JOURNAL OF INTERFERON AND CYTOKINE RESEARCH 18:287-295 (1998) Mary Ann Liebert, Inc. Review The Glycosylation Heterogeneity of Recombinant Human IFN-y ANDREW HOOKER12 and DAVID JAMES2 ABSTRACT The cloning of the cDNA for human interferon-y (IFN-y) has resulted in its expression in Escherichia coli, baculovirus-infected insect cells, Chinese hamster ovary (CHO) cells, and the mammary gland of transgenic mice. Large quantities of highly purified recombinant IFN-y have been generated, aided by the use of highly specific neutralizing monoclonal antibodies, with a view to its production as a human therapeutic protein. The primary source of structural heterogeneity for IFN-y during its production in mammalian expression systems is glycosylation, which can profoundly affect the three-dimensional structure of a glycoprotein and its bio- logical function. A number of analytical approaches have been developed recently to allow a detailed analy- sis of the carbohydrate structures associated with IFN-y, the principal advances being in the areas of capil- lary electrophoresis and mass spectrometry. The implementation of these high-resolution analytical tools to determine the glycosylation profile of IFN-y makes it one of the best characterized recombinant glycopro- teins. Recombinant human IFN-y acts as a model secretory glycoprotein, typifying the intrinsic glycosylation processing events associated with production of a potential therapeutic glycoprotein. INTRODUCTION Virus-induced interferons (IFN-a/ß, type I interferons) were first described in 1957 by Isaacs and Lindenmann.(l) In 1965, Wheelock observed an IFN-like activity in cultures of human leukocytes stimulated by phytohemagglutinin.<2) How- ever, this type II interferon, IFN-y, is unrelated to the type I in- terferons at both the genetic and protein levels, is not virus-in- duced, and is inactivated at low pH. After more than 30 years of research, it is now apparent that IFN-y is primarily a lym- phokine, playing an important role as an immunomodulator, and that its physiological role in vivo as an antiviral agent is minimal, limiting its therapeutic application.(3-5> The cDNA for human IFN-y was reported to be cloned into Escherichia coli and eukaryotic cells in 1982,(6,7) followed rapidly by murine IFN-y.(8) However, recombinant IFN-y pro- duced by E. coli, which has no capacity to glycosylate proteins, results in a susceptibility to proteolysis and low antiviral ac- tivity.<9) Therefore, significant research effort has been focused on the expression of the recombinant human gene in animal cell cultures. The recombinant gene for human IFN-y generates a 1.2-kb mRNA that encodes a 166 amino acid polypeptide and contains a 23 hydrophobic amino acid signal sequence at the amino-ter- minal.<6) Following the proteolytic removal of the signal se- quence in the endoplasmic reticulum before secretion, a mature acidic protein of 143 amino acid residues is produced with a predicted molecular mass of 16,758 Da. Pyroglutamic acid is at the amino-terminus, and the carboxy-terminus is susceptible to posttranslational proteolysis.*10) However, homogeneous in- tact molecules have been reported for recombinant IFN-y ex- pressed in E. co/i(11) IFN-y is physiologically present as a dimer, which is the active form, being held by noncovalent forces in the absence of any cysteine residues for the formation of intramolecular disulfide bonds.<9) Some size analysis studies have suggested that the functional unit is a tetramer.<12,13) X- ray crystallography data on E. co/i-derived human IFN-y indi- cate that the subunits are associated in an antiparallel fashion 'Oxford GlycoSciences (UK) Pic, Abingdon Science Park, Abingdon, 0X14 3YS, United Kingdom. Research School of Biosciences, University of Kent, Canterbury, United Kingdom. 287