Purification and characterization of three forms of differently glycosylated recombinant human granulocyte-macrophage colony-stimulating factor.
We have purified recombinant human granulocyte-macrophage colony-stimulating factor (hGM-CSF) produced in human lymphoblastoid Namalwa cells. From the results of tunicamycin treatment and N-glycosidase F digestion, it was demonstrated that Namalwa-derived hGM-CSF was highly glycosylated at two potential N-glycosylation sites and several O-glycosylation sites as previously shown for naturally occurring hGM-CSF. We classified the hGM-CSF molecules into three groups according to the molecular weight corresponding to the degree of N-glycosylation: the molecules with two N-glycosylation sites occupied (designated 2N), the molecules with either site glycosylated (1N), and the molecules lacking N-glycosylation (0N). Despite such varied degrees of N-glycosylation, almost all molecules were O-glycosylated. To investigate the role of carbohydrate moieties of hGM-CSF, we isolated each form of hGM-CSF and examined its biological properties. The 2N-type showed 200-fold less in vitro specific activity compared with unglycosylated Escherichia coli-derived hGM-CSF, although the activity of the 0N-type was equivalent to that of the E. coli-derived material. The 1N-type showed an intermediate level of activity. However, in terms of clearance from blood circulation in the rat, the 2N-type showed a half-life five times longer than that of the 0N-type and E. coli-derived hGM-CSF. From these findings, we concluded that N-linked carbohydrate moieties of hGM-CSF play conflicting physiological roles in the efficacy of the protein in vivo but that O-linked carbohydrate moieties do not have such effects.
Okamoto
M
M
Takarazuka Research Center, Sumitomo Chemical Co., Ltd., Hyogo, Japan.
Nakai
M
M
Nakayama
C
C
Yanagi
H
H
Matsui
H
H
Noguchi
H
H
Namiki
M
M
Sakai
J
J
Kadota
K
K
Fukui
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D002846:Chromatography, Affinity; D003114:Colony-Forming Units Assay; D015180:Electrophoresis, Gel, Two-Dimensional; D006031:Glycosylation; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000302:isolation & purification* / Q000493:pharmacokinetics / Q000494:pharmacology; D006801:Humans; D008970:Molecular Weight; D011863:Radioimmunoassay; D051381:Rats; D011919:Rats, Inbred Strains; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000493:pharmacokinetics / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor