Andrea Pompa

Zeolin. A New Recombinant Storage Protein Constructed Using Maize γ-Zein and Bean Phaseolin

The major seed storage proteins of maize (Zea mays) and bean (Phaseolus vulgaris), zein and phaseolin, accumulate in the endoplasmic reticulum (ER) and in storage vacuoles, respectively. We show here that a chimeric protein composed of phaseolin and 89 amino acids of γ-zein, including the repeated and the Pro-rich domains, maintains the main characteristics of wild-type γ-zein: It is insoluble unless its disulfide bonds are reduced and forms ER-located protein bodies. Unlike wild-type phaseolin, the protein, which we called zeolin, accumulates to very high amounts in leaves of transgenic tobacco (Nicotiana tabacum). A relevant proportion of the ER chaperone BiP is associated with zeolin protein bodies in an ATP-sensitive fashion. Pulse-chase labeling confirms the high affinity of BiP to insoluble zeolin but indicates that, unlike structurally defective proteins that also extensively interact with BiP, zeolin is highly stable. We conclude that the γ-zein portion is sufficient to induce the formation of protein bodies also when fused to another protein. Because the storage proteins of cereals and legumes nutritionally complement each other, zeolin can be used as a starting point to produce nutritionally balanced and highly stable chimeric storage proteins.

Retention of a Bean Phaseolin/Maize γ-Zein Fusion in the Endoplasmic Reticulum Depends on Disulfide Bond Formation

Most seed storage proteins of the prolamin class accumulate in the endoplasmic reticulum (ER) as large insoluble polymers termed protein bodies (PBs), through mechanisms that are still poorly understood. We previously showed that a fusion between the Phaseolus vulgaris vacuolar storage protein phaseolin and the N-terminal half of the Zea mays prolamin γ-zein forms ER-located PBs. Zeolin has 6 Cys residues and, like γ-zein with 15 residues, is insoluble unless reduced. The contribution of disulfide bonds to zeolin destiny was determined by studying in vivo the effects of 2-mercaptoethanol (2-ME) and by zeolin mutagenesis. We show that in tobacco (Nicotiana tabacum) protoplasts, 2-ME enhances interactions of newly synthesized proteins with the ER chaperone BiP and inhibits the secretory traffic of soluble proteins with or without disulfide bonds. In spite of this general inhibition, 2-ME enhances the solubility of zeolin and relieves its retention in the ER, resulting in increased zeolin traffic. Consistently, mutated zeolin unable to form disulfide bonds is soluble and efficiently enters the secretory traffic without 2-ME treatment. We conclude that disulfide bonds that lead to insolubilization are a determinant for PB-mediated protein accumulation in the ER.

Plastid Proteostasis and Heterologous Protein Accumulation in Transplastomic Plants

In spite of a huge number of reports on successful foreign protein production in plastid, in many cases the expression level of other recombinant proteins in the transplastomic plants appears to be very low or even undetectable. We believe that this is mainly due to our limited knowledge of the mechanisms in plastids influencing the maintenance of protein homeostasis, or proteostasis. Plastids retain a whole series of mechanisms for the preservation of their protein balance, including specific proteases, transcriptional and translational control, as well as molecular chaperones and enzymes useful in protein folding. Therefore, it is important to develop basic studies in factors regulating protein synthesis, stability, folding, targeting, and accumulation in plastids, including the protein quality control which contributes to the functional integrity of proteins. Our intention here is not to provide a comprehensive review of the mechanisms that regulate plastid proteostasis, but to discuss some recent insights into this field which might bring beneficial applications in plastid biotechnology for transgene expression and foreign protein accumulation.

Traffic of Human α-Mannosidase in Plant Cells Suggests the Presence of a New Endoplasmic Reticulum-to-Vacuole Pathway without Involving the Golgi Complex

Noncanonical protein traffic from the endoplasmic reticulum to the vacuole may bypass the Golgi complex altogether. The transport of secretory proteins from the endoplasmic reticulum to the vacuole requires sorting signals as well as specific transport mechanisms. This work is focused on the transport in transgenic tobacco (Nicotiana tabacum) plants of a human α-mannosidase, MAN2B1, which is a lysosomal enzyme involved in the turnover of N-linked glycoproteins and can be used in enzyme replacement therapy. Although ubiquitously expressed, α-mannosidases are targeted to lysosomes or vacuoles through different mechanisms according to the organisms in which these proteins are produced. In tobacco cells, MAN2B1 reaches the vacuole even in the absence of mannose-6-phosphate receptors, which are responsible for its transport in animal cells. We report that MAN2B1 is targeted to the vacuole without passing through the Golgi complex. In addition, a vacuolar targeting signal that is recognized in plant cells is located in the MAN2B1 amino-terminal region. Indeed, when this amino-terminal domain is removed, the protein is retained in the endoplasmic reticulum. Moreover, when this domain is added to a plant-secreted protein, the resulting fusion protein is partially redirected to the vacuole. These results strongly suggest the existence in plants of a new type of vacuolar traffic that can be used by leaf cells to transport vacuolar proteins.

Recombinant human GAD65 accumulates to high levels in transgenic tobacco plants when expressed as an enzymatically inactive mutant

The 65-kDa isoform of glutamic acid decarboxylase (GAD65) is the major autoantigen implicated in the development of type 1 diabetes mellitus (T1DM). The bulk manufacture of GAD65 is a potential issue in the fight against T1DM but current production platforms are expensive. We show that a catalytically inactive form of GAD65 (GAD65mut) accumulates at up to 2.2% total soluble protein in transgenic tobacco leaves, which is more than 10-fold the levels achieved with active GAD65, yet the protein retains the immunogenic properties required to treat T1DM. This higher yield was found to be a result of a higher rate of protein synthesis and not transcript availability or protein stability. We found that targeting GAD65 to the endoplasmic reticulum, a strategy that increases the accumulation of many recombinant proteins expressed in plants, did not improve production of GAD65mut. The production of a catalytically inactive autoantigen that retains its immunogenic properties could be a useful strategy to provide high-quality therapeutic protein for treatment of autoimmune T1DM.

Unconventional pathways of secretory plant proteins from the endoplasmic reticulum to the vacuole bypassing the Golgi complex

Studies on the basic mechanisms that regulate vacuolar delivering of proteins synthesized in the endoplasmic reticulum (ER) have a great importance in plant cell biology. Indeed, many aspects of plant physiology are affected by this intracellular traffic, for example, germination or reaction to biotic stresses due to the accumulation of storage proteins in seeds or enzymes in vegetative tissues, respectively. Up to now, the Golgi complex has been considered the main hub in the sorting of vacuolar secretory proteins; those polypeptides able to reach their final destination without the aid of this organelle are regarded as exceptions to an established route. This mini-review aims to emphasize the existence of several Golgi-independent pathways involved in the trafficking of different types of vacuolar proteins.

An engineered C-terminal disulfide bond can partially replace the phaseolin vacuolar sorting signal.

Seed storage proteins accumulate either in the endoplasmic reticulum (ER) or in vacuoles, and it would appear that polymerization events play a fundamental role in regulating the choice between the two destinies of these proteins. We previously showed that a fusion between the Phaseolus vulgaris vacuolar storage protein phaseolin and the N-terminal half of the Zea mays prolamin gamma-zein forms interchain disulfide bonds that facilitate the formation of ER-located protein bodies. Wild-type phaseolin does not contain cysteine residues, and assembles into soluble trimers that transiently polymerize before sorting to the vacuole. These transient interactions are abolished when the C-terminal vacuolar sorting signal AFVY is deleted, indicating that they play a role in vacuolar sorting. We reasoned that if the phaseolin interactions directly involve the C terminus of the polypeptide, a cysteine residue introduced into this region could stabilize these transient interactions. Biochemical studies of two mutated phaseolin proteins in which a single cysteine residue was inserted at the C terminus, in the presence (PHSL*) or absence (Delta 418*) of the vacuolar signal AFVY, revealed that these mutated proteins form disulphide bonds. PHSL* had reduced protein solubility and a vacuolar trafficking delay with respect to wild-type protein. Moreover, Delta 418* was in part redirected to the vacuole. Our experiments strongly support the idea that vacuolar delivery of phaseolin is promoted very early in the sorting process, when polypeptides are still contained within the ER, by homotypic interactions.

A plant secretory signal peptide targets plastome-encoded recombinant proteins to the thylakoid membrane

Plastids are considered promising bioreactors for the production of recombinant proteins, but the knowledge of the mechanisms regulating foreign protein folding, targeting, and accumulation in these organelles is still incomplete. Here we demonstrate that a plant secretory signal peptide is able to target a plastome-encoded recombinant protein to the thylakoid membrane. The fusion protein zeolin with its native signal peptide expressed by tobacco (Nicotiana tabacum) transplastomic plants was directed into the chloroplast thylakoid membranes, whereas the zeolin mutant devoid of the signal peptide, Δzeolin, is instead accumulated in the stroma. We also show that zeolin folds in the thylakoid membrane where it accumulates as trimers able to form disulphide bonds. Disulphide bonds contribute to protein accumulation since zeolin shows a higher accumulation level with respect to stromal Δzeolin, whose folding is hampered as the protein accumulates at low amounts in a monomeric form and it is not oxidized. Thus, post-transcriptional processes seem to regulate the stability and accumulation of plastid-synthesized zeolin. The most plausible zeolin targeting mechanism to thylakoid is discussed herein.

Unconventional Transport Routes of Soluble and Membrane Proteins and Their Role in Developmental Biology

Many proteins and cargoes in eukaryotic cells are secreted through the conventional secretory pathway that brings proteins and membranes from the endoplasmic reticulum to the plasma membrane, passing through various cell compartments, and then the extracellular space. The recent identification of an increasing number of leaderless secreted proteins bypassing the Golgi apparatus unveiled the existence of alternative protein secretion pathways. Moreover, other unconventional routes for secretion of soluble or transmembrane proteins with initial endoplasmic reticulum localization were identified. Furthermore, other proteins normally functioning in conventional membrane traffic or in the biogenesis of unique plant/fungi organelles or in plasmodesmata transport seem to be involved in unconventional secretory pathways. These alternative pathways are functionally related to biotic stress and development, and are becoming more and more important in cell biology studies in yeast, mammalian cells and in plants. The city of Lecce hosted specialists working on mammals, plants and microorganisms for the inaugural meeting on “Unconventional Protein and Membrane Traffic” (UPMT) during 4–7 October 2016. The main aim of the meeting was to include the highest number of topics, summarized in this report, related to the unconventional transport routes of protein and membranes.

Human α-mannosidase produced in transgenic tobacco plants is processed in human α-mannosidosis cell lines

Deficiency in human lysosomal α-mannosidase (MAN2B1) results in α-mannosidosis, a lysosomal storage disorder; patients present a wide range of neurological, immunological, and skeletal symptoms caused by a multisystemic accumulation of mannose-containing oligosaccharides. Here, we describe the expression of recombinant MAN2B1 both transiently in Nicotiana benthamiana leaves and in the leaves and seeds of stably transformed N. tabacum plants. After purification from tobacco leaves, the recombinant enzyme was found to be N-glycosylated and localized in vacuolar compartments. In the fresh leaves of tobacco transformants, MAN2B1 was measured at 10,200 units/kg, and the purified enzyme from these leaves had a specific activity of 32-45 U/mg. Furthermore, tobacco-produced MAN2B1 was biochemically similar to the enzyme purified from human tissues, and it was internalized and processed by α-mannosidosis fibroblast cells. These results strongly indicate that plants can be considered a promising expression system for the production of recombinant MAN2B1 for use in enzyme replacement therapy.