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Comparative Study
. 1990 Mar 15;265(8):4483-91.

Granulocyte-macrophage colony stimulating factor from human lymphocytes. The effect of glycosylation on receptor binding and biological activity

J Cebon  1 , N Nicola, M Ward, I Gardner, P Dempsey, J Layton, U Dührsen, A W Burgess, E Nice, G Morstyn
Affiliations
  • PMID: 2155231
Free article
Comparative Study

Granulocyte-macrophage colony stimulating factor from human lymphocytes. The effect of glycosylation on receptor binding and biological activity

J Cebon et al. J Biol Chem. .
Free article

Abstract

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.

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