Written by: Nhat Vo
Edited by: Jonathan Keller, Benjamin Eide
(Photo Credit: Jason Lee)
Celiac disease is a common auto-immune disease among Americans. Recent studies suggest there are more than 3 million Americans with Celiac disease. The disease mostly affects people of Northern European descent, but it also occurs in African Americans, Hispanic, and Asian populations. If left untreated, celiac disease patients may develop intestinal inflammation, and their risk of having gastrointestinal cancer is 40 to 100 times higher than the normal population.
Celiac disease patients are gluten intolerant. Gluten, a protein found in wheat, barley, and rye products (Figure 1), is a protein composite of gliadin and glutelin. In gliadin, the amino acids proline and glutamine form a proline-glutamine (P-Q) peptide bond. The P-Q bond protects gluten from being broken down by enzymes in the stomach of people with Celiac disease. The presence of undegraded gluten causes an immune response in the digestive tract, resulting in inflammation. Celiac disease presents a broad range of symptoms, from mild weakness and bone pain, to chronic diarrhea, abdominal bloating, and progressive weight loss.
Much research has been done, but currently the best treatment for celiac disease patients is a gluten-free diet. This poses various problems as gluten-free foods are harder to find and more expensive than gluten-containing products. Some studies have shown that a gluten-free diet can lead to a lower health quality of life.
Many proteins capable of breaking P-Q bonds and degrading gluten, such as prolyl endopeptidase (PEP), have been discovered. But there comes a problem: the majority of those proteins do not function efficiently in the stomach, where gluten needs to be broken down. The acidic pH levels and harsh temperature in the stomach inactivate those proteins (denaturation). Fortunately, in 2011, a group of University of Washington students successfully created a mutant strain of bacteria that produces a type of enzymes that could break down gliadin under gastric conditions. That enzyme was Kumamolisin-As. This discovery led them to win the iGEM 2011 competition and marked a huge milestone in gluten-intolerant genetic therapies.
Kumamolisin-As is an enzyme isolated from a strain of thermoacidophilic bacterium Alicyclobacillus sendaiensis (A. sendaiensis). Thermoacidophilic means “to be in favor of extreme temperatures and acidic environments.” A. sendaiensis is a rod-shaped, spore-forming bacterium that can live in a wide range of temperature (20 – 70 oC) and under acidic environments (pH levels 2.5 – 5). The bacterium is usually found in hot springs. An enzyme of A. sendaiensis, Kumamolisin-As, exhibits high enzymatic activities under pH levels from 2.5 to 5 and 20 – 70 oC temperature. Enzymes able to function normally at these temperatures are rare. One of the primary reasons Kumamolisin-As works so well is that the enzyme has an unusual catalytic triad. A catalytic triad refers to the three amino acid residues found inside of the active site of certain protein-degrading enzymes. A normal triad, such as one found in PEP, is composed of serine, histidine, and aspartate. They are positioned as in figure 3. This normal positioning prevents PEP from degrading proteins at low pH levels. Meanwhile, Kumamolisin-As’ triad includes serine, glutamine, and aspirate. Glutamine replaces histidine in the triad, and the three amino acids are set up in an unusual way that makes the enzyme well suited to a low pH environment. The protein’s maximum activity occurs at 37 oC and 4.0 pH level.
Students of University of Washington then ran mutagenesis (a process of creating mutations) to produce various Kumamolisin-As mutants. Then they tested the mutants’ ability to degrade gluten in gastric environments. Successful mutants were collected and added together to make combinatorial variants. Finally, they synthesized Kumamolisin-As mutant strain N291D that had 784 times better at breaking down P-Q bonds than PEP.
The discovery of N291D mutant strain of Kumamolisin-As hailed a significant mark in the genetic fight against celiac disease. There are many proposals that suggest creating a genetically modified organism that could produce N291D Kumamolisin-As protein in human’s gastrointestinal tracts. Lactobacillus casei appears to be a potential host bacterium, since it is capable of secreting proteins in human stomach and commonly found in yogurt, a widely consumed food. If L. casei successfully shown its ability to function such, then Kumamolisin-As enzyme might be among the most breaking news of the 21st century.
University of Washington. 2011. Gluten Destruction. iGEM Competition 2011. <http://2011.igem.org/Team:Washington/Celiacs/Background>