title: Human serum inactivates non-glycosylated but not glycosylated granulocyte colony stimulating factor by a protease dependent mechanism: significance of carbohydrates on the glycosylated molecule.
abstract: It has previously been reported that the biological activity of the human hematopoetic cytokine granulocyte colony stimulating factor (G-CSF) was reduced following incubation with human serum. The mechanism of action of serum has remained elusive although a number of possible mechanisms have been suggested including inactivation due to binding to the serum protein alpha(2)-macroglobulin (alpha(2)M) and degradation by serum proteases. The aim of this study was to clarify the conditions required by serum to reduce the biological activity of the cytokine and to define the mechanism involved. It has also been noted that G-CSF obtained from a CHO expression system (and therefore considered a glycosylated molecule) was resistant to serum inactivation unlike G-CSF obtained from an E. coli expression system (considered to be non-glycosylated). We used an enzymatic approach to remove the carbohydrate residues from glycosylated G-CSF and tested this material for its stability in serum. We additionally used a mutated G-CSF lacking glycosylation sites. We concluded that glycosylation was important in protecting against serum inactivation. We observed that serum reduced the biological activity of non-glycosylated G-CSF in a dose, and temperature dependent manner and deduced that the mechanism of action was dependent upon alpha(2)M bound serum protease enzymes.
authors.affiliation: Division of Immunobiology, NIBSC, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK.
authors.forename: Clive R D
authors.initials: CR
authors.lastname: Carter
authors.forename: James R
authors.initials: JR
authors.lastname: Keeble
authors.forename: Robin
authors.initials: R
authors.lastname: Thorpe
mesh_terms: D000818:Animals; D001681:Biological Assay; D016466:CHO Cells; D002241:Carbohydrates / Q000737:chemistry*; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006031:Glycosylation; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D044967:Serum / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors; D000511:alpha-Macroglobulins / Q000378:metabolism
chemical_list: D002241:Carbohydrates; D000511:alpha-Macroglobulins; D016179:Granulocyte Colony-Stimulating Factor