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 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.
Sato
C
C
Department of Structural Biology, Biomolecular Engineering Research Institute, Furuedai Suita, Osaka 565-0874, Japan.
Kim
J H
JH
Abe
Y
Y
Saito
K
K
Yokoyama
S
S
Kohda
D
D
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001216:Asparagine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016466:CHO Cells; D002240:Carbohydrate Sequence; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D066246:ErbB Receptors / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D014162:Transfection
D009844:Oligosaccharides; D011994:Recombinant Proteins; D001216:Asparagine; D066246:ErbB Receptors; D008239:Lysine; D003545:Cysteine