Glycosylation does not affect in-vitro biological activity of interleukin-3

Murine interleukin-3 is secreted by activated T cells in three major molecular mass classes, which differ from one another in the extent of their N-linked glycosylation. Experiments were performed to determine whether carbohydrate content of different IL-3 glycoforms will affect their biological activity. IL-3 produced by activated T cells was biosynthetically labeled with 35S-methionine and the three major IL-3 glycoforms forms, with M(r) values of 22,000, 28,000 and 36,000, were purified using antibody affinity chromatography and preparative SDS-PAGE. Portions of these IL-3 glycoforms were enzymatically deglycosylated with N-glycanase and the bioactivity of each IL-3 glycoform and the corresponding deglycosylated fraction was compared in cell proliferation assays. The amount of 35S-label present in the samples was used as an index of protein amount so that equivalent concentrations of the various IL-3 forms could be compared. Our results indicate that the three major glycoforms have identical specific activity and that removal of N-linked carbohydrate does not change the specific in-vitro activity of IL-3. In addition to the three major glycoforms, small amounts of non-glycosylated IL-3 were also recovered from the affinity purified T-cell derived material. Using again the level of incorporated 35S as reference point, no difference in bioactivity compared with glycosylated IL-3 was detected. There is potential heterogeneity in IL-3 receptor complexes present on the many different cell types responsive to IL-3. We therefore tested whether the three IL-3 glycoforms differed in their interaction with various IL-3 responsive cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)