Granulocyte-macrophage colony stimulating factor from human lymphocytes. The effect of glycosylation on receptor binding and biological activity.
Native human granulocyte-macrophage colony stimulating factor (hGM-CSF) has previously been purified using methods which typically required several sequential chromatographic steps and only yielded small amounts of hGM-CSF. We have purified and characterized hGM-CSF using monoclonal antibodies raised against bacterially synthesized hGM-CSF. Activated donor T-lymphocytes grown in interleukin-2 and then reactivated with phytohemagglutinin produce several forms of hGM-CSF which can be purified using immunoaffinity absorption followed by reversed phase high performance liquid chromatography. The purified hGM-CSF consisted of at least nine species ranging in molecular weight (Mr) from 14,500 to 32,000. The higher Mr forms contained one or two N-linked carbohydrate moieties and were more acidic by two-dimensional Western blot analysis, consistent with increasing sialation. N-terminal sequence analysis of high and low molecular weight hGM-CSF fractions corresponded to that predicted by the cDNA sequence. Using the AML 193 [3H]thymidine incorporation assay the specific activity of the heavily glycosylated hGM-CSF was 1 x 10(8) units/mg compared with 6 x 10(8) units/mg for the non-glycosylated hGM-CSF produced by Escherichia coli. The different hGM-CSF forms induced neutrophil superoxide anion production by a variable amount depending on the extent of N-linked glycosylation. Receptor binding studies demonstrated lower receptor affinity for the heavily glycosylated form (KD = 820 pM) compared to less heavily glycosylated (KD = 78 pM) and non-glycosylated hGM-CSF produced by E. coli (KD = 30 pM). These differences are due to differences in the kinetic association rate.
Cebon
J
J
Melbourne Tumour Biology Branch, Ludwig Institute for Cancer Research, Victoria, Australia.
Nicola
N
N
Ward
M
M
Gardner
I
I
Dempsey
P
P
Layton
J
J
Dührsen
U
U
Burgess
A W
AW
Nice
E
E
Morstyn
G
G
D001667:Binding, Competitive; D001681:Biological Assay; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003114:Colony-Forming Units Assay; D003115:Colony-Stimulating Factors / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D003470:Culture Media; D006031:Glycosylation; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D006133:Growth Substances / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D008213:Lymphocyte Activation; D008970:Molecular Weight; D009504:Neutrophils / Q000378:metabolism; D010835:Phytohemagglutinins / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000378:metabolism*; D016184:Receptors, Colony-Stimulating Factor; D013481:Superoxides / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism*
D003115:Colony-Stimulating Factors; D003470:Culture Media; D006133:Growth Substances; D010835:Phytohemagglutinins; D011956:Receptors, Cell Surface; D016184:Receptors, Colony-Stimulating Factor; D013481:Superoxides; D016178:Granulocyte-Macrophage Colony-Stimulating Factor