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Characterization of recombinant murine interleukin 5 expressed in Chinese hamster ovary cells
S Kodama1, T Endo, M Tsujimoto
1Suntory Institute for Biomedical Research, Osaka, Japan.
Abstract:
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.
Insights
Recombinant murine interleukin 5 (rmIL-5) has complex N-linked glycosylation, with Asn-69 being unglycosylated. Tetraantennary oligosaccharides are more prevalent in rmIL-5 than in recombinant human IL-5 (rhIL-5).
Area of Science:
- Biochemistry
- Glycobiology
- Protein Chemistry
Background:
- Recombinant murine interleukin 5 (rmIL-5) is a key cytokine in immune responses.
- Understanding the glycosylation patterns of rmIL-5 is crucial for its biological function and therapeutic potential.
Purpose of the Study:
- To characterize the N-linked glycosylation sites and structures of purified rmIL-5.
- To compare the glycosylation profile of rmIL-5 with that of recombinant human IL-5 (rhIL-5).
Main Methods:
- Purification of rmIL-5 from Chinese hamster ovary cell supernatant.
- Peptide fragmentation and glycosylation site determination using Achromobacter protease I.
- Oligosaccharide analysis via hydrazinolysis, paper electrophoresis, lectin chromatography, gel permeation chromatography, exoglycosidase digestion, and methylation analysis.
Main Results:
- Identified three potential N-linked glycosylation sites (Asn-26, Asn-55, Asn-69), with Asn-69 being unglycosylated.
- Characterized a mixture of complex bi-, tri-, and tetraantennary oligosaccharides, as well as high-mannose type sugar chains.
- Found that rmIL-5 contains a higher proportion of tetraantennary oligosaccharides compared to rhIL-5, with Asn-55 showing a greater abundance of these structures than Asn-26.
Conclusions:
- rmIL-5 exhibits distinct glycosylation patterns compared to rhIL-5, particularly in the prevalence of tetraantennary oligosaccharides.
- The site-specific glycosylation at Asn-26 and Asn-55 contributes to the overall heterogeneity of rmIL-5.
- These findings provide insights into the structural basis of rmIL-5 function and potential therapeutic applications.