Characterization of recombinant murine interleukin 5 expressed in Chinese hamster ovary cells.
We have purified recombinant murine interleukin 5 (rmIL-5) from the supernatant of Chinese hamster ovary cells. Each peptide fragment of the purified rmIL-5 generated by Achromobacter protease I digestion was characterized and glycosylation sites were determined. Although rmIL-5 contains three potential sites of N-linked glycosylation (Asn-26, Asn-55 and Asn-69), Asn-69 is not glycosylated. The oligosaccharides released from the protein by hydrazinolysis were fractionated by paper electrophoresis, lectin column chromatography and gel permeation chromatography, and their structures were analysed by sequential exoglycosidase digestion in combination with methylation analysis. The results indicated that they are a mixture of bi-, tri- and tetraantennary complex-type sugar chains with and without a fucose at the C-6 position of the proximal N-acetylglucosamine residue and high-mannose-type sugar chains. Although > 80% of the sugar chains are neutral oligosaccharides similar to recombinant human IL-5 (rhIL-5; Kodama, S., Endo, T., Tsuroka, N., Tsujimoto, M. and Kobata, A. (1991) J. Biochem., 110, 693-701), rmIL-5 has more tetraantennary oligosaccharides than rhIL-5. A site differential study revealed that Asn-55 has more tetraantennary oligosaccharides than Asn-26.
Kodama
S
S
Suntory Institute for Biomedical Research, Osaka, Japan.
Endo
T
T
Tsujimoto
M
M
Kobata
A
A
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D006224:Cricetinae; D015870:Gene Expression; D006031:Glycosylation; D015848:Interleukin-5 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D015848:Interleukin-5; D009844:Oligosaccharides; D011994:Recombinant Proteins