title: Glycosylation does not affect in-vitro biological activity of interleukin-3.
abstract: 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)
authors.affiliation: Biomedical Research Centre, University of British Columbia, Vancouver, Canada.
authors.forename: H J
authors.initials: HJ
authors.lastname: Ziltener
mesh_terms: D000581:Amidohydrolases; D000818:Animals; D001345:Autoradiography; D002460:Cell Line; D002846:Chromatography, Affinity; D002999:Clone Cells; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D007377:Interleukin-3 / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology*; D008970:Molecular Weight; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D016185:Receptors, Interleukin-3 / Q000378:metabolism; D013462:Sulfur Radioisotopes; D013601:T-Lymphocytes / Q000378:metabolism
chemical_list: D007377:Interleukin-3; D016185:Receptors, Interleukin-3; D013462:Sulfur Radioisotopes; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase