Purification and characterization of natural human interferon omega 1. Two alternative cleavage sites for the signal peptidase.
Human interferon omega 1 (IFN-omega 1 = IFN-alpha II1) is a recently discovered protein structurally related to IFN-alpha and -beta; the biological activities of IFN-omega 1 and its physiological role are not known to date. We have purified IFN-omega 1 from preparations of human leukocyte IFN, derived from peripheral blood leukocytes induced with Sendai virus, by two sequential cycles of monoclonal antibody affinity chromatography. The resulting protein was at least 95% pure as determined by reverse-phase high performance liquid chromatography and showed an Mr of 24,500 upon sodium dodecyl sulfate-gel electrophoresis (theoretical Mr, 19,984). Amino acid sequence analysis revealed that only about 40% of the molecules have the NH2 terminus expected on the basis of the sequence similarity to IFN-alpha, whereas the others contain two additional amino acids. This difference probably results from variable cleavage of the pre-protein by the signal peptidase. No evidence for COOH-terminal heterogeneity was found. Essentially all IFN-omega 1 molecules are glycosylated; enzymatic deglycosylation resulted in a reduction of the Mr to 20,500. Experiments using several plant lectins indicated the presence of biantennary complex oligosaccharides containing neuraminic acid. Two major peaks were observed upon chromatofocusing, with isoelectric points of 8.1 and 8.5. The specific antiviral activity of purified IFN-omega 1 assayed on human cells was determined to be 2.7 x 10(8) IU/mg, similar to that of other human class I IFNs; potent antiviral activity was also observed on cells of bovine and ovine but not of equine or murine origin.
Adolf
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GR
Ernst Boehringer-Institut für Arzneimittelforschung, Bender & Co Ges mbH, Vienna, Austria.
Maurer-Fogy
I
I
Kalsner
I
I
Cantell
K
K
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007118:Immunoassay; D007370:Interferon Type I / Q000302:isolation & purification* / Q000378:metabolism; D007372:Interferons / Q000032:analysis*; D007526:Isoelectric Point; D007962:Leukocytes / Q000032:analysis*; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010222:Parainfluenza Virus 1, Human / Q000502:physiology; D021382:Protein Sorting Signals / Q000378:metabolism; D012697:Serine Endopeptidases
D000911:Antibodies, Monoclonal; D007370:Interferon Type I; D008565:Membrane Proteins; D021382:Protein Sorting Signals; C063743:interferon omega 1; D007372:Interferons; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase