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Structural and Functional Differences Between Glycosylated and Non-glycosylated Forms of Human Interferon-β (IFN-β) (1998)

Runkel, Laura ; Meier, Werner ; Pepinsky, R. Blake ; [et al.]

Springer

Pharmaceutical research 15 (1998), S. 641-649

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ISSN: 1573-904X

Keywords: interferon-β ; AVONEX® ; Betaseron® ; glycosylation ; aggregation ; activity

Source: Springer Online Journal Archives 1860-2000

Topics: Chemistry and Pharmacology

Notes: 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.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1023/A:1011974512425

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Augmented desensitization to epidermal growth factor (EGF) immediate actions: A novel mechanism for altered EGF growth response in mutant A431 cells (1990)

Karasik, Avraham ; Reddy, Sethu S.-K. ; Pepinsky, R. Blake ; [et al.]

New York, NY [u.a.] : Wiley-Blackwell

Journal of Cellular Physiology 142 (1990), S. 231-235

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ISSN: 0021-9541

Keywords: Life and Medical Sciences ; Cell & Developmental Biology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Medicine

Notes: Epidermal growth factor (EGF) may either stimulate or inhibit cell growth. To elucidate the mechanism of these varied effects, we compared EGF action in parental A431 cells in which cell growth is inhibited, and clone 15, a mutant of these cells resistant to EGF growth inhibition. In both lines, EGF receptor was present in similar concentrations and underwent tyrosine phosphorylation to the same extent. Likewise, in both lines, acute exposure to EGF stimulated an increase in free cytoplasmic [Ca2+], as well as a similar increase in phosphoryla-tion of lipocortin 1, a major substrate for the EGF receptor kinase whose phos-phorylation is calcium-dependent. On the other hand, pretreatment of clone 15 cells with EGF for 72 h abolished EGF-induced phosphorylation of lipocortin 1 and led to a loss of the increase in cytoplasmic free [Ca2+], whereas no such desensitization was seen in the parental A431 cells. These data indicate a link between EGF-induced increase in cytoplasmic calcium, lipocortin phosphoryla-tion, and cell growth and suggest that differences in mechanisms of desensitization to these immediate actions of EGF may lead to altered growth response to this hormone.

Additional Material: 4 Ill.