Characterization of the N-oligosaccharides attached to the atypical Asn-X-Cys sequence of recombinant human epidermal growth factor receptor

The extracellular domain of human EGF receptor (sEGFR) produced by CHO cell s has been used in various biophysical studies to elucidate the molecular m echanism of EGF-induced receptor activation. We have found that the CHO sEG FR contains one oligosaccharide chain attached to an atypical N-glycosylati on consensus sequence, Asn(32)-X-33- Cys(34). The oligosaccharide structure at Asn(32) is a mixture of the monosialo and asialo forms of a core fucosy lated biantennary complex-type oligosaccharide, Deletion of this atypical g lycosylation site by replacement of Asn(32) with lysine changed neither the expression nor function of the full length EGFR in CHO cells. The glycosyl ation at Asn(32) in CHO sEGFR was incomplete: 20% of Asn(32) remained unmod ified. Thus, CHO sEGFR itself is heterogeneous with respect to the glycosyl ation at Asn(32), which may cause problems in biophysical studies. An attem pt to remove the oligosaccharide at Asn(32) enzymatically did not succeed u nder nondenaturing conditions. Therefore, sEGFR with the mutation of Asn(32 ) --> Lys(32) is useful for biophysical and biochemical studies, and, parti cularly, for X-ray crystallography.