Secretion of biologically active glycoforms of bovine follicle stimulating hormone in plants

Abstract

We chose the follicle stimulating hormone (FSH), a pituitary heterodimeric glycoprotein hormone, as a model to assess the ability of the plant cell to express a recombinant protein that requires extensive N-glycosylation for subunit folding and assembly, intracellular trafficking, signal transduction and circulatory stability. A tobacco mosaic virus (TMV) based transient expression system was used to express a single-chain (sc) version of bovine FSH in the tobacco related species Nicotiana benthamiana. Preparations of periplasmic proteins from plants infected with recombinant viral RNA contained high levels of sc-bFSH, up to 3% of total soluble proteins. Consistently, in situ indirect immunofluorescence revealed that the plant cell secreted the mammalian secretory protein to the extracellular compartment (EC). By mass spectrometric analysis of immunoaffinity purified sc-bFSH derived from EC fractions, we found two species of the plant paucimannosidic glycan type, truncated forms of complex-type N-glycans. Stimulation of cAMP production in a CHO cell line expressing the porcine FSH receptor acknowledged the native-like structure of sc-bFSH and a sufficient extent of N-glycosylation required for signal transduction. Furthermore, in superovulatory treatments of mice, sc-bFSH displayed significant in vivo bioactivity, although much lower than that of pregnant mare serum gonadotropin. We conclude that plants may have a broad utility as hosts for the recombinant expression of proteins even where glycosylation is essential for function.Keywords: plant; tobacco mosaic virus; recombinant glycoprotein hormone; single-chain FSH.The follicle-stimulating hormone (FSH) is a pituitary glycoprotein hormone that regulates the ovarian follicle and testicular tubule development in all vertebrate species [1,2]. Together with luteinizing hormone (LH), thyroid stimulating hormone (TSH) and chorionic gonadotropin (CG), FSH forms the glycoprotein hormone family, which is the structurally most complex hormone family in the animal kingdom. These hormones are composed of two noncovalently associated subunits, a common a subunit, and an unique b subunit that confers biological specificity to each of these hormones. Each subunit forms intrachain disulfide bridges and carries two N-linked oligosaccharides, which are necessary for proper folding, subunit assembly and secretion of the hormones [3,4]. The carbohydrate components of these hormones are also obligatory for signal transduction, although deglycosylated analogs show preserved receptor binding [5,6]. Moreover, the N-linked carbohydrate chains of natural pituitary FSH exhibit considerable variation in both size and structure, including the degree of terminal sialyation and/or sulfation [7]. The functional significance of this diversity of isoforms is not yet fully understood, but sialic acid seems to be the major determinant for the circulatory stability of FSH by preventing its rapid clearance mediated by the hepatic asialo-glycoprotein receptor [8] (reviewed in [9]). In the case of L...

Development of Cucumber mosaic virus as a vector modifiable for different host species to produce therapeutic proteins

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Stability and biological activities of heterodimeric and single-chain equine LH/chorionic gonadotropin variants

“…The b-a configuration was widely used to produce recombinant single-chain glycoprotein hormones of many species (Narayan et al 1995, Boime & BenMenahem 1999, Dirnberger et al 2001, Fidler et al 2003, Min et al 2004, Jablonka-Shariff et al 2007, sometimes with heterologous CTP from hCG inserted between fused a-and b-subunits to enhance the secretion of these single-chain protein variants (Sugahara et al 1996, Garcia-Campayo et al 1997, Grossmann et al 1997, Fares et al 1998. These earlier studies showed that the CTP played the role of a flexible hydrophilic spacer allowing genetically fused a-and b-subunits to adopt a functional conformation and that secretion in the presence of CTP was enhanced due to its specific O-glycans.…”

Generation of glyco-engineered BY2 cell lines with decreased expression of plant-specific glycoepitopes

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