Related Experiment Videos
Natural murine interferon-gamma. Evidence for post-translational proteolytic processing
Abstract:
Murine interferon-gamma (MuIFN-gamma), produced by the T-cell lymphoma, L12-R4, and stimulated with phorbol myristic acetate, was purified by rat monoclonal antibodies AN-18.17.24. The purified product retained its biologic activity and consisted of two proteins (Mr = 17,500 and 16,500), as determined by sodium dodecyl sulfate-gel electrophoresis. Both species were found to be glycosylated, since their Mr values decreased to 14,500 and 13,500 when the producing cells were stimulated in the presence of tunicamycin. Analysis of intracellular and secreted forms in the presence of [35S] methionine revealed that MuIFN-gamma is synthetized as a single peptide of Mr = 17,500 and undergoes proteolytic cleavage during or after secretion. Peptide mapping by reverse phase high pressure liquid chromatography showed indeed that the peptide profile of both species is very similar, suggesting a high degree of homology in the primary structure. These results demonstrate that the molecular heterogeneity of the MuIFN-gamma is probably the outcome of proteolytic processing during or after secretion.
Insights
Murine interferon-gamma (MuIFN-gamma) exists as two glycosylated protein species due to proteolytic cleavage during secretion. This processing explains the molecular heterogeneity observed in the biologically active MuIFN-gamma.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Murine interferon-gamma (MuIFN-gamma) is a crucial cytokine produced by T-cells.
- Understanding the molecular characteristics of MuIFN-gamma is essential for its therapeutic applications.
Purpose of the Study:
- To purify and characterize the molecular forms of MuIFN-gamma.
- To investigate the source of molecular heterogeneity in MuIFN-gamma.
Main Methods:
- Purification of MuIFN-gamma using rat monoclonal antibodies.
- Sodium dodecyl sulfate-gel electrophoresis (SDS-PAGE) to determine molecular weight.
- Glycosylation analysis using tunicamycin.
- Peptide mapping via reverse-phase high-performance liquid chromatography (RP-HPLC).
Main Results:
- Purified MuIFN-gamma consists of two protein species (Mr = 17,500 and 16,500) with retained biologic activity.
- Both species are glycosylated, with deglycosylated forms showing reduced molecular weights.
- MuIFN-gamma is synthesized as a single 17,500 Mr peptide and undergoes proteolytic cleavage.
- Peptide mapping indicates high homology between the two species, suggesting post-secretory processing.
Conclusions:
- The molecular heterogeneity of MuIFN-gamma arises from proteolytic processing during or after secretion.
- This processing generates distinct glycosylated forms of the cytokine.