Asparagine-linked Glycosylation of the Rat Leukemia Inhibitory Factor Expressed by Simian COS7 Cells

Jun Ichi Aikawa, Ei Ichiro Sato, Shigeru Kyuwa, Eimei Sato, Ken Sasai, Kunio Shiota, Tomoya Ogawa

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Abstract

The leukemia inhibitory factor (LIF) is a secretory glycoprotein and a pluripotent growth factor which acts in diverse cell systems. LIF has been reported to be heavily glycosylated. In this paper, we examine the transient expression of rat LIF (rLIF) in COS? cells and its glycosylation by a PNGaseF treatment and lectin blot. rLIF expression in COS7 cells resulted in seven molecular species being produced with zero to six N-glycosyl moieties. Mutated rLIF proteins with substitutions at the seven possible N-glycosylation sites were also expressed. An analysis of the molecular weight of the mutated rLIF confirmed the six N-glycosylation sites. Bioassays of mouse leukemia cell lines were performed to analyze the contribution of the glycosyl moieties to their functions. We found that the glycosyl moieties at each of the N-glycosylation sites were not essential to their function of the protein, but the reduced functions to promote the proliferation of DA-1a cells that had been observed for some mutants suggests a biochemical role for the in vitro function.

Original languageEnglish
Pages (from-to)1318-1325
Number of pages8
JournalBioscience, Biotechnology and Biochemistry
Volume62
Issue number7
Publication statusPublished - 1998 Jul
Externally publishedYes

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Keywords

  • Asparagine-linked glycosylation (N-glycosylation)
  • COS7 cells
  • DA-1a cells
  • Leukemia inhibitory factor (LIF)
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Food Science
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Biotechnology
  • Chemistry (miscellaneous)
  • Applied Microbiology and Biotechnology
  • Bioengineering

Cite this

Aikawa, J. I., Sato, E. I., Kyuwa, S., Sato, E., Sasai, K., Shiota, K., & Ogawa, T. (1998). Asparagine-linked Glycosylation of the Rat Leukemia Inhibitory Factor Expressed by Simian COS7 Cells. Bioscience, Biotechnology and Biochemistry, 62(7), 1318-1325.