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Plant heat shock protein 70 as carrier for immunization against a plant-expressed reporter antigen

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Abstract

Mammalian Heat Shock Proteins (HSP), have potent immune-stimulatory properties due to the natural capability to associate with polypeptides and bind receptors on antigen presenting cells. The present study was aimed to explore whether plant HSP, and in particular HSP70, share similar properties. We wanted in particular to evaluate if HSP70 extracted in association to naturally bound polypeptides from plant tissues expressing a recombinant “reporter” antigen, carry antigen-derived polypeptides and can be used to activate antigen-specific immune responses. This application of HSP70 has been very poorly investigated so far. The analysis started by structurally modeling the plant protein and defining the conditions that ensure maximal expression levels and optimal recovery from plant tissues. Afterwards, HSP70 was purified from Nicotiana benthamiana leaves transiently expressing a heterologous “reporter” protein. The purification was carried out taking care to avoid the release from HSP70 of the polypeptides chaperoned within plant cells. The evaluation of antibody titers in mice sera subsequent to the subcutaneous delivery of the purified HSP70 demonstrated that it is highly effective in priming humoral immune responses specific to the plant expressed “reporter” protein. Overall results indicated that plant-derived HSP70 shares structural and functional properties with the mammalian homologue. This study paves the way to further investigations targeted at determining the properties of HSP70 extracted from plants expressing foreign recombinant antigens as a readily available immunological carrier for the efficient delivery of polypeptides derived from these antigens.

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References

  • Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modeling. Bioinformatics 22:195–201

    CAS  Google Scholar 

  • Basu S, Matsutake T (2004) Heat shock protein-antigen presenting cell interactions. Methods 32:38–41

    CAS  PubMed  Google Scholar 

  • Batten JS, Yoshinari S, Hemenway C (2003) Potato virus X: a model system for virus replication, movement and gene expression. Mol Plant Pathol 4:125–131

    CAS  PubMed  Google Scholar 

  • Baulcombe DC, Chapman S, Santa Cruz S (1995) Jellyfish green fluorescent protein as a reporter for virus infections. Plant J 6:1045–1053

    Google Scholar 

  • Blachere NE, Li Z, Chandawarkar RY, Suto R, Jaikaria NS, Basu S, Udono H, Srivastava PK (1997) Heat shock protein-peptide complexes, reconstituted in vitro, elicit peptide-specific cytotoxic T lymphocyte response and tumor immunity. J Exp Med 186:1315–1323

    CAS  PubMed  PubMed Central  Google Scholar 

  • Calderwood SK, Theriault JR, Gong J (2005) Message in a bottle: role of the 70-kDa heat shock protein family in anti-tumor immunity. Eur J Immunol 35:2518–2527

    CAS  PubMed  Google Scholar 

  • Calderwood SK, Mambula SS, Gray PJ Jr (2007) Extracellular heat shock proteins in cell signaling and immunity. Ann NY Acad Sci 1113:28–39

    CAS  PubMed  Google Scholar 

  • Chang HC, Tang YC, Hayer-Hartl M, Hartl FU (2007) Snapshot: molecular chaperones, part I. Cell 128:212

    PubMed  Google Scholar 

  • Chang YW, Sun YJ, Wang C, Hsia CD (2008) Crystal structures of the 70-kDa heat shock protein in domain disjoining conformation. J Biol Chem 283:15502–15511

    CAS  PubMed  PubMed Central  Google Scholar 

  • Cho EK, Hong CB (2004) Molecular cloning and expression pattern analysis of heat shock protein 70 genes from Nicotiana tabacum. J Plant Biol 47:149–159

    CAS  Google Scholar 

  • Chothia C, Lesk AM (1986) The relation between the divergence of sequence and structure in proteins. EMBO J 5:823–826

    CAS  PubMed  PubMed Central  Google Scholar 

  • Donini M, Lico C, Baschieri S, Conti S, Magliani W, Polonelli L, Benvenuto E (2005) Production of an engineered killer peptide in Nicotiana benthamiana using a Potato virus X expression system. Appl Environ Microb 71:6360–6367

    CAS  Google Scholar 

  • Esser C, Alberti S, Hofeld J (2004) Cooperation of molecular chaperones with the ubiquitin/proteasome system. Biochim Biophys Acta 1695:171–188

    CAS  PubMed  Google Scholar 

  • Facciponte JG, MacDonald IJ, Wang XY, Kim H, Manjili MH, Subjeck JR (2005) Heat shock proteins and scavenger receptors: role in adaptive immune responses. Immunol Invest 34:325–342

    CAS  PubMed  Google Scholar 

  • Ginalski K (2006) Comparative modelling for protein structure prediction. Curr Opin Struct Biol 16:172–177

    CAS  PubMed  Google Scholar 

  • Goloubinoff P, De Los Rios P (2007) The mechanism of HSP70 chaperones: (entropic) pulling the models together. Trends Biochem Sci 32:372–380

    CAS  PubMed  Google Scholar 

  • Goodin MM, Zaitlin D, Naidu RA, Lommel SA (2008) Nicotiana benthamiana: its history and future as a model for plant-pathogen interactions. Mol Plant Microbe Interact 21:1015–1026

    CAS  PubMed  Google Scholar 

  • Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-PDBViewer: an environment for comparative protein modelling. Electrophoresis 18:2714–2723

    CAS  PubMed  Google Scholar 

  • Guy B (2007) The perfect mix: recent progress in adjuvant research. Nat Rev Microbiol 5:505–517

    CAS  PubMed  Google Scholar 

  • Hartl FU, Hayer-Hartl M (2009) Converging concepts of protein folding in vitro and in vivo. Nat Struct Mol Biol 16:574–581

    CAS  PubMed  Google Scholar 

  • Heikema A, Agsteribbe E, Wilschut J, Huckriede A (1997) Generation of heat shock protein-based vaccine by intracellular loading of gp96 with antigenic peptides. Immunol Lett 57:69–74

    CAS  PubMed  Google Scholar 

  • Hohfeld J, Cyr DM, Patterson C (2001) From the cradle to the grave: molecular chaperones that may choose between folding and degradation. EMBO Rep 2:885–890

    CAS  PubMed  PubMed Central  Google Scholar 

  • Hooft RW, Vriend G, Sander C, Abola EE (1996) Errors in protein structures. Nature 381:272

    CAS  PubMed  Google Scholar 

  • Javid B, MacAry PA, Lehner PJ (2007) Structure and function: heat shock proteins and adaptive immunity. J Immunol 179:2035–2040

    CAS  PubMed  Google Scholar 

  • Jiang J, Lafer EM, Sousa R (2006) Crystallization of a functionally intact HSC70 chaperone. Acta Crystallogr Sect F Struct Biol Cryst Commun 62:39–43

    CAS  PubMed  Google Scholar 

  • Kumaraguru U, Gouffon CA, Ivey RA, Barry TR, Barry DB (2003) Antigenic peptides complexed to phylogenically diverse HSP70 s induce differential immune responses. Cell Stress Chap 8:134–143

    CAS  Google Scholar 

  • Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Cryst 26:283–291

    CAS  Google Scholar 

  • Li Z (2004) In vitro reconstitution of heat shock protein-peptide complexes for generating peptide-specific vaccines against cancers and infectious diseases. Methods 32:25–28

    CAS  PubMed  Google Scholar 

  • Li Z, Ménoret A, Srivastava P (2002) Roles of heat-shock proteins in antigen presentation and cross-presentation. Curr Op Immunol 14:45–51

    CAS  Google Scholar 

  • Lico C, Mancini C, Italiani P, Betti C, Boraschi D, Benvenuto E, Baschieri S (2009) Plant-produced Potato virus X chimeric particles displaying an influenza virus-derived peptide activate specific CD8+ T cells in mice. Vaccine 27:5069–5076

    CAS  PubMed  Google Scholar 

  • Lombardi R, Circelli P, Villani ME, Buriani G, Nardi L, Coppola V, Bianco L, Benvenuto E, Donini M, Marusic C (2009) High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle virus. BMC Biotech 9:96

    Google Scholar 

  • Lu R, Malcuit I, Moffet P, Ruiz MT, Peart J, Wu AJ, Rathjen JP, Bendahmane A, Day L, Baulcombe DC (2003) High throughput virus-induced gene silencing implicates heat shock protein 90 in plant disease resistance. EMBO J 22:5690–5699

    CAS  PubMed  PubMed Central  Google Scholar 

  • Lüthy R, Bowie JU, Eisenberg D (1992) Assessment of protein models with three-dimensional profiles. Nature 356:83–85

    PubMed  Google Scholar 

  • Ma J, Chikwamba R, Sparrow P, Fischer R, Mahoney R, Twyman R (2005) Plant-derived pharmaceuticals-the road forward. Trends Plant Sci 10:580–585

    CAS  PubMed  Google Scholar 

  • Marusic C, Rizza P, Lattanzi L, Mancini C, Spada M, Belardelli F, Benvenuto E, Capone I (2001) Chimeric plant virus particles as immunogens for inducing murine and human immune responses against human immunodeficiency virus type 1. J Virol 75:8434–8449

    CAS  PubMed  PubMed Central  Google Scholar 

  • Marusic C, Nuttall J, Buriani G, Lico C, Lombardi R, Baschieri S, Benvenuto E, Frigerio L (2007) Expression, intracellular targeting and purification of HIV Nef variants in tobacco cells. BMC Biotechnol 7:12

    PubMed  PubMed Central  Google Scholar 

  • Mayer MP, Bukau B (2005) HSP70 chaperones: cellular functions and molecular mechanisms. Cell Mol Life Sci 62:670–684

    CAS  PubMed  PubMed Central  Google Scholar 

  • Ménoret A (2004) Purification of recombinant and endogenous HSP70 s. Methods 32:7–12

    PubMed  Google Scholar 

  • Morshauser RC, Wang H, Flynn GC, Zuiderweg ER (1995) The peptide-binding domain of the chaperone protein HSC70 has an unusual secondary structure topology. Biochemistry 34:6261–6266

    CAS  PubMed  Google Scholar 

  • Morshauser RC, Hu W, Wang H, Pang Y, Flynn GC, Zuiderweg ER (1999) High-resolution solution structure of the 18 kDa substrate-binding domain of the mammalian chaperone protein HSC70. J Mol Biol 289:1387–1403

    CAS  PubMed  Google Scholar 

  • Roman E, Moreno C (1996) Synthetic peptides non-covalently bound to bacterial hsp 70 elicit peptide-specific T-cell responses in vivo. Immunology 88:487–492

    CAS  PubMed  PubMed Central  Google Scholar 

  • Rutherford SL (2003) Between genotype and phenotype: protein chaperones and evolvability. Nat Rev Genet 4:263–274

    CAS  PubMed  Google Scholar 

  • Sette A, Fikes J (2003) Epitope-based vaccines: an update on epitope identification, vaccine design and delivery. Curr Opin Immunol 15:461–470

    CAS  PubMed  Google Scholar 

  • Singh-Jasuja H, Hilf N, Arnol-Schild D, Schild H (2001) The role of heat shock proteins and their receptors in the activation of the immune system. Biol Chem 382:629–636

    CAS  PubMed  Google Scholar 

  • Sparkes IA, Runions J, Kearns A, Hawes C (2006) Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Protoc 1:2019–2025

    CAS  Google Scholar 

  • Srivastava P (2002) Roles of heat-shock proteins in innate and adaptive immunity. Nat Rev Immunol 2:185–194

    CAS  PubMed  Google Scholar 

  • Streatfield SJ (2007) Approaches to achieve high-level heterologous protein production in plants. Plant Biotech J 5:2–15

    CAS  Google Scholar 

  • Swain JF, Schulz EG, Gierasch LM (2006) Direct comparison of a stable isolated HSP70 substrate binding domain in the empty and substrate-bound states. J Biol Chem 281:1605–1611

    CAS  PubMed  Google Scholar 

  • Tague BW, Mantis J (2006) In plantaAgrobacterium-mediated transformation by vacuum infiltration. Methods Mol Biol 323:215–223

    PubMed  Google Scholar 

  • Tang YC, Chang HC, Hayer-Hartl M, Hartl FU (2007) Snapshot: molecular chaperones, part II. Cell 128:412

    CAS  PubMed  Google Scholar 

  • Udono H, Srivastava PK (1994) Comparison of tumor-specific immunogenicities of stress-induced proteins gp96, hsp90, and hsp70. J Immunol 152:5398–5403

    CAS  PubMed  Google Scholar 

  • Wickner S, Maurizi MR, Gottesman S (1999) Posttranslational quality control: folding, refolding, and degrading proteins. Science 286:1888–1893

    CAS  PubMed  Google Scholar 

  • Worrall LJ, Walkinshaw MD (2007) Crystal structure of the C-terminal three-helix bundle subdomain of C. elegans HSP70. Biochem Biophys Res Commun 357:105–110

    CAS  PubMed  Google Scholar 

  • Wu X, Yano M, Washida H, Kido H (2004) The second metal-binding site of 70 kDa heat-shock protein is essential for ADP binding, ATP hydrolysis and ATP synthesis. Biochem J 378:793–799

    CAS  PubMed  PubMed Central  Google Scholar 

  • Zhu X, Zhao X, Burkholder WF, Gragerov A, Ogata CM, Gottesman ME, Hendrickson WA (1996) Structural analysis of substrate binding by the molecular chaperone DnaK. Science 72:1606–1614

    Google Scholar 

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Acknowledgements

We thank Linda Bianco and Gaetano Perrotta for mass spectrometry analysis and Veronica Morea, and Chiara Lico for critical reading of the manuscript. The work was partially supported by a Grant of the Italian Foreign Affairs Ministry in the framework of Bilateral Projects of Great Relevance.

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  1. Technical Unit Radiation Biology and Human Health, Biotechnologies Laboratory, ENEA C.R. Casaccia, Via Anguillarese 301, 00123, Rome, Italy

    Giampaolo Buriani, Camillo Mancini, Eugenio Benvenuto & Selene Baschieri

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  1. Giampaolo Buriani
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  2. Camillo Mancini
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  3. Eugenio Benvenuto
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  4. Selene Baschieri
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Correspondence to Selene Baschieri.

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Buriani, G., Mancini, C., Benvenuto, E. et al. Plant heat shock protein 70 as carrier for immunization against a plant-expressed reporter antigen. Transgenic Res 20, 331–344 (2011). https://doi.org/10.1007/s11248-010-9418-1

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  • DOI: https://doi.org/10.1007/s11248-010-9418-1

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