Asparagine-linked glycosylation of the rat leukemia inhibitory factor expressed by simian COS7 cells.
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 COS7 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.
Laboratory of Synthetic Cellular Chemistry, RIKEN (the Institute of Physical and Chemical Research), Saitama, Japan. akemi@postman.riken.go.jp
J
J
Aikawa
E
E
Sato
S
S
Kyuwa
E
E
Sato
K
K
Sasai
K
K
Shiota
T
T
Ogawa
D000581:Amidohydrolases / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000737:chemistry*; D001483:Base Sequence; D019556:COS Cells; D002455:Cell Division / Q000235:genetics; D006031:Glycosylation; D006131:Growth Inhibitors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D000882:Haplorhini; D015850:Interleukin-6; D037102:Lectins; D053605:Leukemia Inhibitory Factor; D008222:Lymphokines / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D051381:Rats; D014407:Tumor Cells, Cultured
D006131:Growth Inhibitors; D015850:Interleukin-6; D037102:Lectins; D053605:Leukemia Inhibitory Factor; C507838:Lif protein, mouse; D008222:Lymphokines; D001216:Asparagine; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase