Title : Characterization of
the N-oligosaccharides attached to the atypical Asn-X-Cys
sequence of recombinant human
epidermal growth factor receptor
Abstract :
- The extracellular domain of human EGF receptor ( sEGFR ) produced by CHO cells has been used in various biophysical studies to elucidate the molecular mechanism of EGF-induced receptor activation
- We have found that the CHO sEGFR contains one oligosaccharide chain attached to an atypical N-glycosylation 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 fucosylated biantennary complex-type oligosaccharide
- Deletion of this atypical glycosylation site by replacement of Asn(32 ) with lysine changed neither the expression nor function of the full length EGFR in CHO cells
- The glycosylation at Asn(32 ) in CHO sEGFR was incomplete: 20% of Asn(32 ) remained unmodified
- Thus, CHO sEGFR itself is heterogeneous with respect to the glycosylation at Asn(32 ) , which may cause problems in biophysical studies
- An attempt to remove the oligosaccharide at Asn(32 ) enzymatically did not succeed under nondenaturing conditions
- Therefore, sEGFR with the mutation of Asn(32) -> Lys(32 ) is useful for biophysical and biochemical studies, and, particularly, for X-ray crystallography