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Department of Molecular Biology and Microbiology, University of Central Florida, Biomolecular Science, Orlando, Florida, USA
2Correspondence: University of Central Florida, 4000 Central Florida Blvd., Department of Molecular Biology and Microbiology, Biomolecular Science, Bldg. #20, Rm. 336, Orlando FL 32816-2364, USA. E-mail: daniell{at}mail.ucf.edu
SPECIFIC AIMS
The major limitation in the efficient delivery of plant-expressed therapeutic proteins across the intestinal epithelial layer is their permeability. Receptor-mediated oral delivery might serve as a possible route to deliver vaccine antigens and biopharmaceutical proteins. To test the concept of receptor-mediated oral delivery of foreign proteins, we have fed mice with leaves expressing cholera toxin B-green fluorescent protein (CTB-GFP) fusion protein with a furin cleavage site between CTB and GFP and elucidated the path of CTB and GFP in the circulatory system.
PRINCIPAL FINDINGS
1. Generation of transgenic lines expressing CTB-GFP
Nicotiana tabacum cv. Petit Havana leaves were bombarded with the pLD-CTB-GFP vector, and the leaves were grown on selection medium containing 500 mg/l spectinomycin. The resultant shoots were then screened for chloroplast transformants by polymerase chain reaction and Southern analysis. Western blot analysis was performed to investigate the expression of the CTB-GFP fusion protein in transgenic tobacco chloroplasts. The amount of CTB-GFP in the transgenic plants ranged from 19.09 to 21.3% total soluble protein as determined by ELISA. GM1 binding assay showed that pentamers of CTB-GFP were formed. This finding confirms the correct folding and disulfide bond formation of CTB pentamers within transgenic chloroplasts because only the pentameric form of CTB can bind to GM1.
2. Fluorescent microscopy to detect the presence of GFP in tissues
In mice fed with CTB-GFP expressing plant leaf material, fluorescence microscopy and anti-GFP antibodies showed the presence of GFP in intestinal mucosa and submucosa, the hepatocytes of the liver, as well as various cells of the spleen. In the mice fed with wild-type (untransformed) leaf material or green fluorescent protein-interferon fusion protein (IFN-GFP) expressing plant leaf material, no GFP fluorescence was observed. Detection of GFP in the liver and spleen, following oral delivery of CTB-GFP expressing plant leaf material, confirms the successful delivery of the foreign protein across the intestinal lumen into the systemic circulation. Moreover, the lack of detection of significant amount of GFP in the liver and spleen of mice fed with IFN-GFP expressing plants suggests that a transmucosal carrier such as CTB is required for delivery of adequate amount of a foreign protein across the intestinal lumen into the systemic circulation.
3. Immunohistochemistry
To confirm the fluorescent microscopy findings, immunostaining was performed with both CTB and GFP antibodies. In the intestine of the mice fed with CTB-GFP, anti-GFP antibody (Ab) detected GFP inside the epithelial cells of the villi of the intestine, in the crypts, as well as in the submucosal tissue (Fig. 1
A, C), suggesting GFP uptake by lymphoid cells as well as the circulation. These results confirmed the previous microscopy findings and showed the presence of GFP in various tissues, establishing that GFP was successfully delivered to blood when transgenic leaf material was orally fed to mice. GFP immunoreactivity was detected in the liver and spleen (Fig. 1E and H
) in a similar pattern to that seen with fluorescence microscopy. In the case of the mice fed with wild-type leaf material, no GFP was detected in any of the tissues (Fig. 1F, I
). In the mice fed with plants expressing IFN-GFP, GFP was not detected in the liver or spleen cells (Fig. 1G, J
).
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To study the route of CTB in the body, we performed immunohistochemistry using anti-CTB antibodies, which showed that CTB was detected in the intestinal cells as well as inside the villi (Fig. 6A in full-length article online) in the lamina propia and the submucosa. CTB was not detected in the liver (Fig. 6E in full-length article online), indicating that GFP is cleaved away from CTB. An in vitro furin cleavage assay was performed on the CTB-GFP expressing plant extract to show that the engineered cleavage site (Arg-Ala-Arg-Arg) was recognized by furin. A 26 kDa polypeptide that corresponded with the recombinant GFP protein was observed in the samples that were incubated with furin. While GFP leaves the cell, CTB probably is translocated to the basolateral membrane of the cell.
To localize the GFP and/or CTB in the gut associated lymphoid tissue (GALT) and other tissues, double staining for antigen presenting cells such as macrophages or dendritic cells was performed. A double staining with F4/80 Ab for macrophages showed the presence of CTB inside macrophages (Fig. 6C in full-length article online). Fig. 6G in full-length online article shows macrophages associated with GFP; Fig. 6I in full-length article online shows dendritic cells taking up the GFP. In either case, there are associations of GFP with these antigen-presenting cells. Most of the macrophages were not associated with GFP, which is perhaps due to uptake by the blood and lymph circulation, while the CTB is translocated to the basolateral membrane and is associated with macrophages.
CONCLUSIONS AND SIGNIFICANCE
In this study, detection of GFP and CTB in the intestinal mucosa (Fig. 1 and Fig 6 in full-length article online) suggests that CTB–GFP has been taken up by the enterocytes and the gut-associated lymphoid tissue (GALT). The CTB domain of the CTB-GFP forms the pentameric structure within chloroplasts through disulfide bond formation; pentameric form binds to the GM1 receptors on enterocytes and is endocytosed into the intestinal cells as endosomes. After endocytosis, the CTB-GM1 complex trafficking occurs retrogradely through Golgi cisternae and/or trans-Golgi network (TGN) into the lumen of the endoplasmic reticulum (ER). The GM1-CTB-GFP complex in the lipid rafts, targeted to the TGN, loses its endosomal covering. Abundant experimental evidence indicates the cleavage of GFP from CTB by furin, either within the endosome or in the TGN. The CTB is taken into the ER and from there to the baso-lateral surface of the cell (transcytosis), where it remains membrane-bound to the GM1 receptor. The GFP molecule getting out of the TGN (presumably membrane bound) is exocytosed through the basolateral membrane and finds its way into extracellular fluid and into the submucosal vessels, including the lymphatic system. Besides the entry of CTB-GFP through the GM1 receptor, the M cells in intestinal epithelium covering the GALT in the digestive tract also serve as a port of entry of macromolecules and microorganisms by pinocytosis. Therefore, a small amount of CTB-GFP could be taken up by the GALT. This is shown in our study by the presence of CTB and GFP in the antigen-presenting cells, including the macrophages as well as the dendritic cells in the intestinal lamina propia and submucosa. Similarly, the small amount of GFP associated with macrophages in the intestine of the IFN-GFP fed mice is likely to be taken up by the M cells. The IFN-GFP fusion protein also contains a furin cleavage site but, due to limited uptake by the intestinal epithelial cells, there is no significant GFP transport to the tissues of the IFN-GFP fed mice. The amount of CTB-GFP reaching the enterocytes via GM1 receptor is very high compared with the entry of IFN-GFP through M cells. This condition is quite evident due to the GFP detected in various organs of the CTB-GFP fed mice. Presence of GFP and not CTB in the liver of CTB-GFP treated mice in our study suggests the cleavage of the CTB-GFP fusion protein in enterocytes and uptake of GFP into the vasculature of the lamina propia and the submucosa. CTB however, might be translocated to the basolateral cell membrane and remain bound to GM1.
One of the most challenging problems of human health management is the high cost of prescription drugs in developed countries and their lack of availability in developing countries. Such high cost of therapeutic proteins can be attributed to their production in fermentation-based systems, expensive purification and processing methods, low temperature storage, transportation, and sterile delivery using syringes through health professionals. Most of these expenses could be avoided by expressing therapeutic proteins in plant cells and through their oral delivery. This study shows internalization of CTB-GFP by the mouse intestinal mucosal cells as well as the antigen-presenting cells in the intestinal mucosa and submucosa. We also show the presence of GFP but not CTB in the liver of mice following oral delivery of CTB-GFP leaf material. Several vaccine antigens and human blood proteins have been expressed in transgenic chloroplasts and shown to be fully functional. The ability to express high levels of foreign proteins in plastids present within edible plant parts and the rapid turnover of intestinal epithelial cells for recycling GM1 receptors make this approach a reality. This study opens the door for low-cost production and delivery of human therapeutic proteins.
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FOOTNOTES
1 These authors contributed equally to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5134fje
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