Journal
JOURNAL OF BIOCHEMISTRY
Volume 127, Issue 1, Pages 65-72Publisher
JAPANESE BIOCHEMICAL SOC
DOI: 10.1093/oxfordjournals.jbchem.a022585
Keywords
Asn-X-Cys; CHO cells; epidermal growth factor receptor; EGF; glycosylation
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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.
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