Andrea Pompa

Publications (10) View all

• Article: Traffic of human α-mannosidase in plant cells suggests the presence of a new endoplasmic reticulum to vacuole pathway without involving the Golgi complex.

  Francesca De Marchis, Michele Bellucci, Andrea Pompa
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  ABSTRACT: Transport of secretory proteins from the endoplasmic reticulum to the vacuole requires sorting signals as well as specific transport mechanisms. The present work is focused on the transport in transgenic tobacco 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, alpha-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 which is recognized in plant cells is located in the MAN2B1 N-terminal region. Indeed, when this N-terminus domain is removed, the protein is retained in the ER. 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 which can be used by leaf cells to transport vacuolar proteins.
  Plant physiology 02/2013; · 6.53 Impact Factor
• Article: Plastid proteostasis and heterologous protein accumulation in transplastomic plants.

  Francesca De Marchis, Andrea Pompa, Michele Bellucci
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  ABSTRACT: 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.
  Plant physiology 08/2012; 160(2):571-81. · 6.53 Impact Factor
• Article: A plant secretory signal peptide targets plastome-encoded recombinant proteins to the thylakoid membrane.

  Francesca De Marchis, Andrea Pompa, Roberta Mannucci, Tomas Morosinotto, Michele Bellucci
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  ABSTRACT: 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.
  Plant Molecular Biology 07/2011; 76(3-5):427-41. · 4.15 Impact Factor
• Article: Human α-mannosidase produced in transgenic tobacco plants is processed in human α-mannosidosis cell lines.

  Francesca De Marchis, Chiara Balducci, Andrea Pompa, Hilde M F Riise Stensland, Marco Guaragno, Rita Pagiotti, Anna R Menghini, Emanuele Persichetti, Tommaso Beccari, Michele Bellucci
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  ABSTRACT: 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.
  Plant Biotechnology Journal 06/2011; 9(9):1061-73. · 5.44 Impact Factor
• Article: Recombinant human GAD65 accumulates to high levels in transgenic tobacco plants when expressed as an enzymatically inactive mutant.

  Linda Avesani, Alessandro Vitale, Emanuela Pedrazzini, Maddalena Devirgilio, Andrea Pompa, Alessandra Barbante, Elisa Gecchele, Paola Dominici, Francesca Morandini, Annalisa Brozzetti, Alberto Falorni, Mario Pezzotti
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  ABSTRACT: 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.
  Plant Biotechnology Journal 03/2010; 8(8):862-72. · 5.44 Impact Factor