Title : Natural human interferon-alpha 2 is O-glycosylated
Abstract :
Natural human interferon alpha 22 (IFN-alpha 2 ) was isolated from a preparation of partially purified human leucocyte IFN by monoclonal-antibody immunoaffinity chromatography
The purified protein had a specific activity of 1.5 x 10(8) i.u./mg; it was estimated to constitute 10-20% of the total antiviral activity of leucocyte IFN
N-Terminal amino-acid-sequence analysis identified the subspecies IFN-alpha 2b and/or IFN-alpha 2c, whereas IFN-alpha 2a was not detectable
The structure of natural IFN-alpha 2 was found to differ from that of its recombinant (Escherichia coli-derived) equivalent
First, reverse-phase h.p.l.c. showed that natural IFN-alpha 2 was significantly more hydrophilic then expected
Secondly, the apparent molecular mass of the natural protein determined by SDS /PAGE was higher than that of recombinant IFN-alpha 2 ; incubation under mild alkaline conditions known to eliminate O-linked carbohydrates resulted in a reduction of the apparent molecular mass to that of the recombinant protein
On sequence analysis of proteolytic peptides, Thr-106 was found to be modified
These results suggested that Thr-106 of natural IFN-alpha 2 carries O-linked carbohydrates
Reverse-phase h.p.l.c. as well as SDS /PAGE of natural IFN-alpha 2 showed that glycosylation is heterogeneous
For characterization of the carbohydrate moieties, the protein was treated with neuraminidase and/or O-glycanase and analysed by gel electrophoresis; in addition, glycopeptides obtained by proteinase digestion and separated by h.p.l.c. were characterized by sequence analysis and m.s. Further information on the com position of the glycans was obtained by monosaccharide analysis
The results indicate that natural IFN-alpha 2 contains the disaccharide galactosyl-N-acetylgalactosamine (Gal-GalNAc) linked to Thr-106
In part of the molecules, this core carbohydrate carries (alpha-)N-acetylneuraminic acid, whereas a disaccharide, probably N-acetyl-lactosamine, is bound to Gal-GalNAc in another proportion of the protein
Further glycosylation isomers are present in small amounts
As IFN-alpha 2 is the only IFN-alpha species with a threonine residue at position 106 , it may represent the only O-glycosylated human IFN-alpha protein