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Characterization of the N-Oligosaccharides Attached to the Atypical Asn-X-Cys Sequence of Recombinant Human Epidermal Growth Factor Receptor

2000; Oxford University Press; Volume: 127; Issue: 1 Linguagem: Inglês

10.1093/oxfordjournals.jbchem.a022585

1756-2651

Chiaki Sato, Jae‐Hoon Kim, Yoshito Abe, Kazuki Saito, Shigeyuki Yokoyama, Daisuke Kohda,

Monoclonal and Polyclonal Antibodies Research

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 oligosac-charide chain attached to an atypical N-glycosylation consensus sequence, Asn32 -X33 -Cys34. The oligosaccharide structure at Asn32 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 Asn32 with lysine changed neither the expression nor function of the full length EGFR in CHO cells. The glycosylation at Asn32 in CHO sEGFR was incomplete: 20% of Asn32 remained unmodified. Thus, CHO sEGFR itself is heterogeneous with respect to the glycosylation at Asn32, which may cause problems in biophysical studies. An attempt to remove the oligosaccharide at Asn32 enzymat-ically did not succeed under nondenaturing conditions. Therefore, sEGFR with the mutation of Asn32→Lys32 is useful for biophysical and biochemical studies, and, particularly, for X-ray crystallography.