Natural human interferon-alpha 2 is O-glycosylated

Area of Science:

• Biochemistry
• Immunology
• Glycobiology

Background:

• Natural human interferon alpha 2 (IFN-alpha 2) is a key antiviral protein.
• Previous studies focused on recombinant IFN-alpha 2, with limited understanding of natural IFN-alpha 2 structure.
• Leukocyte-derived IFN preparations contain multiple IFN-alpha subtypes, necessitating purification for detailed analysis.

Purpose of the Study:

• To isolate and characterize natural human IFN-alpha 2.
• To investigate structural differences between natural and recombinant IFN-alpha 2.
• To identify and characterize the post-translational modifications of natural IFN-alpha 2.

Main Methods:

• Monoclonal-antibody immunoaffinity chromatography for IFN-alpha 2 purification.
• N-terminal amino-acid sequencing and SDS/PAGE for protein identification and mass determination.
• Reverse-phase high-performance liquid chromatography (h.p.l.c.) for hydrophilicity analysis.
• Enzymatic treatment (neuraminidase, O-glycanase) and mass spectrometry (m.s.) for glycan characterization.

Main Results:

• Purified natural IFN-alpha 2 (1.5 x 10(8) i.u./mg) comprised 10-20% of leukocyte IFN antiviral activity.
• Natural IFN-alpha 2 was more hydrophilic and had a higher apparent molecular mass than recombinant IFN-alpha 2.
• O-linked glycosylation at Thr-106 was identified as the cause of structural differences.
• Heterogeneous glycosylation was observed, including galactosyl-N-acetylgalactosamine (Gal-GalNAc) and N-acetylneuraminic acid or N-acetyl-lactosamine.

Conclusions:

• Natural human IFN-alpha 2 is O-glycosylated at Thr-106, a unique modification among IFN-alpha species.
• Glycosylation significantly alters the physicochemical properties of IFN-alpha 2 compared to its recombinant counterpart.
• These findings provide crucial insights into the structural heterogeneity and biological function of natural interferons.