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Viral and murine interleukin-10 are correctly processed and retain their biological activity when produced in tobacco.

Bortesi L, Rossato M, Schuster F, Raven N, Stadlmann J, Avesani L, Falorni A, Bazzoni F, Bock R, Schillberg S, Pezzotti M - BMC Biotechnol. (2009)

Bottom Line: The best yields using this strategy in T1 plants were 10.8 and 37.0 microg/g fresh leaf weight for viral and murine IL-10, respectively.Tobacco plants are able to correctly process viral and murine IL-10 into biologically active dimers, therefore representing a suitable platform for the production for these cytokines.The accumulation levels obtained are high enough to allow delivery of an immunologically relevant dose of IL-10 in a reasonable amount of leaf material, without extensive purification.

View Article: PubMed Central - HTML - PubMed

Affiliation: Scientific and Technologic Department, University of Verona, Strada Le Grazie 15, Verona, Italy. luisa.bortesi@univr.it

ABSTRACT

Background: Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine, with therapeutic applications in several autoimmune and inflammatory diseases. Oral administration of this cytokine alone, or in combination with disease-associated autoantigens could confer protection form the onset of a specific autoimmune disease through the induction of oral tolerance. Transgenic plants are attractive systems for production of therapeutic proteins because of the ability to do large scale-up at low cost, and the low maintenance requirements. They are highly amenable to oral administration and could become effective delivery systems without extensive protein purification. We investigated the ability of tobacco plants to produce high levels of biologically-active viral and murine IL-10.

Results: Three different subcellular targeting strategies were assessed in transient expression experiments, and stable transgenic tobacco plants were generated with the constructs that yielded the highest accumulation levels by targeting the recombinant proteins to the endoplasmic reticulum. The best yields using this strategy in T1 plants were 10.8 and 37.0 microg/g fresh leaf weight for viral and murine IL-10, respectively. The recombinant proteins were purified from transgenic leaf material and characterized in terms of their N-glycan composition, dimerization and biological activity in in vitro assays. Both molecules formed stable dimers, were able to activate the IL-10 signaling pathway and to induce specific anti-inflammatory responses in mouse J774 macrophage cells.

Conclusion: Tobacco plants are able to correctly process viral and murine IL-10 into biologically active dimers, therefore representing a suitable platform for the production for these cytokines. The accumulation levels obtained are high enough to allow delivery of an immunologically relevant dose of IL-10 in a reasonable amount of leaf material, without extensive purification. This study paves the way to performing feeding studies in mouse models of autoimmune diseases, that will allow the evaluation the immunomodulatory properties and effectiveness of the viral IL-10 in inducing oral tolerance compared to the murine protein.

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Related in: MedlinePlus

Liquid chromatography-mass spectrometry (LC-MS) of the murine IL-10 glycosylated peptide EDNNCTHFPVGQSHMLLELR. Murine IL-10 purified from stable transgenic leaf material was digested with trypsin and the peptides subjected to LC-MS analysis. The spectrum of the glycosylated peptide is shown. See  for an explanation of N-glycan abbreviations.
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Figure 4: Liquid chromatography-mass spectrometry (LC-MS) of the murine IL-10 glycosylated peptide EDNNCTHFPVGQSHMLLELR. Murine IL-10 purified from stable transgenic leaf material was digested with trypsin and the peptides subjected to LC-MS analysis. The spectrum of the glycosylated peptide is shown. See for an explanation of N-glycan abbreviations.

Mentions: The mature mIL-10 polypeptide has two potential N-glycosylation acceptor sites, whereas vIL-10 has a single site. To determine the glycan composition, both plant-derived proteins were digested with trypsin and analyzed by mass spectrometry (MS). Despite the presence of a glycan acceptor site at Asn127, the plant-derived vIL-10 was not glycosylated. In contrast, mIL-10 was glycosylated, but only one of the two potential acceptor sites was used, as is the case for the native protein [20,21]. Unexpectedly for a protein bearing a SEKDEL tag, oligo-mannose-type (OMT) N-glycans accounted for only 46% of the total N-glycan population, with Man7 being the most abundant glycoform (Figure 4, N-glycan abbreviations according to Proglycan [22]). Complex type N-glycans accounted for 44% of the total, with GnGnXF and GnGnX being the most prominent. GnMX was present at lower levels, while the other complex type N-glycans were found in only trace amounts.


Viral and murine interleukin-10 are correctly processed and retain their biological activity when produced in tobacco.

Bortesi L, Rossato M, Schuster F, Raven N, Stadlmann J, Avesani L, Falorni A, Bazzoni F, Bock R, Schillberg S, Pezzotti M - BMC Biotechnol. (2009)

Liquid chromatography-mass spectrometry (LC-MS) of the murine IL-10 glycosylated peptide EDNNCTHFPVGQSHMLLELR. Murine IL-10 purified from stable transgenic leaf material was digested with trypsin and the peptides subjected to LC-MS analysis. The spectrum of the glycosylated peptide is shown. See  for an explanation of N-glycan abbreviations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667500&req=5

Figure 4: Liquid chromatography-mass spectrometry (LC-MS) of the murine IL-10 glycosylated peptide EDNNCTHFPVGQSHMLLELR. Murine IL-10 purified from stable transgenic leaf material was digested with trypsin and the peptides subjected to LC-MS analysis. The spectrum of the glycosylated peptide is shown. See for an explanation of N-glycan abbreviations.
Mentions: The mature mIL-10 polypeptide has two potential N-glycosylation acceptor sites, whereas vIL-10 has a single site. To determine the glycan composition, both plant-derived proteins were digested with trypsin and analyzed by mass spectrometry (MS). Despite the presence of a glycan acceptor site at Asn127, the plant-derived vIL-10 was not glycosylated. In contrast, mIL-10 was glycosylated, but only one of the two potential acceptor sites was used, as is the case for the native protein [20,21]. Unexpectedly for a protein bearing a SEKDEL tag, oligo-mannose-type (OMT) N-glycans accounted for only 46% of the total N-glycan population, with Man7 being the most abundant glycoform (Figure 4, N-glycan abbreviations according to Proglycan [22]). Complex type N-glycans accounted for 44% of the total, with GnGnXF and GnGnX being the most prominent. GnMX was present at lower levels, while the other complex type N-glycans were found in only trace amounts.

Bottom Line: The best yields using this strategy in T1 plants were 10.8 and 37.0 microg/g fresh leaf weight for viral and murine IL-10, respectively.Tobacco plants are able to correctly process viral and murine IL-10 into biologically active dimers, therefore representing a suitable platform for the production for these cytokines.The accumulation levels obtained are high enough to allow delivery of an immunologically relevant dose of IL-10 in a reasonable amount of leaf material, without extensive purification.

View Article: PubMed Central - HTML - PubMed

Affiliation: Scientific and Technologic Department, University of Verona, Strada Le Grazie 15, Verona, Italy. luisa.bortesi@univr.it

ABSTRACT

Background: Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine, with therapeutic applications in several autoimmune and inflammatory diseases. Oral administration of this cytokine alone, or in combination with disease-associated autoantigens could confer protection form the onset of a specific autoimmune disease through the induction of oral tolerance. Transgenic plants are attractive systems for production of therapeutic proteins because of the ability to do large scale-up at low cost, and the low maintenance requirements. They are highly amenable to oral administration and could become effective delivery systems without extensive protein purification. We investigated the ability of tobacco plants to produce high levels of biologically-active viral and murine IL-10.

Results: Three different subcellular targeting strategies were assessed in transient expression experiments, and stable transgenic tobacco plants were generated with the constructs that yielded the highest accumulation levels by targeting the recombinant proteins to the endoplasmic reticulum. The best yields using this strategy in T1 plants were 10.8 and 37.0 microg/g fresh leaf weight for viral and murine IL-10, respectively. The recombinant proteins were purified from transgenic leaf material and characterized in terms of their N-glycan composition, dimerization and biological activity in in vitro assays. Both molecules formed stable dimers, were able to activate the IL-10 signaling pathway and to induce specific anti-inflammatory responses in mouse J774 macrophage cells.

Conclusion: Tobacco plants are able to correctly process viral and murine IL-10 into biologically active dimers, therefore representing a suitable platform for the production for these cytokines. The accumulation levels obtained are high enough to allow delivery of an immunologically relevant dose of IL-10 in a reasonable amount of leaf material, without extensive purification. This study paves the way to performing feeding studies in mouse models of autoimmune diseases, that will allow the evaluation the immunomodulatory properties and effectiveness of the viral IL-10 in inducing oral tolerance compared to the murine protein.

Show MeSH
Related in: MedlinePlus