Purification and carbohydrate structure of natural murine interferon‐β

Abstract

Mouse interferon$ (Mu-IFN-P) induced in C-243 cells with Newcastle disease virus was purified in four steps including ammonium sulfate fractionation, DEAE-cellulose, monoclonal Mu-IFN-P antibody affinity and Mono-S cation-exchange chromatographies. Specific activity of the purified Mu-IFN-P ranged over 1.1 -1.4 x lo9 NIH units/mg protein. This preparation was submitted to pronase digestion and gel filtration on Fractogel TSK HW-40. The permethylated and acetylated glycopeptide fraction was analyzed by chemical-ionization (ammonia) mass spectrometry. The major glycopeptide is composed of Gal, Man, GlcNAc and NeuAc with a molar ratio of 2.0: 3.6: 3.4: 0.5. The GLC pattern of methyl derivatives obtained by methanolysis and acetylation of fully methylated glycopeptide identified 2,3,4,6-tetra-O-methylgalactose; 3,4,6-tri-O-methyl-mannose; 2,3,4-and 2,4,6-tri-0-methyl galactose; 2,4,di-O-methyl mannose and 3,6-di-O-rnethylglucosamine.These results when compared with data on N-glycans suggest the following structure for the carbohydrate moiety of Mu-IFN-P:Murine interferons a and fl are released from various cells in response to virus infection [l-41 or upon induction by double-stranded polyribonucleotide [5]. Murine interferon-? is produced by lymphocytes exposed to a variety of antigens or mitogen stimulations [6]. These three types of IFN are glycosylated proteins which are distinct in their antigenicityThe potential therapeutic application of IFNs for treating viral infections or tumor proliferation [lo] established a priority in the cloning of human IFNs [ll]. Nevertheless, interferon's antiproliferative and pharmacodynamic effects requiring in vivo experimentation had to be performed in well defined systems such as isogenic mouse strains.For these reasons, recombinant murine IFNs have been obtained and their primary structure have been determined (see [12] for review). These studies showed that Mu-IFN-a polypeptides are the products of at least ten different genes which seem to be highly conserved during evolution [13]. Sequence analysis of seven of them indicated that the mature Mu-IFN-a proteins vary in size from 162 to 167 amino acids; all except two possess a single N-glycosylation site (Asn-AlaThr) at position 78.[7-91.Correspondence to J. Doly, Biologie Moleculaire des Interfkrons, Institut de Recherches sur le Cancer, 7, Rue Guy-Mocquet, F-94802 Villejuif Cedex, FranceAbbreviations. Mu-IFN, murine interferon; Hu-IFN, human interferon; MEM, minimal essential medium; NDV, Newcastle disease virus; VSV, vesicular stomatitis virus; PBS, 0.02 M phosphate buffer pH 7.3 and 0.15 NaCl; CHO, Chinese hamster ovary. M a n LY I

The Function of the Human Interferon-β1a Glycan Determined in Vivo

“…For example, low heterogeneity of the glycans of Chinese hamster ovary cell-derived rhIFN-␤1a has been reported, and 95% of the structures were found to be biantennary complex glycans containing terminating galactose sialylation and core fucosylation with the overall carbohydrate residue composition NeuAc 2 Gal 2 Man 3 GlcNAc 4 Fuc 1 (Conradt et al, 1987). In addition, the glycans of Chinese hamster ovary cell-derived IFN-␤ show high similarity to the naturally occurring human (Kagawa et al, 1988) and murine (Civas et al, 1988) counterpart. Protein-conjugated carbohydrates are increasingly being recognized as functional modulators of gene products or by exhibiting independent functions affecting the stability, solubility, clearance, and receptor-binding properties of proteins (Oh-eda et al, 1990;Kodama et al, 1993;Logsdon et al, 2004).…”

“…Protein-conjugated carbohydrates are increasingly being recognized as functional modulators of gene products or by exhibiting independent functions affecting the stability, solubility, clearance, and receptor-binding properties of proteins (Oh-eda et al, 1990;Kodama et al, 1993;Logsdon et al, 2004). Although, the IFN-␤1a glycan has been hypothesized to be involved in receptor binding (Civas et al, 1988), it is generally accepted that the carbohydrate moiety is involved in solubility and stability of the protein. This was supported by a 10 times higher antiviral activity of the glycosylated rhIFN-␤1a compared with the nonglycosylated variant as well as altered physicochemical properties, e.g., increased propensity of aggregation of the deglycosylated rhIFN-␤1a (Watanabe and Kawade, 1983;Conradt et al, 1987;Runkel et al, 1998).…”