M. Dolors Ludevid

Two Structural Domains Mediate Two Sequential Events in [gamma]-Zein Targeting: Protein Endoplasmic Reticulum Retention and Protein Body Formation.

[gamma]-Zein is a maize storage protein synthesized by endosperm cells and stored together with [alpha]- and [beta]-zeins in specialized organelles called protein bodies. Previous studies have shown that in maize there is only one type of protein body and it is derived directly from the endoplasmic reticulum (ER). In this article, we describe the domains of [gamma]-zein involved in ER retention and the domains involved in protein body formation. To identify the signal responsible for [gamma]-zein retention in ER-derived protein bodies, DNAs encoding various deletion mutants of [gamma]-zein were constructed and introduced into Arabidopsis as a heterologous system. By using pulse-chase experiments and immunoelectron microscopy, we demonstrated that the deletion of a proline-rich domain at the N terminus of [gamma]-zein puts an end to its retention in the ER; this resulted in the secretion of the mutated protein. The amino acid sequence of [gamma]-zein necessary for ER retention is the repeat domain composed of eight units of the hexapeptide PPPVHL. In addition, we observed that only those [gamma]-zein mutants that contained both the proline-rich repeat domain and the C-terminal cysteine-rich domain were able to form ER-derived protein bodies. We suggest that the retention of [gamma]-zein in the ER could be a result of a protein-protein association or a transient interaction of the repeat domain with ER membranes.

Eukaryotic protein production in designed storage organelles

BackgroundProtein bodies (PBs) are natural endoplasmic reticulum (ER) or vacuole plant-derived organelles that stably accumulate large amounts of storage proteins in seeds. The proline-rich N-terminal domain derived from the maize storage protein γ zein (Zera) is sufficient to induce PBs in non-seed tissues of Arabidopsis and tobacco. This Zera property opens up new routes for high-level accumulation of recombinant proteins by fusion of Zera with proteins of interest. In this work we extend the advantageous properties of plant seed PBs to recombinant protein production in useful non-plant eukaryotic hosts including cultured fungal, mammalian and insect cells.ResultsVarious Zera fusions with fluorescent and therapeutic proteins accumulate in induced PB-like organelles in all eukaryotic systems tested: tobacco leaves, Trichoderma reesei, several mammalian cultured cells and Sf9 insect cells. This accumulation in membranous organelles insulates both recombinant protein and host from undesirable activities of either. Recombinant protein encapsulation in these PBs facilitates stable accumulation of proteins in a protected sub-cellular compartment which results in an enhancement of protein production without affecting the viability and development of stably transformed hosts. The induced PBs also retain the high-density properties of native seed PBs which facilitate the recovery and purification of the recombinant proteins they contain.ConclusionThe Zera sequence provides an efficient and universal means to produce recombinant proteins by accumulation in ER-derived organelles. The remarkable cross-kingdom conservation of PB formation and their biophysical properties should have broad application in the manufacture of non-secreted recombinant proteins and suggests the existence of universal ER pathways for protein insulation.

Molecular cloning of cDNAs encoding a putative cell wall protein from Zea mays and immunological identification of related polypeptides

Copy DNAs corresponding to a highly repetitive, proline-rich protein from maize have been cloned by differential screening of a coleoptile cDNA library. The deduced amino acid sequence contains a single repetitive element of carrot extensin (Ser-Pro-Pro-Pro-Pro). The related mRNAs have a defined distribution in tissues of the plant and are accumulated mainly in the coleoptile node and root tip. A peptide that corresponds to one of the repetitive elements of the protein has been synthesized and antisera have been obtained in rabbits. These antibodies react against crude preparations of coleoptile cell wall and against polypeptides extracted following the protocols described for the extraction of extensin. From these data it is concluded that the cDNAs correspond to a family of cell wall glycoproteins from maize.

Expression of genes for cell-wall proteins in dividing and wounded tissues of Zea mays L.

Hydroxyproline-rich glycoproteins (HRGPs) fromZea mays have been immunolocalized in the cell wall of root tip cells using ultrathin sections and antibodies ellicited against the purified protein. The accumulation of mRNA corresponding to this protein was studied using the cDNA probe. Maximum accumulation of the mRNA was found in tissues with a high proportion of dividing cells such as those in the root tip of young maize seedlings and a close relationship with cellular division was also observed in in-vitro cultures. However, the level of the mRNA in elongating tissues was minimal, as shown by studies carried out on the elongation zones of root tips and coleoptiles. The mRNA was induced by stress conditions, particularly by wounding young leaves and coleoptiles. It is concluded that in maize this group of proline-rich cell-wall proteins accumulates during cell division and not during cell elongation or differentiation, and participates in the stress-response mechanisms of the plant.

Protein Body Induction: A New Tool to Produce and Recover Recombinant Proteins in Plants

Stable accumulation of storage proteins, lipids and carbohydrates is a hallmark of the plant seed, and is a characteristic that is typically deficient in existing platforms for recombinant protein manufacture. One of the biological sequestration mechanisms that facilitate the folding, assembly and stabilization of plant seed storage proteins involve the de novo formation of unique intracellular organelles, the endoplasmic reticulum (ER)-derived protein bodies (PBs). In cereals, such as maize, PBs are formed directly in the lumen of the ER of endosperm cells and contain zeins, a group of polypeptides, which account for more than half of the total seed protein mass. The 27 kD gamma zein protein localizes to the periphery of the PBs surrounding aggregates of other zeins (including a zein and delta zein). Heterologous expression of gamma zein has been shown to result in the formation of PB-like structures, and the N-terminal proline-rich domain of gamma zein (Zera), containing eight PPPVHL repeats and a Pro-X sequence is by itself capable of directing ER retention and PB formation in non-seed tissues. We present a novel approach to produce recombinant proteins in plants based on the ability of gamma zein-Zera domain to store recombinant proteins inside PBs. Zera domain fused to several proteins, including a enhanced cyan fluorescent protein (ECFP), calcitonin (Ct) and epidermal growth factor (EGF), were cloned into vectors for transient or stable transformation of tobacco plants. In tobacco leaves, we observed the formation of dense, ER-localized structures containing high concentrations of the respective target proteins. The intact synthetic organelles containing Zera fusions were readily isolated from cellular material using density-based separation methods.

Relevant Elements of a Maize γ-Zein Domain Involved in Protein Body Biogenesis

The N-terminal proline-rich domain of γ-zein (Zera) plays an important role in protein body (PB) formation not only in the original host (maize seeds) but in a broad spectrum of eukaryotic cells. However, the elements within the Zera sequence that are involved in the biogenesis of PBs have not been clearly identified. Here, we focused on amino acid sequence motifs that could be involved in Zera oligomerization, leading to PB-like structures in Nicotiana benthamiana leaves. By using fusions of Zera with fluorescent proteins, we found that the lack of the repeat region (PPPVHL)8 of Zera resulted in the secretion of the fusion protein but that this repeat by itself did not form PBs. Although the repeat region containing eight units was the most efficient for Zera self-assembly, shorter repeats of 4–6 units still formed small multimers. Based on site-directed mutagenesis of Zera cysteine residues and analysis of multimer formation, we conclude that the two N-terminal Cys residues of Zera (Cys7 and Cys9) are critical for oligomerization. Immunoelectron microscopy and confocal studies on PB development over time revealed that early, small, Zera-derived oligomers were sequestered in buds along the rough ER and that the mature size of the PBs could be attained by both cross-linking of preformed multimers and the incorporation of new chains of Zera fusions synthesized by active membrane-bound ribosomes. Based on these results and on the behavior of the Zera structure determined by molecular dynamics simulation studies, we propose a model of Zera-induced PB biogenesis.

D-SAP: a new, noncytotoxic, and fully protease resistant cell-penetrating peptide.

Protease resistant cell‐penetrating peptides (CPPs) are promising carriers for drugs unable to cross the cell membrane. As these CPPs are stable in vivo for much longer periods of time compared to other classes of therapeutic peptides, noncytotoxicity is a property sine qua non for their pharmacological development. Described herein is a fully protease resistant CPP that is noncytotoxic at concentrations up to 1 mM. Proteolytic stability was obtained by chiral inversion of the residues of a known self‐assembling CPP—from all L‐amino acids to all D‐amino acids—and then assessed against trypsin and human serum. Circular dichroism studies confirmed the enantiomeric structure of the analogue, and transmission electron microscopy (TEM) studies indicated that the new inverso analogue retains the ability of the original peptide to self‐assemble. The results of uptake experiments indicate that the protease‐stable (that is, D‐amino acid) analogue of the peptide is internalised by cells to the same extent as the protease‐susceptible (that is, L‐amino acid) parent peptide. Also reported herein are the results of studies on the cellular internalisation mechanism of the all‐D analogue, which reveal the steps followed by the peptide upon its entry into the cell.

Differential expression of a hydroxyproline-rich cell-wall protein gene in embryonic tissues of Zea mays L.

A hydroxyproline-rich glycoprotein (HRGP) component of the maize cell wall was shown to be present in different organs of the plant by extraction of cell wall proteins and detection by Western blotting and immunocytochemistry. Antibodies raised against the protein or against synthetic peptides designed from the protein sequence immunoprecipitated a proline-rich polypeptide which was synthesized in-vitro from poly(A) + RNA extracted from different tissues of the plant and from the complete in-vitro-transcribed mRNA. A very low amount of the protein was found in immature embryos. In particular, the protein could not be detected in the scutellum either by Western blotting or by immunocytochemistry. In agreement with this finding, HRGP mRNA was barely detected in the scutellum, in contrast to its accumulation in the embryo axis. Our results indicate the existence of a unique cell wall structure in embryonic tissues from maize as well as a tissuespecific component of the control of maize HRGP gene expression, distinct to others already described such as cell division.

The Expression of a Xylanase Targeted to ER-Protein Bodies Provides a Simple Strategy to Produce Active Insoluble Enzyme Polymers in Tobacco Plants

Background Xylanases deserve particular attention due to their potential application in the feed, pulp bleaching and paper industries. We have developed here an efficient system for the production of an active xylanase in tobacco plants fused to a proline-rich domain (Zera) of the maize storage protein γ-zein. Zera is a self-assembling domain able to form protein aggregates in vivo packed in newly formed endoplasmic reticulum-derived organelles known as protein bodies (PBs). Methodology/Principal Findings Tobacco leaves were transiently transformed with a binary vector containing the Zera-xylanase coding region, which was optimized for plant expression, under the control of the 35S CaMV promoter. The fusion protein was efficiently expressed and stored in dense PBs, resulting in yields of up to 9% of total protein. Zera-xylanase was post-translationally modified with high-mannose-type glycans. Xylanase fused to Zera was biologically active not only when solubilized from PBs but also in its insoluble form. The resistance of insoluble Zera-xylanase to trypsin digestion demonstrated that the correct folding of xylanase in PBs was not impaired by Zera oligomerization. The activity of insoluble Zera-xylanase was enhanced when substrate accessibility was facilitated by physical treatments such as ultrasound. Moreover, we found that the thermostability of the enzyme was improved when Zera was fused to the C-terminus of xylanase. Conclusion/Significance In the present work we have successfully produced an active insoluble aggregate of xylanase fused to Zera in plants. Zera-xylanase chimeric protein accumulates within ER-derived protein bodies as active aggregates that can easily be recovered by a simple density-based downstream process. The production of insoluble active Zera-xylanase protein in tobacco outlines the potential of Zera as a fusion partner for producing enzymes of biotechnological relevance. Zera-PBs could thus become efficient and low-cost bioreactors for industrial purposes.

Proteomic characterisation of endoplasmic reticulum-derived protein bodies in tobacco leaves

BackgroundThe N-terminal proline-rich domain (Zera) of the maize storage protein γ-zein, is able to induce the formation of endoplasmic reticulum (ER)-derived protein bodies (PBs) when fused to proteins of interest. This encapsulation enables a recombinant fused protein to escape from degradation and facilitates its recovery from plant biomass by gradient purification. The aim of the present work was to evaluate if induced PBs encapsulate additional proteins jointly with the recombinant protein. The exhaustive analysis of protein composition of PBs is expected to facilitate a better understanding of PB formation and the optimization of recombinant protein purification approaches from these organelles.ResultsWe analysed the proteome of PBs induced in Nicotiana benthamiana leaves by transient transformation with Zera fused to a fluorescent marker protein (DsRed). Intact PBs with their surrounding ER-membrane were isolated on iodixanol based density gradients and their integrity verified by confocal and electron microscopy. SDS-PAGE analysis of isolated PBs showed that Zera-DsRed accounted for around 85% of PB proteins in term of abundance. Differential extraction of PBs was performed for in-depth analysis of their proteome and structure. Besides Zera-DsRed, 195 additional proteins were identified including a broad range of proteins resident or trafficking through the ER and recruited within the Zera-DsRed polymer.ConclusionsThis study indicates that Zera-protein fusion is still the major protein component of the new formed organelle in tobacco leaves. The analysis also reveals the presence of an unexpected diversity of proteins in PBs derived from both the insoluble Zera-DsRed polymer formation, including ER-resident and secretory proteins, and a secretory stress response induced most likely by the recombinant protein overloading. Knowledge of PBs protein composition is likely to be useful to optimize downstream purification of recombinant proteins in molecular farming applications.