The Changing Fate of a Secretory Glycoprotein in Developing Maize Endosperm

Arcalís, Elsa; Stadlmann, Johannes; Marcel, Sylvain; Drakakaki, Georgia; Winter, Verena; Rodrı́guez, Julián; Fischer, Rainer; Altmann, Friedrich; Stöger, Eva

doi:10.1104/pp.109.152363

Cited by 41 publications

(59 citation statements)

References 35 publications

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“…The same difference between immature and mature seeds (i.e. a stronger tendency toward ER-derived structures in later stages of maturation) was also observed in maize (Zea mays) seeds expressing a recombinant protein (Arcalis et al, 2010).…”

Section: Table III In Vitro Virus Neutralization Activity Of Seed-prsupporting

confidence: 55%

Expression of Antibody Fragments with a ControlledN-Glycosylation Pattern and Induction of Endoplasmic Reticulum-Derived Vesicles in Seeds of Arabidopsis

et al. 2011

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Intracellular trafficking and subcellular deposition are critical factors influencing the accumulation and posttranslational modifications of proteins. In seeds, these processes are not yet fully understood. In this study, we set out to investigate the intracellular transport, final destination, N-glycosylation status, and stability of the fusion of recombinant single-chain variable fragments to the crystallizing fragment of an antibody (scFv-Fc) of two antiviral monoclonal antibodies (2G12 and HA78). The scFv-Fcs were expressed in Arabidopsis (Arabidopsis thaliana) seeds and leaves both as secretory molecules and tagged with an endoplasmic reticulum (ER) retention signal. We demonstrate differential proteolytic degradation of scFv-Fcs in leaves versus seeds, with higher degradation in the latter organ. In seeds, we show that secretory versions of HA78 scFv-Fcs are targeted to the extracellular space but are deposited in newly formed ER-derived vesicles upon KDEL tagging. These results are in accordance with the obtained N-glycosylation profiles: complex-type and ER-typical oligomannosidic N-glycans, respectively. HA78 scFv-Fcs, expressed in seeds of an Arabidopsis glycosylation mutant lacking plant-specific N-glycans, exhibit custommade human-type N-glycosylation. In contrast, 2G12 scFv-Fcs carry exclusively ER-typical oligomannosidic N-glycans and were deposited in newly formed ER-derived vesicles irrespective of the targeting signals. HA78 scFv-Fcs exhibited efficient virus neutralization activity, while 2G12 scFv-Fcs were inactive. We demonstrate the efficient generation of scFv-Fcs with a controlled N-glycosylation pattern. However, our results also reveal aberrant subcellular deposition and, as a consequence, unexpected N-glycosylation profiles. Our attempts to elucidate intracellular protein transport in seeds contributes to a better understanding of this basic cell biological mechanism and is a step toward the versatile use of Arabidopsis seeds as an alternative expression platform for pharmaceutically relevant proteins.Recombinant monoclonal antibodies (mAbs) are of high therapeutic potential and have thus become a major product of the pharmaceutical industry (Aggarwal, 2009). In addition to full-length mAbs, antibodies are being engineered to alter their size, pharmacokinetics, specificity, valency, effector functions, etc. in order to better suit the intended applications (for review, see Filpula, 2007;Harmsen and De Haard, 2007). Of particular interest among these engineered fragments are single-chain variable fragments (scFvs), fusions of variable heavy and variable light domains that retain an antigen-binding function. Due to their smaller size as compared with their full-length counterparts, these molecules penetrate target tissues better and can even bind to intracellular targets. Furthermore, multimerization via disulfide bonds and/or multimerization domains allows for the production of divalent or higher order antibody-like molecules, where increased avidity

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Section: Table III In Vitro Virus Neutralization Activity Of Seed-prsupporting

confidence: 55%

Expression of Antibody Fragments with a ControlledN-Glycosylation Pattern and Induction of Endoplasmic Reticulum-Derived Vesicles in Seeds of Arabidopsis

et al. 2011

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“…The expression and accumulation pattern of zeins and other storage proteins have been extensively studied in the maize starchy endosperm (Marks et al, 1985;Lending and Larkins, 1989;Or et al, 1993;Wang and Messing, 1998;Woo et al, 2001;Shewry and Halford, 2002;Marzá bal et al, 2008;Arcalis et al, 2010). Despite the importance of the aleurone layer for endosperm development and seed germination, the expression and trafficking of storage proteins in this tissue has not been analyzed in detail.…”

Section: Discussion Although Zein Genes Are Transcribed In Aleurone Cmentioning

confidence: 99%

“…Maize seeds also contain smaller quantities of legumin-1 and a-globulin (Woo et al, 2001;Yamagata et al, 2003). These storage proteins accumulate in vacuoles during the early stages of endosperm development and are then retained in ER protein bodies at later stages (Arcalis et al, 2010).…”

mentioning

confidence: 99%

Delivery of Prolamins to the Protein Storage Vacuole in Maize Aleurone Cells

et al. 2011

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Zeins, the prolamin storage proteins found in maize (Zea mays), accumulate in accretions called protein bodies inside the endoplasmic reticulum (ER) of starchy endosperm cells. We found that genes encoding zeins, a-globulin, and legumin-1 are transcribed not only in the starchy endosperm but also in aleurone cells. Unlike the starchy endosperm, aleurone cells accumulate these storage proteins inside protein storage vacuoles (PSVs) instead of the ER. Aleurone PSVs contain zeinrich protein inclusions, a matrix, and a large system of intravacuolar membranes. After being assembled in the ER, zeins are delivered to the aleurone PSVs in atypical prevacuolar compartments that seem to arise at least partially by autophagy and consist of multilayered membranes and engulfed cytoplasmic material. The zein-containing prevacuolar compartments are neither surrounded by a double membrane nor decorated by AUTOPHAGY RELATED8 protein, suggesting that they are not typical autophagosomes. The PSV matrix contains glycoproteins that are trafficked through a Golgi-multivesicular body (MVB) pathway. MVBs likely fuse with the multilayered, autophagic compartments before merging with the PSV. The presence of similar PSVs also containing prolamins and large systems of intravacuolar membranes in wheat (Triticum aestivum) and barley (Hordeum vulgare) starchy endosperm suggests that this trafficking mechanism may be common among cereals.The cereal endosperm consists of three main cell types: an inner mass of starchy endosperm cells, one to three layers of epidermal aleurone cells, and the transfer cells that contact the maternal vascular tissue (Olsen, 2004). The starchy endosperm accounts for 80 to 90% of the grain weight and contains large amounts of storage proteins and starch. These cells undergo programmed cell death during maturation. Aleurone cells are rich in protein storage vacuoles (PSVs), minerals, and lipid bodies and remain alive during seed development. It is assumed that the breakdown of proteins localized to PSVs in aleurone cells provides an essential source of the amino acids necessary for the synthesis of hydrolytic enzymes required for mobilizing food stored in the starchy endosperm (Filner and Varner, 1967;Jacobsen et al., 1988;Bethke et al., 1998).Besides its biological relevance as a model to study plant development, cell differentiation, and programmed cell death, the cereal endosperm is very important in terms of its nutritional value. Cereal grains contain less protein than do legume seeds, but because cereals are produced and consumed in much larger quantities, they are the main source of protein for the nutrition of humans and livestock (Shewry and Halford, 2002). The major storage proteins in maize (Zea mays) kernels are the alcoholsoluble prolamins, a type of storage proteins present only in grasses. Maize prolamins, referred to as zeins, are divided into different types: a-, b-, g-, and d-zeins (Coleman and Larkins, 1999) that differ in amino acid composition and structural properties (Shewry and Halford, 2002)...

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“…[64][65][66][67][68] Typically, the presence of an N-terminal signal peptide with no further targeting information results in the secretion of a recombinant protein to the apoplast, but the same proteins expressed in endosperm cells are likely to accumulate within the cell. For example, human lysozyme expressed in rice endosperm with a functional signal peptide accumulated in PSVs, accompanied by the distortion of protein body morphology.…”

confidence: 99%

“…75 Finally, the localization of a recombinant protein may also be influenced by the developmental stage of a seed, as reported for a model glycoprotein in maize. 66 These studies indicate that the specific properties of seed tissues in different species must be taken into account when considering strategies for molecular farming in seeds. Further studies are required to fully elucidate the mechanisms of protein trafficking in storage cells and allow the behavior of recombinant proteins in seeds to be predicted reliably.…”

confidence: 99%