Celiac disease is a disorder of the small intestine caused by an improper immune response to wheat gluten and related proteins of barley and rye. Gluten is rich in proline and glutamine residues resistant to proteases and peptidases of the gut. Currently the only available treatment is a strict diet that does not have gluten, hence the need for alternative treatments. The latest advances have enhanced the understanding of the molecular cause of this disorder and there are several attractive targets for new treatments. Oral enzyme supplementation is intended to speed up gastrointestinal degradation of proline-rich gluten, especially its proteolytically stable antigenic peptides. Complementary strategies aiming to hinder with activation of gluten-reactive T cells include the inhibition of intestinal tissue transglutaminase activity to prevent selective deamidation of gluten peptides, and blocking the binding of gluten peptides to the HLA-DQ2 or HLA-DQ8 molecules. Other possible treatments include cytokine therapy, and selective adhesion molecule inhibitors that interfere within flammatory reactions, some of which are already showing promise in the clinic for other gastrointestinal diseases. Prolyl endopeptidases (PEP) have potential for treating coeliac sprue .Prolyl endopeptidases, are a family of serine proteases with the unique ability to hydrolyse the peptide bond on the carboxyl side of a proline residue. These proline specific enzymes are widely distributed in bacteria, fungi, animals and plants .
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The potential to use PEPs as a cure for celiac disease is attractive because their specificity complements gastrointestinal proteolytic processes. Not only does all PEP-catalyzed cleavages generate one new amino and carboxyl terminus, but it also truncates the long-peptide end products of gastric and duodenal gluten metabolism. Both of these outcomes give residual smaller peptides that are suitable substrates for the intestinal brush-border aminopeptidases and carboxypeptidases. Microorganisms and plants including cereals such as maize and rice express various PEPs (Piper et al., 2004). There is also a human PEP, but this is expressed only in the cytosol and is therefore unlikely to have a physiological role in the degradation of gluten peptides. It has been proposed that oral administration of a therapeutic dose of a suitably formulated PEP might counter the toxic effects of moderate quantities of ingested gluten, This hypothesis is supported by extensive in vitro, in vivo (in rats) and ex vivo (using biopsy-derived T cells)studies on synthetic gluten peptides, recombinant gliadin molecules and whole gluten as obtained in a grocery store. In each case, dose dependent breakdown of immunotoxic gluten peptides by a Flavobacterium meningosepticum PEP was observed. This proteolysis action was also reflected in a concomitant reduction of immunogenicity, as judged by reduced stimulation of most polyclonal T-cell lines from patients with celiac disease. Together, these results set the stage for controlled studies in patients with celiac disease.( Marti et al.,2005)
Although the adaptive immune response is often considered to be the primary trigger of the flattened mucosa that characterizes Celiac Spree other hypotheses of Celiac Sprue pathogenesis have been presented .Regardless of the primary mechanism, the essential prerequisite is the initiating role of the digestive-resistant intact gliadin peptide fragments, which must persist in the intestinal lumen to have deleterious effects. Previous in vitro studies have suggested digestive resistance of gliadin in Celiac Sprue patients. More recent studies have demonstrated that a region of gliadin harboring tandem repeat sequences of PQPQLPY is resistant to digestion by gastric, pancreatic, and intestinal brush-border peptidases, and harbors multiple copies of highly immunogenic epitopes (Hausch et al., 2002; ) To be therapeutically useful, a PEP enzyme must be capable of proteolyzing whole gluten, a complex protein mixture that consists of hundreds of distinct, but related, gliadin and glutenin polypeptides. Gliadin is the alcohol soluble fraction of wheat gluten and the storage protein of wheat endosperm rich. It is especially rich in glutamine and proline content .( Marti et al.,2005)
Monomeric gliadins are divided based upon sequence into three classes:a, g, and o. Successive digestion of food-grade gluten with pepsin, pancreatic proteases, and FMPEP greatly diminishes its potential for inducing the proliferation of patient-derived inflammatory T cells and also avoids the onset of gluten-induced malabsorption in Celiac Sprue patients However, the challenge remains of developing a suitable delivery mechanism for a PEP enzyme into the small intestine where it can work in concert with the pancreatic proteases. Specifically, the PEP must be protected from the acidic and proteolytic environment of the stomach, and then released rapidly as gluten containing food is emptied into the duodenum, neutralized and mixed with pancreatic enzyme secretions.. In conjunction with pancreatic enzymes (Yamamoto et al 1991) , MX PEP breaks down whole gluten into a product mixture that is virtually indistinguishable from that generated by the FM PEP as judged by chromatographic assays. Our results support the evaluation of this PEP capsule as an oral gluten digesting supplement, first in an appropriate animal model and subsequently in healthy adult volunteers.Earlier studies evaluating the ability of PEP to detoxify gluten were conducted with the FM enzyme 2004;( Shan et al., 2002, 2004). Notwithstanding its efficacy, the low fermentation yield and consequent high cost of FM PEP precludes further clinical development. In contrast, as summarized above, the MX PEP can be produced more economically and to a higher degree of purity. Therefore, a detailed comparison of the activities of two PEPs against whole gluten was warranted. ( Marti et al.,2005).
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Celiac disease has a prevalence of 1% in adults and is caused by the principle protein in staple foods such as wheat , barley and rye. The resulting intestinal inflammation often causes symptoms related to malabsorption, such as diarrhoea and failure to thrive in infants. At present the only available cure is a gluten exclusion diet but recently research has been done using PEP from Myxococcus Xanthus and Flavobacterium meningosepticum and these have been able to cleave gluten peptides in vitro to give short short oligopeptdes that do not elicit an inflammatory response .Various other plants such as the cereals maize and rice also contain similar forms of PEP ,taking oral supplementation of such forms of PEP would speed up gastrointestinal degradation of proline-rich gluten
To Express Prolyl Endopeptidase from cereals
1.To make multiple copies of Gene for Propyl Endopeptidase from cereals
2.To insert the gene into a plasmid
3.To transform plasmid into E.coli
4 Heterogeneous Expression of PEP
The only present treatment for colic spree is a gluten exclusion diet but cereal plants have the potential to produce therapeutically viable form of PEP
The fully developed leaves of these plants will be harvested and total RNA will be extracted using SV Total RNA Isolation Kit from Promega, following the instructions. See appendix A
Preparation of competent cells
A culture of commercial JM109 cells will be prepared to test for their cell competency as outlined in appendix B
Gene amplification will be achieved through the polymerase chain reaction and the presence of the plasmid will be confirmed by running a 0.8% TAE agarose gel.
Polymerase chain reaction
RT-PCR will be performed using the total RNA extracted from the ground tissues in a 50μl RT-PCR reaction volume according to the protocols described in Promega RT-PCR kit. 0.8% agarose gel will be prepared and amplification will be confirmed on it.
Ligation and Transformation
Complimentary DNA will be ligated to T/A vector as described by QIAGEN PCR cloning kit.
The recombinant vector will be transformed to E.coli cells following the protocols that are described in the handbook.
Colony, Screening and Sequencing
Transformed insert will be inoculated on /LB/AMP plates and transformed colonies will be used for Miniprep. The E.coli cells were harvested and lysed to release the cDNA using Plasmid DNA Purification kit (Macherey-Nagel) according to alkaline lysis Mini-Prep protocol. See appendix C. Colony PCR will be carried out and amplicons will be run on 0.8% agarose gel for gene confirmation. Sequencing of the cDNA will be carried out at the Central Analytical Facilities at Stellenboch University, using ABI PrismTM BigDyeTM Terminator cycle sequencing Ready Reaction Kit (PE Applied Biosystems).
For heterogonous expression of PEP, a plasmid containing the PEP will be used for inhibitory activity of API In vitro. A fluorescent labeled substrate assay will be used to measure the proteolytic activity of PEP and the inhibition activity of the expressed API's.
Shan L Lu SHAN, Thomas Marti, Ludvig M. SOLLID, Gary M. GRAY and Chaitan KHOSLA . (2004) Comparative biochemical analysis of three bacterial prolyl endopeptidases: implications for coeliac sprue. Biochem J 383: 311-318
Marti T, Molberg O, Li Q, Gray GM, Khosla C, Sollid LM. 2005. Prolyl endopeptidase mediated destruction of T cell epitopes in whole gluten: Chemical and immunological characterization. J Pharmacol Exp Ther 312(1):19-26.
Hausch F, Shan L, Santiago NA, Gray GM, Khosla C. 2002. Intestinal digestive resistance of immune dominant gliadin peptides. AmJ Physiol Gastrointestinal Liver Physiol 283(4):G996-G1003
Yoshimoto, T., Kanatani, A., Shimoda, T., Inaoka, T., Kokubo, T. and Tsuru, D. (1991) Prolyl endopeptidase from Flavobacterium meningosepticum: cloning and sequencing of the enzyme gene. J. Biochem. (Tokyo) 110, 873-878
Piper JL, Gray GM, Khosla C. 2004. Effect of prolyl endopeptidase on digestive-resistant gliadin peptides in vivo. J Pharmacol Exp Ther
Ahnen DJ, Santiago NA, Cezard JP, and Gray GM (1982) Intestinal aminooligopeptidase. In vivo synthesis on intracellular membranes of rat jejunum. J Biol Chem
Sollid LM. 2002. Coeliac disease: Dissecting a complex inflammatory disorder. Nat Rev Immunol 2(9):647-655.
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Pyle GG, Paaso B, Anderson BE, Marti T, Li Q, Siegel M, Khosla C, Gray GM. 2005b. Pre-treatment of food gluten with prolyl endopeptidase
(PEP) avoids gluten-induced malabsorption in Celiac Sprue. Clin Gastroenterol Hepatol :687-694.
Yamamoto, Y., Watabe, S., Kageyama, T., Takahashi, S.Y., 1999. Proregion of Bombyx mori cysteine proteinase functions as an intramolecular chaperone to promote proper folding of the mature enzyme. Arch. Insect Biochem. Physiol. 42, 167-178.
SV Total RNA Isolation Protocol
175µL of RNA Lyses Buffer was placed in an autoclaved tube. The sample to be lysed was prepared and immediately placed into the lyses buffer and mixed thoroughly by inversion. 350µL of SV RNA dilution buffer was added to the 175µL lysate and mixed by inversion. This was then placed in a water bath or heat block at 70 degrees Celsius for 3 minutes then centrifuged for 10 minutes. The clear lysate was then transferred to a fresh microcentrifuge tube. 200µL of 95% ethanol was then added to the clear lysate and mixed well. The mixture was then transferred to the spin basket assembly and centrifuged for 1 minute. The eluate was discarded and the spin basket was put back into the column. 600µL of RNA wash solution was then added to the spin column assembly and then centrifuged for 1 minute. The collection tube was then emptied as before and set in a rack. DNase incubation mix was prepared by combining 40µL Yellow Core Buffer, 5µL 0.09M MnCl2 and 5µL of DNase 1 enzyme per sample in a sterile tube. 50µL of the DNase incubation mix was then directly applied to the membrane inside the spin basket and incubated for 15 minutes at room temperature. 200µL of DNase stop solution was added to the spin basket and centrifuged for 1 minute. 600µL of RNA Wash Solution was added and centrifuged for 1 minute. Thereafter, the collection tube was emptied and 250µL RNA Wash Solution was added and centrifuged for 2 minutes. The spin basket was then transferred to an elution tube and 100µL Nuclease-Free Water was added to the membrane. The spin basket assemblies were placed in the centrifuge with the lids of elution tubes facing out and centrifuged for 1 minute. The spin basket was then removed and discarded whilst the elution tube was capped containing the purified RNA and stored at -70 degrees Celsius.
Preparation of competent JM109 E.coli cell cultures
A starter culture was prepared by inoculating a 2µL aliquot of commercially prepared cells into 5mL of LB medium and grown for 4-6 hours at 37 degrees Celsius with shaking. Two millimeters of the starter culture was inoculated into 200mL of LB medium and grown at 37 degrees Celsius with shaking to an OD600 of approximately 0.4.
Following this, this culture was then placed on ice and the cells were harvested by centrifugation for 15min at 2500-g, 4 degrees Celsius. After centrifugation, the supernatant was discarded carefully and the cell pellet re-suspended gently in 100mL of sterile, ice cold 100mM CaCl2. The re-suspended pellet was centrifuged again for 15min at 2500-g, 4 degrees Celsius and the supernatant discarded carefully. The pellet was gently re-suspended in 50mL of sterile, ice cold 100mM CaCl2 and centrifuged for 15min at 2500-g, 4 degrees Celsius. Following centrifugation, the cell pellet was gently re-suspended in 5mL of sterile; ice cold 100Mm CaCl2, 20% glycerol and 100µL of the competent cells aliquoted into pre-chilled, sterile polypropylene tubes. The competent cells were then frozen in liquid nitrogen and immediately stored at -80 degrees Celsius.
Mini-prep plasmid extraction
600µL of bacterial culture was added to a 1.5mL microcentrifuge tube. 100µL of cell lyses buffer was added and mixed by inversion. After 2 minutes, 350µL of cold neutralization solution was added and again mixed by inversion. This was then centrifuged at maximum speed for 3 minutes. The supernatant was then transferred to a minicolumn without disturbing the cell debris pellet. The minicolumn was placed into a collecting tube and centrifuged at maximum speed in a microcentrifuge for 15 seconds. The flow through was discarded and the minicolumn was placed into the same collecting tube. 200µL of endotoxin removal wash added to the minicolumn. This was then centrifuged at maximum speed for 15 seconds. The flow through was discarded and 400µL of column wash solution was added to the minicolumn and again, centrifuged at maximum for 30 seconds. The minicolumn was then transferred to a clean 1.5mL microcentrifuged tube and 30µL of elution buffer or nuclease free-water was added directly onto the minicolumn matrix. This was allowed to stand for 1 minute at room temperature before it was centrifuged for 15 seconds to elude the plasmid DNA. The microcentrifuge tube was then capped and the eluted plasmid DNA was stored at -20 degrees Celsius.