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 raise...
Gespeichert in:
Veröffentlicht in: | The Journal of biological chemistry 1990-03, Vol.265 (8), p.4483-4491 |
---|---|
Hauptverfasser: | , , , , , , , , , |
Format: | Artikel |
Sprache: | eng |
Schlagworte: | |
Online-Zugang: | Volltext |
Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
Zusammenfassung: | 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. |
---|---|
ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/s0021-9258(19)39589-4 |