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

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

研究成果: ジャーナルへの寄稿記事

49 引用 (Scopus)

抄録

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, 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 enzymatically 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.

元の言語英語
ページ(範囲)65-72
ページ数8
ジャーナルJournal of biochemistry
127
発行部数1
DOI
出版物ステータス出版済み - 1 1 2000

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Glycosylation
Oligosaccharides
Epidermal Growth Factor Receptor
CHO Cells
X ray crystallography
X Ray Crystallography
Consensus Sequence
Epidermal Growth Factor
Lysine
Chemical activation
Mutation

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

これを引用

Characterization of the N-oligosaccharides attached to the atypical Asn- X-Cys sequence of recombinant human epidermal growth factor receptor. / Sato, Chiaki; Kim, Jae Hoon; Abe, Yoshito; Saito, Kazuki; Yokoyama, Shigeyuki; Kohda, Daisuke.

:: Journal of biochemistry, 巻 127, 番号 1, 01.01.2000, p. 65-72.

研究成果: ジャーナルへの寄稿記事

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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, 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 enzymatically 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.",
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T1 - Characterization of the N-oligosaccharides attached to the atypical Asn- X-Cys sequence of recombinant human epidermal growth factor receptor

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AU - Kim, Jae Hoon

AU - Abe, Yoshito

AU - Saito, Kazuki

AU - Yokoyama, Shigeyuki

AU - Kohda, Daisuke

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N2 - 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, 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 enzymatically 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.

AB - 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, 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 enzymatically 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.

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