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Structural and Functional Differences Between Glycosylated and Non-glycosylated Forms of Human Interferon-β (IFN-β)

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Abstract

Purpose. Two recombinant IFN-β products have been approved for the treatment of multiple sclerosis, a glycosylated form with the predicted natural amino acid sequence (IFN-β-la) and a non-glycosylated form that has a Met-1 deletion and a Cys-17 to Ser mutation (IFN-β-lb). The structural basis for activity differences between IFN-β-la and IFN-β-lb, is determined.

Methods. In vitro antiviral, antiproliferative and immunomodulatory assays were used to directly compare the two IFN-β products. Size exclusion chromatography (SEC), SDS-PAGE, thermal denaturation, and X-ray crystallography were used to examine structural differences.

Results. IFN-β- la was 10 times more active than IFN-β- Ib with specific activities in a standard antiviral assay of 20 × 107 lU/mg for IFN-β-la and 2 × 107 lU/mg for IFN-β-lb. Of the known structural differences between IFN-β-la and IFN-β-lb, only glycosylation affected in vitro activity. Deglycosylation of IFN-β-la produced a decrease in total activity that was primarily caused by the formation of an insoluble disulfide-linked IFN precipitate. Deglycosylation also resulted in an increased sensitivity to thermal denaturation. SEC data for IFN-β-lb revealed large, soluble aggregates that had reduced antiviral activity (approximated at 0.7 × 107 lU/mg). Crystallographic data for IFN-β-la revealed that the glycan formed H-bonds with the peptide backbone and shielded an uncharged surface from solvent exposure.

Conclusions. Together these results suggest that the greater biological activity of IFN-β-la is due to a stabilizing effect of the carbohydrate on structure.

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  1. Biogen, Inc., 14 Cambridge Center, Cambridge, Massachusetts, 02142

    Laura Runkel, Werner Meier, R. Blake Pepinsky, Michael Karpusas, Adrian Whitty, Kathleen Kimball, Margot Brickelmaier, Celine Muldowney, Wendy Jones & Susan E. Goelz

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  1. Laura Runkel
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Runkel, L., Meier, W., Pepinsky, R.B. et al. Structural and Functional Differences Between Glycosylated and Non-glycosylated Forms of Human Interferon-β (IFN-β). Pharm Res 15, 641–649 (1998). https://doi.org/10.1023/A:1011974512425

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