"
Team:Washington/Celiacs
From 2011.igem.org
| Line 3: | Line 3: | ||
Celiac disease is a genetic autoimmune disorder of the small intestine. For the 2 million people in the United States with celiac disease, the presence of gliadin and similar prolamines (parts of gluten) in the small intestine causes an inappropriate T cell-mediated response against ingested gluten. This results in intestinal damage, most notably inflammation, the loss of absorptive villi, and hyperplasia of the crypts. This damage to the small intestine causes malabsorption of nutrients due to the loss of villi. More visible symptoms could include diarrhea, fatigue, and weight loss. This autoimmune reaction is caused by a failure of the tight-junction system between the intestine and the immune system; a genetic defect could let undigested, immune-system-sensitive gluten proteins create a reinforcing immune response. | Celiac disease is a genetic autoimmune disorder of the small intestine. For the 2 million people in the United States with celiac disease, the presence of gliadin and similar prolamines (parts of gluten) in the small intestine causes an inappropriate T cell-mediated response against ingested gluten. This results in intestinal damage, most notably inflammation, the loss of absorptive villi, and hyperplasia of the crypts. This damage to the small intestine causes malabsorption of nutrients due to the loss of villi. More visible symptoms could include diarrhea, fatigue, and weight loss. This autoimmune reaction is caused by a failure of the tight-junction system between the intestine and the immune system; a genetic defect could let undigested, immune-system-sensitive gluten proteins create a reinforcing immune response. | ||
| - | Currently, the only known therapy for celiac disease is the complete avoidance of gluten-containing foods. However, research on this disorder has found enzymes capable of breaking apart gluten using prolyl endopeptidases | + | Currently, the only known therapy for celiac disease is the complete avoidance of gluten-containing foods. However, research on this disorder has found enzymes capable of breaking apart gluten using prolyl endopeptidases, which are able to cleave at internal proline residues in proteolytically resistant gluten epitopes. Unfortunately, such enzymes only produce catalytic activity at relatively neutral pH, like that of the small intestine; this would allow gluten to simultaneously react with the immune system, and could still trigger an inflammatory response. An optimal protease would be able to conduct its activity at low pH in the stomach to prevent the opportunity for inflammation. |
| - | Thus, we identified a serine-carboxyl peptidase from the literature, Kumamolisin-As, with promise of gliadin cleaving activity. Isolated from the thermoacidophilic soil bacterium ''Alicyclobacillus sendaiensis'', Kumamolisin-As was characterized as thermally stable up to 70<sup>o</sup>C, an optimal activity at pH 4, and having specificity for proline-arginine epitopes, though about 10% of that catalytic activity was seen when exposed to proline-glutamine epitopes, a common occurrence in gliadin peptides. | + | Thus, we identified a serine-carboxyl peptidase from the literature, Kumamolisin-As, with promise of gliadin cleaving activity. Isolated from the thermoacidophilic soil bacterium ''Alicyclobacillus sendaiensis'', Kumamolisin-As was characterized as thermally stable up to 70<sup>o</sup>C, an optimal activity at pH 4, and having specificity for proline-arginine epitopes, though about 10% of that catalytic activity was seen when exposed to proline-glutamine epitopes, a common occurrence in gliadin peptides. Using computational methods, we designed, built, and tested over 100 mutants of Kumamolisin-As and achieved a 30-fold increase in specificity for the PQLP epitope. |
Latest revision as of 06:00, 4 September 2011
Celiac disease is a genetic autoimmune disorder of the small intestine. For the 2 million people in the United States with celiac disease, the presence of gliadin and similar prolamines (parts of gluten) in the small intestine causes an inappropriate T cell-mediated response against ingested gluten. This results in intestinal damage, most notably inflammation, the loss of absorptive villi, and hyperplasia of the crypts. This damage to the small intestine causes malabsorption of nutrients due to the loss of villi. More visible symptoms could include diarrhea, fatigue, and weight loss. This autoimmune reaction is caused by a failure of the tight-junction system between the intestine and the immune system; a genetic defect could let undigested, immune-system-sensitive gluten proteins create a reinforcing immune response.
Currently, the only known therapy for celiac disease is the complete avoidance of gluten-containing foods. However, research on this disorder has found enzymes capable of breaking apart gluten using prolyl endopeptidases, which are able to cleave at internal proline residues in proteolytically resistant gluten epitopes. Unfortunately, such enzymes only produce catalytic activity at relatively neutral pH, like that of the small intestine; this would allow gluten to simultaneously react with the immune system, and could still trigger an inflammatory response. An optimal protease would be able to conduct its activity at low pH in the stomach to prevent the opportunity for inflammation.
Thus, we identified a serine-carboxyl peptidase from the literature, Kumamolisin-As, with promise of gliadin cleaving activity. Isolated from the thermoacidophilic soil bacterium Alicyclobacillus sendaiensis, Kumamolisin-As was characterized as thermally stable up to 70oC, an optimal activity at pH 4, and having specificity for proline-arginine epitopes, though about 10% of that catalytic activity was seen when exposed to proline-glutamine epitopes, a common occurrence in gliadin peptides. Using computational methods, we designed, built, and tested over 100 mutants of Kumamolisin-As and achieved a 30-fold increase in specificity for the PQLP epitope.
