Structural characterisation of N‐linked and O‐linked oligosaccharides derived from interferon‐α2b and interferon‐α14c produced by Sendai‐virus‐induced human peripheral blood leukocytes

Abstract

We have previously isolated and partially characterised the components of a highly purified interferon-A (IFN-A) preparation produced by Sendai-virus-induced human peripheral blood leukocytes. Nine IFN-A species were identified, and two of these were found to be glycosylated [Nyman, T. A., Tölö, H., Parkkinen, J. & Kalkkinen, N. (1998) Identification of nine interferon-A subtypes produced by Sendaivirus-induced human peripheral blood leukocytes, Biochem. J. 329, 295Ϫ302]. Here, we isolated the Nlinked oligosaccharides of IFN-A14c and the O-linked chains of IFN-A2b, and the glycans were characterised by electrospray tandem mass spectrometry and by specific glycosidase digestions monitored by matrix-assisted laser desorption ionisation time of flight mass spectrometry. The IFN-A14c N-glycans were shown to exhibit core-fucosylated biantennary glycans, with about 10% carrying an additional A1,3-linked fucose unit at the antennae. The IFN-A2b was shown to carry about 50% core type-1 disialyltetrasaccharides, 30% core type-1 monosialyltrisaccharides and 20% core type-2 monosialylpentasaccharides.Keywords : interferon-A; glycosylation; matrix-assisted-laser-desorption ionisation time of flight; electrospray-ionisation collision-induced dissociation; glycosidase. The oligosaccharide structures in a glycoprotein can have profound effects on its biological properties [12]. However, the the mRNA level in leukocytes induced by Sendai virus [2], and it has previously been shown [3] that at least nine different sub-structural characterisation of glycoprotein glycans has been hampered by the microheterogeneity usually present in a given types are also produced at the protein level. The biological significance of the expression of several closely similar IFN-A pro-glycosylation site, and by the lack of efficient chromatographic means to isolate pure oligosaccharide species for enzymatic and teins is not known, but several reports suggest that they have quantitatively distinct patterns of antiviral, growth inhibitory and spectrometric analyses. Mass spectrometry has been widely used for structural analyses of glycoprotein glycans. Extensive studies killer-cell-stimulatory activities [4Ϫ9]. Two IFN-A variants, IFN-A2a and IFN-A2b, are mass produced in Escherichia coli by of oligosaccharides with fast-atom bombardment and liquid-secondary-ion mass spectrometry provided the basis of the straterecombinant technology and marketed as drugs. Unlike natural IFN-A, these recombinant IFN-A products have proved immuno-gies for obtaining sequence and linkage information [13Ϫ16], and more recently, matrix-assisted laser desorption/ionisation genic in some patients [10, 11], which could be due to unnatural (MALDI) and electrospray ionisation (ESI) mass spectrometry forms of IFN-A proteins. Therefore, it may be important for the have been successfully employed in glycan analyses [17Ϫ19]. development of IFN-A drugs to not only identify the IFN-A subBy complementing MS analyses with specific glycosidase treattypes and variants expressed in no...

Altering the central carbon metabolism of HEK293 cells: Impact on recombinant glycoprotein quality

“…The second most abundant mass appears to be composed of the mature IFNα2b peptide chain containing a core type-2 disialylated O-glycan (peaks d and g), this glycoform possibly being the one the O-glycosidase from Diplococcus pneumoniae enzyme failed to remove during the sequential digestion experiments. These mass spectrometry results are in good agreement with previous glycosylation characterization of IFNα2b isolated from human leukocytes (Nyman et al, 1998). The spectra also show the occurrence of amino acids loss by proteolytic cleavage of the IFNα2b peptide chain.…”

“…It should be noted that the affinity of a lectin toward a specific glycan can be influenced by its linkage but also by the nature of the surrounding glycans (Haseley et al, 1999). The human IFNα2b can be sialylated either in α2,6-or in α2,3-linkage (Nyman et al, 1998). In our SPR assays, considering the specificity of the SNA lectin employed, it was mainly the interaction with α2,6-linked sialic acid that accounted for the SPR signal.…”

Biological characterisation of a recombinant Atlantic salmon type I interferon synthesized in Escherichia coli

“…In eukaryotes, N-linked glycosylation, being the most abundant form of protein modification, is vital for normal cellular function and is believed to have an effect on the stability and folding kinetics of proteins [32]. Although only two out of the 13 human IFN-a subtypes are glycosylated, the mouse and chicken IFNs may possess up to four glycosylation sites [15,33,34]. All known fish IFNs have been found to contain between one and four potential Nlinked glycosylation sites, except for zebrafish IFN [4,18].…”

Characterization of the Murine Alpha Interferon Gene Family

“…In humans, although IFN-␤ is known to be N-glycosylated, only 2 out of 13 IFN-␣ subtypes have been found to carry glycosylations. IFN-␣2 was shown to be O-glycosylated, and IFN-␣14 was shown to be N-glycosylated (1,31).…”