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

J. Cebon, N. Nicola, M. Ward, I. Gardner, P. Dempsey, J. Layton, U. Duhrsen, A. W. Burgess, E. Nice, G. Morstyn

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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 ll 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 (M(r)) from 14,500 to 32,000. The higher M(r) 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 [ 3 H]thymidine incorporation assay the specific activity of the heavily glucosylated 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 (K(D) = 820 pM) compared to less heavily glycosylated (K(D) = 78 pM) and non-glycosylated hGM-CSF produced by E. coli (K(D) = 30 pM). These differences are due to differences in the kinetic association rate.

Original languageEnglish
Pages (from-to)4483-4491
Number of pages9
JournalJournal of Biological Chemistry
Issue number8
Publication statusPublished - 30 Mar 1990
Externally publishedYes

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