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 (proConAΔ9) was expressed in BY-2 cells. In contrast to proConA, proConAΔ9 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 determinan

Arginine/Lysine Residues in the Cytoplasmic Tail Promote ER Export of Plant Glycosylation Enzymes

Plant N-glycan processing enzymes are arranged along the early secretory pathway, forming an assembly line to facilitate the step-by-step modification of oligosaccharides on glycoproteins. Thus, these enzymes provide excellent tools to study signals and mechanisms, promoting their localization and retention in the endoplasmic reticulum (ER) and Golgi apparatus. Herein, we focused on a detailed investigation of amino acid sequence motifs present in their short cytoplasmic tails in respect to ER export. Using site-directed mutagenesis, we determined that single arginine/lysine residues within the cytoplasmic tail are sufficient to promote rapid Golgi targeting of Golgi-resident N-acetylglucosaminyltransferase I (GnTI) and α-mannosidase II (GMII). Furthermore, we reveal that an intact ER export motif is essential for proper in vivofunction of GnTI. Coexpression studies with Sar1p provided evidence for COPII-dependent transport of GnTI to the Golgi. Our data provide evidence that efficient ER export of Golgi-resident plant N-glycan processing enzymes occurs through a selective mechanism based on recognition of single basic amino acids present in their cytoplasmic tails

Ricin B chain targeted to the endoplasmic reticulum of tobacco protoplasts is degraded by a CDC48- and vacuole-independent mechanism

The B chain of ricin was expressed and delivered to the endoplasmic reticulum of tobacco protoplasts where it disappeared with time in a manner consistent with degradation. This turnover did not occur in the vacuoles or upon secretion. Indeed, several lines of evidence indicate that, in contrast to the turnover of endoplasmic reticulum-targeted ricin A chain in the cytosol, the bulk of expressed ricin B chain was degraded in the secretory pathway

Differential effects of human and plant N-acetylglucosaminyltransferase I (GnTI) in plants

In plants and animals, the first step in complex type N-glycan formation on glycoproteins is catalyzed by N-acetylglucosaminyltransferase I (GnTI). We show that the cgl1-1 mutant of Arabidopsis, which lacks GnTI activity, is fully complemented by YFP-labeled plant AtGnTI, but only partially complemented by YFP-labeled human HuGnTI and that this is due to post-transcriptional events. In contrast to AtGnTI-YFP, only low levels of HuGnTI-YFP protein was detected in transgenic plants. In protoplast co-transfection experiments all GnTI-YFP fusion proteins co-localized with a Golgi marker protein, but only limited co-localization of AtGnTI and HuGnTI in the same plant protoplast. The partial alternative targeting of HuGnTI in plant protoplasts was alleviated by exchanging the membrane-anchor domain with that of AtGnTI, but in stably transformed cgl1-1 plants this chimeric GnTI still did not lead to full complementation of the cgl1-1 phenotype. Combined, the results indicate that activity of HuGnTI in plants is limited by a combination of reduced protein stability, alternative protein targeting and possibly to some extend to lower enzymatic performance of the catalytic domain in the plant biochemical environment

Combinatorial Analysis of Secretory Immunoglobulin A (sIgA) Expression in Plants

Delivery of secretory immunoglobulin A (sIgA) to mucosal surfaces as a passive immunotherapy agent is a promising strategy to prevent infectious diseases. Recombinant sIgA production in plants requires the co-expression of four transcriptional units encoding the light chain (LC), heavy chain (HC), joining chain (JC) and secretory component (SC). As a way to optimize sIgA production in plants, we tested the combinatorial expression of 16 versions of a human sIgA against the VP8* rotavirus antigen in Nicotiana benthamiana, using the recently developed GoldenBraid multigene assembly system. Each sIgA version was obtained by combining one of the two types of HC (alpha 1 and alpha 2) with one of the two LC types (k and lambda) and linking or not a KDEL peptide to the HC and/or SC. From the analysis of the anti-VP8* activity, it was concluded that those sIgA versions carrying HC alpha 1 and LC lambda provided the highest yields. Moreover, ER retention significantly increased antibody production, particularly when the KDEL signal was linked to the SC. Maximum expression levels of 32.5 mu g IgA/g fresh weight (FW) were obtained in the best performing combination, with an estimated 33% of it in the form of a secretory complex.This work has been funded by Grant BIO2010-15384 from Plan Nacional I + D of the Spanish Ministry of Science. Juarez P. is a recipient of a FPU fellowship, and Sarrion-Perdigones A. and Huet-Trujillo E. are recipients of a FPI fellowship. We want to thank Monedero for kindly providing scFv and VP8* clones.Juárez Ortega, P.; Huet Trujillo, E.; Sarrion-Perdigones, A.; Falconi, E.; Granell Richart, A.; Orzáez Calatayud, DV. (2013). Combinatorial Analysis of Secretory Immunoglobulin A (sIgA) Expression in Plants. International Journal of Molecular Sciences. 14(3):6205-6222. https://doi.org/10.3390/ijms14036205S62056222143Reichert, J. M., & Valge-Archer, V. E. (2007). Development trends for monoclonal antibody cancer therapeutics. Nature Reviews Drug Discovery, 6(5), 349-356. doi:10.1038/nrd2241Corthésy, B. (2010). Role of secretory immunoglobulin A and secretory component in the protection of mucosal surfaces. Future Microbiology, 5(5), 817-829. doi:10.2217/fmb.10.39Corthésy, B. (2003). Recombinant Secretory Immunoglobulin A in Passive Immunotherapy: Linking Immunology and Biotechnology. Current Pharmaceutical Biotechnology, 4(1), 51-67. doi:10.2174/1389201033378020Corthësy, B. (2008). Secretory immunoglobulin A: well beyond immune exclusion at mucosal surfaces. Immunopharmacology and Immunotoxicology, 31(2), 174-179. doi:10.1080/08923970802438441Sarrion-Perdigones, A., Juarez, P., Granell, A., & Orzaez, D. (2011). Production of Antibodies in Plants. Cell Engineering, 143-164. doi:10.1007/978-94-007-1257-7_7Paul, M., Dolleweerd, C. van, Drake, P. M. W., Reljic, R., Thangaraj, H., Barbi, T., … Ma, J. K.-C. (2011). Molecular pharming. Human Vaccines, 7(3), 375-382. doi:10.4161/hv.7.3.14456Fischer, R., Stoger, E., Schillberg, S., Christou, P., & Twyman, R. M. (2004). Plant-based production of biopharmaceuticals. Current Opinion in Plant Biology, 7(2), 152-158. doi:10.1016/j.pbi.2004.01.007Twyman, R. M., Stoger, E., Schillberg, S., Christou, P., & Fischer, R. (2003). Molecular farming in plants: host systems and expression technology. Trends in Biotechnology, 21(12), 570-578. doi:10.1016/j.tibtech.2003.10.002Saint-Jore-Dupas, C., Faye, L., & Gomord, V. (2007). From planta to pharma with glycosylation in the toolbox. Trends in Biotechnology, 25(7), 317-323. doi:10.1016/j.tibtech.2007.04.008Ma, J., Hiatt, A., Hein, M., Vine, N., Wang, F., Stabila, P., … Lehner, T. (1995). Generation and assembly of secretory antibodies in plants. Science, 268(5211), 716-719. doi:10.1126/science.7732380Ma, J. K.-C., Hikmat, B. Y., Wycoff, K., Vine, N. D., Chargelegue, D., Yu, L., … Lehner, T. (1998). Characterization of a recombinant plant monoclonal secretory antibody and preventive immunotherapy in humans. Nature Medicine, 4(5), 601-606. doi:10.1038/nm0598-601Weintraub, J. A., Hilton, J. F., White, J. M., Hoover, C. I., Wycoff, K. L., Yu, L., … Featherstone, J. D. B. (2005). Clinical Trial of a Plant-Derived Antibody on Recolonization of Mutans Streptococci. Caries Research, 39(3), 241-250. doi:10.1159/000084805Wycoff, K. (2005). Secretory IgA Antibodies from Plants. Current Pharmaceutical Design, 11(19), 2429-2437. doi:10.2174/1381612054367508Nicholson, L., Gonzalez-Melendi, P., Van Dolleweerd, C., Tuck, H., Perrin, Y., Ma, J. K.-C., … Stoger, E. (2004). A recombinant multimeric immunoglobulin expressed in rice shows assembly-dependent subcellular localization in endosperm cells. Plant Biotechnology Journal, 3(1), 115-127. doi:10.1111/j.1467-7652.2004.00106.xWieland, W. H., Lammers, A., Schots, A., & Orzáez, D. V. (2006). Plant expression of chicken secretory antibodies derived from combinatorial libraries. Journal of Biotechnology, 122(3), 382-391. doi:10.1016/j.jbiotec.2005.12.020Larrick, J. W., Yu, L., Naftzger, C., Jaiswal, S., & Wycoff, K. (2001). Production of secretory IgA antibodies in plants. Biomolecular Engineering, 18(3), 87-94. doi:10.1016/s1389-0344(01)00102-2FOLEY, R. C., RAISONS, R. L., & BEH, K. J. (1991). Monoclonal Antibody Against Sheep Kappa Light Chain. Hybridoma, 10(4), 507-515. doi:10.1089/hyb.1991.10.507Furtado, P. B., Whitty, P. W., Robertson, A., Eaton, J. T., Almogren, A., Kerr, M. A., … Perkins, S. J. (2004). Solution Structure Determination of Monomeric Human IgA2 by X-ray and Neutron Scattering, Analytical Ultracentrifugation and Constrained Modelling: A Comparison with Monomeric Human IgA1. Journal of Molecular Biology, 338(5), 921-941. doi:10.1016/j.jmb.2004.03.007De Muynck, B., Navarre, C., & Boutry, M. (2010). Production of antibodies in plants: status after twenty years. Plant Biotechnology Journal, 8(5), 529-563. doi:10.1111/j.1467-7652.2009.00494.xMa, J. K.-C., Drake, P. M. W., & Christou, P. (2003). The production of recombinant pharmaceutical proteins in plants. Nature Reviews Genetics, 4(10), 794-805. doi:10.1038/nrg1177De Muynck, B., Navarre, C., Nizet, Y., Stadlmann, J., & Boutry, M. (2009). Different subcellular localization and glycosylation for a functional antibody expressed in Nicotiana tabacum plants and suspension cells. Transgenic Research, 18(3), 467-482. doi:10.1007/s11248-008-9240-1D�ring, K., Hippe, S., Kreuzaler, F., & Schell, J. (1990). Synthesis and self-assembly of a functional monoclonal antibody in transgenic Nicotiana tabacum. Plant Molecular Biology, 15(2), 281-293. doi:10.1007/bf00036914Wilde, C. D., Rycke, R. D., Beeckman, T., Neve, M. D., Montagu, M. V., Engler, G., & Depicker, A. (1998). Accumulation Pattern of IgG Antibodies and Fab Fragments in Transgenic Arabidopsis thaliana Plants. Plant and Cell Physiology, 39(6), 639-646. doi:10.1093/oxfordjournals.pcp.a029416Schouten, A., Roosien, J., van Engelen, F. A., (Ineke) de Jong, G. A. M., (Tanja) Borst-Vrenssen, A. W. M., Zilverentant, J. F., … Bakker, J. (1996). The C-terminal KDEL sequence increases the expression level of a single-chain antibody designed to be targeted to both the cytosol and the secretory pathway in transgenic tobacco. Plant Molecular Biology, 30(4), 781-793. doi:10.1007/bf00019011Bencurova, M. (2004). Specificity of IgG and IgE antibodies against plant and insect glycoprotein glycans determined with artificial glycoforms of human transferrin. Glycobiology, 14(5), 457-466. doi:10.1093/glycob/cwh058Gomord, V., Fitchette, A.-C., Menu-Bouaouiche, L., Saint-Jore-Dupas, C., Plasson, C., Michaud, D., & Faye, L. (2010). Plant-specific glycosylation patterns in the context of therapeutic protein production. Plant Biotechnology Journal, 8(5), 564-587. doi:10.1111/j.1467-7652.2009.00497.xSarrion-Perdigones, A., Falconi, E. E., Zandalinas, S. I., Juárez, P., Fernández-del-Carmen, A., Granell, A., & Orzaez, D. (2011). GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules. PLoS ONE, 6(7), e21622. doi:10.1371/journal.pone.0021622Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE, 6(2), e16765. doi:10.1371/journal.pone.0016765Monedero, V., Rodriguez-Diaz, J., Viana, R., Buesa, J., & Perez-Martinez, G. (2004). Selection of Single-Chain Antibodies against the VP8* Subunit of Rotavirus VP4 Outer Capsid Protein and Their Expression in Lactobacillus casei. Applied and Environmental Microbiology, 70(11), 6936-6939. doi:10.1128/aem.70.11.6936-6939.2004Juárez, P., Presa, S., Espí, J., Pineda, B., Antón, M. T., Moreno, V., … Orzaez, D. (2011). Neutralizing antibodies against rotavirus produced in transgenically labelled purple tomatoes. Plant Biotechnology Journal, 10(3), 341-352. doi:10.1111/j.1467-7652.2011.00666.xLangley, R., Wines, B., Willoughby, N., Basu, I., Proft, T., & Fraser, J. D. (2005). The Staphylococcal Superantigen-Like Protein 7 Binds IgA and Complement C5 and Inhibits IgA-FcαRI Binding and Serum Killing of Bacteria. The Journal of Immunology, 174(5), 2926-2933. doi:10.4049/jimmunol.174.5.2926Sack, M., Paetz, A., Kunert, R., Bomble, M., Hesse, F., Stiegler, G., … Rademacher, T. (2007). Functional analysis of the broadly neutralizing human anti-HIV-1 antibody 2F5 produced in transgenic BY-2 suspension cultures. The FASEB Journal, 21(8), 1655-1664. doi:10.1096/fj.06-5863comRamessar, K., Rademacher, T., Sack, M., Stadlmann, J., Platis, D., Stiegler, G., … Christou, P. (2008). Cost-effective production of a vaginal protein microbicide to prevent HIV transmission. Proceedings of the National Academy of Sciences, 105(10), 3727-3732. doi:10.1073/pnas.0708841104Brandtzaeg, P., & Prydz, H. (1984). Direct evidence for an integrated function of J chain and secretory component in epithelial transport of immunoglobulins. Nature, 311(5981), 71-73. doi:10.1038/311071a0Johansen, Braathen, & Brandtzaeg. (2000). Role of J Chain in Secretory Immunoglobulin Formation. Scandinavian Journal of Immunology, 52(3), 240-248. doi:10.1046/j.1365-3083.2000.00790.xBraathen, R., Hohman, V. S., Brandtzaeg, P., & Johansen, F.-E. (2007). Secretory Antibody Formation: Conserved Binding Interactions between J Chain and Polymeric Ig Receptor from Humans and Amphibians. The Journal of Immunology, 178(3), 1589-1597. doi:10.4049/jimmunol.178.3.1589Murthy, A. K., Chaganty, B. K. R., Troutman, T., Guentzel, M. N., Yu, J.-J., Ali, S. K., … Arulanandam, B. P. (2011). Mannose-Containing Oligosaccharides of Non-Specific Human Secretory Immunoglobulin A Mediate Inhibition of Vibrio cholerae Biofilm Formation. PLoS ONE, 6(2), e16847. doi:10.1371/journal.pone.0016847Mathias, A., & Corthésy, B. (2011). N-Glycans on secretory component. Gut Microbes, 2(5), 287-293. doi:10.4161/gmic.2.5.18269Hu, L., Crawford, S. E., Czako, R., Cortes-Penfield, N. W., Smith, D. F., Le Pendu, J., … Prasad, B. V. V. (2012). Cell attachment protein VP8* of a human rotavirus specifically interacts with A-type histo-blood group antigen. Nature, 485(7397), 256-259. doi:10.1038/nature10996Yu, X., Dang, V. T., Fleming, F. E., von Itzstein, M., Coulson, B. S., & Blanchard, H. (2012). Structural Basis of Rotavirus Strain Preference toward N-Acetyl- or N-Glycolylneuraminic Acid-Containing Receptors. Journal of Virology, 86(24), 13456-13466. doi:10.1128/jvi.06975-11Haselhorst, T., Fleming, F. E., Dyason, J. C., Hartnell, R. D., Yu, X., Holloway, G., … von Itzstein, M. (2008). Sialic acid dependence in rotavirus host cell invasion. Nature Chemical Biology, 5(2), 91-93. doi:10.1038/nchembio.134Bonner, A., Almogren, A., Furtado, P. B., Kerr, M. A., & Perkins, S. J. (2008). The Nonplanar Secretory IgA2 and Near Planar Secretory IgA1 Solution Structures Rationalize Their Different Mucosal Immune Responses. Journal of Biological Chemistry, 284(8), 5077-5087. doi:10.1074/jbc.m807529200Yoo, E. M., & Morrison, S. L. (2005). IgA: An immune glycoprotein. Clinical Immunology, 116(1), 3-10. doi:10.1016/j.clim.2005.03.010Karnoup, A. S., Turkelson, V., & Anderson, W. H. K. (2005). O-Linked glycosylation in maize-expressed human IgA1. Glycobiology, 15(10), 965-981. doi:10.1093/glycob/cwi077Ramsland, P. A., Willoughby, N., Trist, H. M., Farrugia, W., Hogarth, P. M., Fraser, J. D., & Wines, B. D. (2007). Structural basis for evasion of IgA immunity by Staphylococcus aureus revealed in the complex of SSL7 with Fc of human IgA1. Proceedings of the National Academy of Sciences, 104(38), 15051-15056. doi:10.1073/pnas.0706028104Almogren, A., Senior, B. W., & Kerr, M. A. (2007). A comparison of the binding of secretory component to immunoglobulin A (IgA) in human colostral S-IgA1 and S-IgA2. Immunology, 120(2), 273-280. doi:10.1111/j.1365-2567.2006.02498.xOrzaez, D., Mirabel, S., Wieland, W. H., & Granell, A. (2006). Agroinjection of Tomato Fruits. A Tool for Rapid Functional Analysis of Transgenes Directly in Fruit. Plant Physiology, 140(1), 3-11. doi:10.1104/pp.105.06822

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), α-1,2 mannosidase I (ManI) and N-acetylglucosaminyltransferase I (GNTI) both targeted to the ER and Golgi, and β-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

Transient co-expression for fast and high-yield production of antibodies with human-like N -glycans in plants

International audiencePlant-based transient expression is potentially the most rapid and cost-efficient system for the production of recombinant pharmaceutical proteins, but safety concerns associated with plant-specific N-glycosylation have hampered its adoption as a commercial production system. In this article, we describe an approach based on the simultaneous transient co-expression of an antibody, a suppressor of silencing and a chimaeric human β 1,4-galactosyltransferase targeted for optimal activity to the early secretory pathway in agroinfiltrated Nicotiana benthamiana leaves. This strategy allows fast and high-yield production of antibodies with human-like N-glycans and, more generally, provides solutions to many critical problems posed by the large-scale production of therapeutic and vaccinal proteins, specifically yield, volume and quality