title: Structural and functional differences between glycosylated and non-glycosylated forms of human interferon-beta (IFN-beta).
abstract: OBJECTIVE
  Two recombinant IFN-beta products have been approved for the treatment of multiple sclerosis, a glycosylated form with the predicted natural amino acid sequence (IFN-beta-1a) and a non-glycosylated form that has a Met-1 deletion and a Cys-17 to Ser mutation (IFN-beta-1b). The structural basis for activity differences between IFN-beta-1a and IFN-beta-1b, is determined.
  
  
  METHODS
  In vitro antiviral, antiproliferative and immunomodulatory assays were used to directly compare the two IFN-beta products. Size exclusion chromatography (SEC), SDS-PAGE, thermal denaturation, and X-ray crystallography were used to examine structural differences.
  
  
  RESULTS
  IFN-beta-1a was 10 times more active than IFN-beta-1b with specific activities in a standard antiviral assay of 20 x 10(7) IU/mg for IFN-beta-1a and 2 x 10(7) IU/mg for IFN-beta-1b. Of the known structural differences between IFN-beta-1a and IFN-beta-1b, only glycosylation affected in vitro activity. Deglycosylation of IFN-beta-1a 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-beta-1b revealed large, soluble aggregates that had reduced antiviral activity (approximated at 0.7 x 10(7) IU/mg). Crystallographic data for IFN-beta-1a 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-beta-1a is due to a stabilizing effect of the carbohydrate on structure.
authors.affiliation: Biogen, Inc., Cambridge, Massachusetts 02142, USA. LauraRunkel@Biogen.com
authors.forename: L
authors.initials: L
authors.lastname: Runkel
authors.forename: W
authors.initials: W
authors.lastname: Meier
authors.forename: R B
authors.initials: RB
authors.lastname: Pepinsky
authors.forename: M
authors.initials: M
authors.lastname: Karpusas
authors.forename: A
authors.initials: A
authors.lastname: Whitty
authors.forename: K
authors.initials: K
authors.lastname: Kimball
authors.forename: M
authors.initials: M
authors.lastname: Brickelmaier
authors.forename: C
authors.initials: C
authors.lastname: Muldowney
authors.forename: W
authors.initials: W
authors.lastname: Jones
authors.forename: S E
authors.initials: SE
authors.lastname: Goelz
mesh_terms: D000276:Adjuvants, Immunologic / Q000737:chemistry / Q000494:pharmacology*; D000998:Antiviral Agents / Q000494:pharmacology*; D001483:Base Sequence; D018188:Cardiovirus Infections / Q000188:drug therapy; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry; D004686:Encephalomyocarditis virus / Q000187:drug effects; D006031:Glycosylation; D006801:Humans; D000068556:Interferon beta-1a; D000068576:Interferon beta-1b; D016899:Interferon-beta / Q000737:chemistry / Q000494:pharmacology*; D008285:Major Histocompatibility Complex / Q000187:drug effects; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology*; D012694:Serine / Q000737:chemistry
chemical_list: D000276:Adjuvants, Immunologic; D000998:Antiviral Agents; D011994:Recombinant Proteins; D000068576:Interferon beta-1b; D012694:Serine; D016899:Interferon-beta; D003545:Cysteine; D000068556:Interferon beta-1a