Date Published: March 12, 2019
Publisher: Public Library of Science
Author(s): Tarlan Mamedov, Ilaha Musayeva, Rabia Acsora, Nilufer Gun, Burcu Gulec, Gulshan Mammadova, Kader Cicek, Gulnara Hasanova, Debasis Chakrabarty.
A plant expression platform with eukaryotic post-translational modification (PTM) machinery has many advantages compared to other protein expression systems. This promising technology is useful for the production of a variety of recombinant proteins including, therapeutic proteins, vaccine antigens, native additives, and industrial enzymes. However, plants lack some of the important PTMs, including furin processing, which limits this system for the production of certain mammalian complex proteins of therapeutic value. Furin is a ubiquitous proprotein convertase that is involved in the processing (activation) of a wide variety of precursor proteins, including blood coagulation factors, cell surface receptors, hormones and growth factors, viral envelope glycoproteins, etc. and plays a critical regulatory role in a wide variety of cellular events. In this study, we engineered the human furin gene for expression in plants and demonstrated the production of a functional active recombinant truncated human furin in N. benthamiana plant. We demonstrate that plant produced human furin is highly active both in vivo and in vitro and specifically cleaved the tested target proteins, Factor IX (FIX) and Protective Antigen (PA83). We also demonstrate that both, enzymatic deglycosylation and proteolytic processing of target proteins can be achieved in vivo by co-expression of deglycosylating and furin cleavage enzymes in a single cell to produce deglycosylated and furin processed target proteins. It is highly expected that this strategy will have many potential applications in pharmaceutical industry and can be used to produce safe and affordable therapeutic proteins, antibodies, and vaccines using a plant expression system.
In recent years, numerous studies have demonstrated the plant transient expression systems as a promising expression platform with high expression capacity, which provide safe, cost-effective production of a variety of biologically active proteins in a relatively short period of time [1–5]. Since plants possess eukaryotic PTM machinery, this technology is especially useful for the production of mammalian complex proteins, where PTMs play a critical role in the proper folding and functional activity . Despite the significant similarities between mammalian and plant cell PTM machinery, plants lack the ability to make a number of important PTMs present in mammalian cells . Furin is a mammalian subtilisin/kex2p like endoprotease, belonging to the proprotein convertase family. It is responsible for the post-translational cleavage of a number of precursor proteins. Although the existence of a kex2p like pathway was reported earlier in Nicotiana tabacum plant , no furin cleavage activity was observed in plants . The human furin protein is a 794 amino acid long, membrane-associated protease. It possesses a signal peptide, propeptide, and subtilisin-like catalytic domain characterized by a catalytic triad of three amino acids: aspartate, histidine and serine. Furin also contains a cysteine rich domain, homo B domain, which is essential for catalytic activity. It is anchored in the plasma membrane by a transmembrane domain and the cytoplasmic domain, which regulates the localization of furin in the cellular trans-Golgi network . The luminal and extracellular domains of human furin share a homology with other members of the proprotein convertase (PC) family. Notably, the highest sequence similarity is found in the subtilisin-like catalytic domain, where aspartate, histidine and serine residues form a strictly conserved catalytic triad . The catalytic domain of furin is 54–70% identical to other PCs . Furin cleavage of propeptides is essential for maturation of the precursor proteins. For example, furin processing is essential for the gamma carboxylation of glutamic acid residues , disulfide bridge formation [12, 13], regulating the synthesis of multiple mature peptides [14, 15], and directing intracellular targeting . Furin is involved in the cleavage of serum proteins including blood clotting factors, cell surface receptors, hormones, growth factors and their receptors [9, 17] mainly at Arg-X-Lys/Arg-Arg (RXK/RR) consensus sequence [18,19]. A number of studies have reported that a mutation found at the furin cleavage site of certain precursor proteins is associated with the onset of the various types of diseases . For example, the envelope proteins of influenza virus , HIV , dengue fever  and several filoviruses including ebola and marburg virus  must be cleaved by furin-like proteases to become functionally active. Furin also functions in the cleavage of papillomaviruses , anthrax toxin  and pseudomonad exotoxin  during entry into the host cells. It should be noted that, recombinant blood clotting factors VII, VIII, IX, and protein C, which are currently used in the treatment of a number of diseases are mainly prepared from donated human blood and therefore, have a viral contamination risk and also very expensive [27–29]. Defects in factor IX (FIX) synthesis result in hemophilia B (Christmas disease), an X-linked disorder. Different expression systems have been used to produce recombinant FIX , however, all attempts were impeded by limitations in PTM, safety, and high costs. A plant transient expression system could be an alternative expression system for the production of safe and affordable blood clotting factors, such as FIX to be used as a hemophilia B treatment. As previously stated, plants lack a number of important mammalian PTMs, such as gamma carboxylation, furin processing, sialylation, mannose-6-phosphate modification, sulfation, and etc. . Therefore, engineering of plants that can produce recombinant proteins in their native forms would be very important for their functionality. FIX is expressed as a precursor polypeptide that requires posttranslational processing. In order to produce functionally active FIX in plants, FIX must be cleaved in vivo by PACE (Paired basic Amino acid Cleaving Enzyme)/furin processing enzyme. Given that furin cleavage is involved in many different cellular events, production of functionally active furin is necessary for the production of a variety of pharmaceutically valuable precursor proteins. Since human furin is a transmembrane protein, it therefore, would be challenging to produce a highly soluble and fully functional active human furin in plants. At this point, although co-expression of full length human furin and latent transforming growth factor-b1 in N. benthamiana plants was recently reported , there was no direct confirmation of furin expression; for example, western blot analysis using a specific antibody of plant produced human furin was not reported in the study . Additionally, there were no reports regarding the isolation of furin from plants or the in vitro assessment of plant produced recombinant furin. In this study, we engineered a human furin gene for production in plants. For the first time we have shown that this truncated version of recombinant human furin is highly expressed, soluble and functionally active in N. benthamiana plants. We also described a method for producing furin processed proteins in plant cells by co-expressing human furin with target proteins of interest in vivo. We transiently co-expressed a hemophilia B therapeutic candidate, FIX, and an anthrax vaccine candidate, protective antigen (PA) of B. anthracis along side human furin in N. benthamiana. Our results showed that human furin cleaved all target proteins. We also demonstrated that both the enzymatic deglycosylation and proteolytic processing of target proteins were achieved in vivo by introducing and co-expressing deglycosylating and furin cleavage enzymes in the same plant cells.
Recently a series of strategies and approaches have been utilized to successfully develop a system for the efficient recombinant production and subsequent PTM of proteins in plants, which are important for proper folding, functionality and stability [4, 44, 47, 48]. In this study, we engineered the human furin gene for expression in N. benthamiana plants resulting in the production of a highly soluble, functionally active enzyme. We show that plant produced furin has about 75% relative activity of commercial human furin in vitro. The major goal of this study was to achieve furin processing in plants and apply the technology to the PTM of proteins, in which furin cleavage is necessary for maturation and activation. Moreover, we demonstrate that plant produced truncated form of human furin is active in vivo and specifically cleaved target proteins, FIX and PA83. As described above, furin modifies proteins of vitamin K-dependent coagulation Factors (Factors VII, IX and protein C). Defects in FIX synthesis result in hemophilia B (Christmas disease), an X-linked disorder. Currently, patients with hemophilia B are mainly treated with FIX, obtained from concentrates made from human blood and recombinant FIX produced in CHO cells. However, such preparations of FIX are extremely expensive and difficult to obtain, especially in developing countries. To date, all attempts at producing recombinant Factor IX using different expression systems have been hampered by limitations in post-translation processing, safety concerns, and high cost. There remains an urgent need for a safe and affordable therapeutic treatment for hemophilia B. Thus, the findings in this study may open the door for the production of affordable, safe (pathogen free), functionally active human clotting factors, such as FIX, Factor VII and protein C in plants using transient expression technology. In this study, we also demonstrate that both enzymatic deglycosylation and proteolytic processing of PA83 protein were achieved in vivo by introducing the respective enzymatic repertoire into a eukaryotic system. The PA83 protein does not carry N-linked glycans in the native hosts, but contains potential N-linked glycosylation sites, which are aberrantly glycosylated during expression in plants [4, 44, 47, 48]. Glycosylated PA83 has no biological activity, and therefore, cannot form a lethal toxin [32, 44]. It has low immunogenicity compared to the deglycosylated form [4, 32, 44], and is also highly unstable especially at elevated temperatures . Thus, deglycosylation of PA83 or PA63 is important for functional activity. Plant produced, PNGase F [44, 47] or Endo H deglycosylated forms of PA83  were more stable compared to the glycosylated counterpart and had a superior immunogenicity. However, further improvement in the potency, immunogenicity and stability of the anthrax vaccine is still needed. In vivo processing of PA83 the protein and production of deglycosylated PA63 protein in plants could potentially be used to develop a new vaccine candidate against anthrax, based on heptamerized PA63, which can be produced in vitro (without the need of costly commercial cleavage enzymes, i.e. furin or trypsin) or in vivo. This technology has potential applications in molecular farming and can be used to produce subunit vaccines, therapeutic proteins, and antibodies in eukaryotic system. Recombinant human furin has not been previously produced in plants, therefore, the technology developed in this study supports the utility of plants as an efficient expression system for the production of active, endotoxin-free recombinant human furin.