ATR3 encodes a diflavin reductase essential for Arabidopsis embryo development.

*The Arabidopsis genome possesses two confirmed Cytochrome P450 Reductase (CPR) genes, ATR1 and ATR2, together with a third putative homologue, ATR3, which annotation is questionable. *Phylogenetic analysis classified ATR3 as a CPR-like protein sharing homologies with the animal cytosolic dual flavin reductases, NR1 and Fre-1, distinct from the microsomal CPRs, ATR1 and ATR2. Like NR1 and Fre-1, ATR3 lacks the N-terminal endoplasmic reticulum (ER) anchor domain of CPRs and is localized in the cytoplasm. Recombinant ATR3 in plant soluble extracts was able to reduce cytochrome c but failed to reduce the human P450 CYP1A2. *Loss of ATR3 function resulted in early embryo lethality indicating that this reductase activity is essential. A yeast 2-hybrid screen identified a unique interaction of ATR3 with the homologue of the human anti-apoptotic CIAPIN1 and the yeast Dre2 protein. *This interaction suggests two possible roles for ATR3 in the control of cell death and in chromosome segregation at mitosis. Consistent with these results, the promoter of ATR3 is activated during cell cycle progression. Together these results demonstrated that ATR3 belongs to the NR1 subfamily of diflavin reductases whose characterized members are involved in essential cellular functions.

Plant-specific glycosylation patterns in the context of therapeutic protein production.

While N-glycan synthesis in the endoplasmic reticulum (ER) is relatively well conserved in eukaryotes, N-glycan processing and O-glycan biosynthesis in the Golgi apparatus are kingdom specific and result in different oligosaccharide structures attached to glycoproteins in plants and mammals. With the prospect of using plants as alternative hosts to mammalian cell lines for the production of therapeutic glycoproteins, significant progress has been made towards the humanization of protein N-glycosylation in plant cells. To date, successful efforts in this direction have mainly focused on the targeted expression of therapeutic proteins, the knockout of plant-specific N-glycan-processing genes, and/or the introduction of the enzymatic machinery catalyzing the synthesis, transport and addition of human sugars. By contrast, very little attention has been paid until now to the O-glycosylation status of plant-made therapeutic proteins, which is surprising considering that hundreds of human proteins represent good candidates for Hyp-O glycosylation when produced in a plant expression system. This review describes protein N- and O-linked glycosylation in plants and highlights the limitations and advantages of plant-specific glycosylation on plant-made biopharmaceuticals.

Cytosolic N-terminal arginine-based signals together with a luminal signal target a type II membrane protein to the plant ER.

BACKGROUND: In eukaryotic cells, the membrane compartments that constitute the exocytic pathway are traversed by a constant flow of lipids and proteins. This is particularly true for the endoplasmic reticulum (ER), the main "gateway of the secretory pathway", where biosynthesis of sterols, lipids, membrane-bound and soluble proteins, and glycoproteins occurs. Maintenance of the resident proteins in this compartment implies they have to be distinguished from the secretory cargo. To this end, they must possess specific ER localization determinants to prevent their exit from the ER, and/or to interact with receptors responsible for their retrieval from the Golgi apparatus. Very few information is available about the signal(s) involved in the retention of membrane type II protein in the ER but it is generally accepted that sorting of ER type II cargo membrane proteins depends on motifs mainly located in their cytosolic tails. RESULTS: Here, using Arabidopsis glucosidase I as a model, we have identified two types of signals sufficient for the location of a type II membrane protein in the ER. A first signal is located in the luminal domain, while a second signal corresponds to a short amino acid sequence located in the cytosolic tail of the membrane protein. The cytosolic tail contains at its N-terminal end four arginine residues constitutive of three di-arginine motifs (RR, RXR or RXXR) independently sufficient to confer ER localization. Interestingly, when only one di-arginine motif is present, fusion proteins are located both in the ER and in mobile punctate structures, distinct but close to Golgi bodies. Soluble and membrane ER protein markers are excluded from these punctate structures, which also do not colocalize with an ER-exit-site marker. It is hypothesized they correspond to sites involved in Golgi to ER retrotransport. CONCLUSION: Altogether, these results clearly show that cytosolic and luminal signals responsible for ER retention could coexist in a same type II membrane protein. These data also suggest that both retrieval and retention mechanisms govern protein residency in the ER membrane. We hypothesized that mobile punctate structures not yet described at the ER/Golgi interface and tentatively named GERES, could be involved in retrieval mechanisms from the Golgi to the ER.

Production of recombinant proteins in suspension-cultured plant cells.

Plants have emerged in the past decade as a suitable alternative to the current production systems for recombinant pharmaceutical proteins and, today their potential for low-cost production of high quality, much safer and biologically active mammalian proteins is largely documented. Among various plant expression systems being explored, genetically modified suspension-cultured plant cells offer a promising system for production of biopharmaceuticals. Indeed, when compared to other plant-based production platforms that have been explored, suspension-cultured plant cells have the advantage of being totally devoid of problems associated with the vagaries of weather, pest, soil and gene flow in the environment. Because of short growth cycles, the timescale needed for the production of recombinant proteins in plant cell culture can be counted in days or weeks after transformation compared to months needed for the production in transgenic plants. Moreover, recovery and purification of recombinant proteins from plant biomass is an expensive and technically challenging business that may amount to 80-94% of the final product cost. One additional advantage of plant cell culture is that the recombinant protein fused with a signal sequence can be expressed and secreted into the culture medium, and therefore recovered and purified in the absence of large quantities of contaminating proteins. Consequently, the downstream processing of proteins extracted from plant cell culture medium is less expensive, which may/does balance the higher costs of fermentation. When needed for clinical use, recombinant proteins are easily produced in suspension-cultured plant cells under certified, controllable and sterile conditions that offer improved safety and provide advantages for good manufacturing practices and regulatory compliance. In this chapter, we present basic protocols for rapid generation of transgenic suspension-cultured cells of Nicotiana tabacum, Oriza sativa and Arabidopis thaliana. These systems are powerful tools for plant-made pharmaceuticals production in highly controlled conditions.

Nucleocytoplasmic transit of human alpha1-antichymotrypsin in tobacco leaf epidermal cells.

Recently, we have observed a nuclear localization for human alpha(1)-antichymotrypsin (AACT) expressed in the cytosol of transgenic Bright Yellow-2 (BY-2) tobacco cultured cells (see accompanying paper: Benchabane, M., Saint-Jore-Dupas, C., Bardor, M., Faye, L., Michaud, D. and Gomord, V. (2008a) Targeting and post-translational processing of human alpha(1)-antichymotrypsin in BY-2 tobacco cultured cells. Plant Biotechnol. J. doi: 10.1111/j.1467-7652.2008.00382.x). In the present article, we assess whether the intrinsic DNA-binding activity of AACT can explain its nuclear localization, and whether this same activity has an impact on its protease inhibitory potency and stability in planta. An engineered form of AACT with no DNA-binding activity, rAACTDeltaK, was compared with the wild-type polypeptide, rAACT, in terms of chymotrypsin inhibitory potency, stability in planta and distribution in tobacco cells. In accordance with available data reporting distinct sites for protease inhibition and DNA binding, rAACT and rAACTDeltaK showed similar antichymotrypsin activity, similar to the activity of native AACT purified from human plasma. As observed for AACT in BY-2 tobacco cells, a green fluorescent protein (GFP)-AACT fusion transiently expressed in the cytosol of tobacco leaf epidermal cells was detected mainly in the nucleus by confocal laser microscopy. By contrast, rAACTDeltaK expressed as a GFP fusion showed a balanced distribution between the cytosol and the nucleus, similar to the distribution pattern of free GFP exhibiting no DNA-binding affinity. In line with immunodetection data showing higher accumulation levels for GFP-AACT in tobacco leaf cells, rAACTDeltaK was more susceptible than rAACT to tryptic digestion in the presence of DNA. Overall, these observations suggest the following: (i) a retention effect of DNA on AACT in the nucleus; and (ii) a stabilizing effect of the AACT-DNA interaction on rAACT challenged with non-target proteases, which, possibly, may be useful in protecting this protein in plant expression platforms.

Targeting and post-translational processing of human alpha1-antichymotrypsin in BY-2 tobacco cultured cells.

The post-translational processing of human alpha(1)-antichymotrypsin (AACT) in Bright Yellow-2 (BY-2) tobacco cells was assessed in relation to the cellular compartment targeted for accumulation. As determined by pulse-chase labelling experiments and immunofluorescence microscopy, AACT sent to the vacuole or the endoplasmic reticulum (ER) was found mainly in the culture medium, similar to a secreted form targeted to the apoplast. Unexpectedly, AACT expressed in the cytosol was found in the nucleus under a stable, non-glycosylated form, in contrast with secreted variants undergoing multiple post-translational modifications during their transit through the secretory pathway. All secreted forms of AACT were N-glycosylated, with the presence of complex glycans as observed naturally on human AACT. Proteolytic trimming was also observed for all secreted variants, both during their intracellular transit and after their secretion in the culture medium. Overall, the targeting of human AACT to different compartments of BY-2 tobacco cells led to the production of two protein products: (i) a stable, non-glycosylated protein accumulated in the nucleus; and (ii) a heterogeneous mixture of secreted variants resulting from post-translational N-glycosylation and proteolytic processing. Overall, these data suggest that AACT is sensitive to resident proteases in the ER, the Golgi and/or the apoplast, and that the production of intact AACT in the plant secretory pathway will require innovative approaches to protect its structural integrity in vivo. Studies are now needed to assess the activity of the different AACT variants, and to identify the molecular determinants for the nuclear localization of AACT expressed in the cytosol.

BFA effects are tissue and not just plant specific.

Brefeldin A (BFA) is one of the most popular drugs used by researchers for studies on secretion and endocytosis because it interferes with specific vesicle coat proteins via action on a guanine nucleotide exchange factor. Due to its range of morphological effects on the Golgi apparatus in a variety of plant tissues, we believe that there is more to the BFA response than the primary molecular targets so far identified.

From planta to pharma with glycosylation in the toolbox.

Plant-specific glycosylation has long been a major limitation to the extensive use of plant-made pharmaceuticals in human therapy. Our goal here is to highlight the progress recently made towards humanization of N-glycosylation in plants and to illustrate that plant-typical N- and O-glycosylation progressively emerge as additional advantages for using this promising expression system.

Plant N-glycan processing enzymes employ different targeting mechanisms for their spatial arrangement along the secretory pathway.

The processing of N-linked oligosaccharides in the secretory pathway requires the sequential action of a number of glycosidases and glycosyltransferases. We studied the spatial distribution of several type II membrane-bound enzymes from Glycine max, Arabidopsis thaliana, and Nicotiana tabacum. Glucosidase I (GCSI) localized to the endoplasmic reticulum (ER), alpha-1,2 mannosidase I (ManI) and N-acetylglucosaminyltransferase I (GNTI) both targeted to the ER and Golgi, and beta-1,2 xylosyltransferase localized exclusively to Golgi stacks, corresponding to the order of expected function. ManI deletion constructs revealed that the ManI transmembrane domain (TMD) contains all necessary targeting information. Likewise, GNTI truncations showed that this could apply to other type II enzymes. A green fluorescent protein chimera with ManI TMD, lengthened by duplicating its last seven amino acids, localized exclusively to the Golgi and colocalized with a trans-Golgi marker (ST52-mRFP), suggesting roles for protein-lipid interactions in ManI targeting. However, the TMD lengths of other plant glycosylation enzymes indicate that this mechanism cannot apply to all enzymes in the pathway. In fact, removal of the first 11 amino acids of the GCSI cytoplasmic tail resulted in relocalization from the ER to the Golgi, suggesting a targeting mechanism relying on protein-protein interactions. We conclude that the localization of N-glycan processing enzymes corresponds to an assembly line in the early secretory pathway and depends on both TMD length and signals in the cytoplasmic tail.

Targeting of proConA to the plant vacuole depends on its nine amino-acid C-terminal propeptide.

Concanavalin A (ConA) is a well characterized and extensively used lectin accumulated in the protein bodies of jack bean cotyledons. ConA is synthesized as an inactive precursor proConA. The maturation of inactive proConA into biologically active ConA is a complex process including the removal of an internal glycopeptide and a C-terminal propeptide (CTPP), followed by a head-to-tail ligation of the two largest polypeptides. The cDNA encoding proConA was cloned and expressed in tobacco BY-2 cells. ProConA was slowly transported to the vacuole where its maturation into ConA was similar to that in jack bean cotyledons, apart from an incomplete final ligation. To investigate the role of the nine amino acid CTPP, a truncated form lacking the propeptide (proConADelta9) was expressed in BY-2 cells. In contrast to proConA, proConADelta9 was rapidly chased out of the endoplasmic reticulum (ER) and secreted into the culture medium. The CTPP was then fused to the C-terminal end of a secreted form of green fluorescent protein (secGFP). When expressed in tobacco BY-2 cells and leaf protoplasts, the chimaeric protein was located in the vacuole whereas secGFP was located in the culture medium and in the vacuole. Altogether, our results show we have isolated a new C-terminal vacuolar sorting determinant.