surface areas mainly made up of nonaromatic hydrocarbon groups.