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Team:UC Davis/Project
From 2009.igem.org
What is Celiac Disease?
Celiac Disease is an
autoimmune
disorder that occurs inside the small
intestine. When the body cannot properly digest gliadin (a component of
gluten),
this leads to an immune response on the surface of the small intestine
(14).
Different people have varying degrees of immune
response
such as bloating, diarrhea, and weight loss (14). An estimated one out
of 133 Americans currently suffer from this
disease (11). Considering the estimated 3.08 million people in America,
this isn't a minor issue. In fact, it was one of our teammate's friends
suffering from celiac who initially sparked our interest in designing
this project.
What happens in the normal small
intestine?
The inside surface of the small
intestine is covered with small microvilli, constituting the brush
border membrane. In the normal small intestine, the brush border
membrane is where polysaccharides, proteins, and fat droplets are
digested into smaller parts that then get absorbed into the
bloodstream.
The brush border
characteristic to people with celiac disease allows large molecules
such as gliadin to go straight
into the
blood stream without directing them through the transcellular route.
Once
gliadin passes through, Antigen Presenting Cells (APCs) recognize
gliadin as a
foreign object and attack. This immune response results in the
inflammation of
the surface of the intestine with loss of the normal cells required for
absorption of sugars, protein, and fat from the diet.
In addition to the array of
symptoms illustrated above, the immune response may also damage
intestinal villi, which are important
for
absorbing nutrients. The destruction of villi eventually leads to a
lack of
absorption of different nutrients, which can lead to malnutrition (14).
Some
people affected with this disorder can also suffer from autoimmune
thyroid
disease, autoimmune liver disease, and rheumatoid arthritis (diseases
in
which body immune system attacks healthy cells/tissues) (14).
Current
treatments:
Currently, no cure has been found
for this illness. The only way to avoid
the symptoms of celiac disease is by adopting a gluten-free diet
(14, 12,
and 11). However, you are highly likely to find gluten in your everyday
diet (in foods such as grains), and
people with celiac disease must purchase gluten-free substances from
specialized grocery stores. "Oral supplementation with prolyl
oligopeptidases that can digest and detoxify gluten has therefore been
proposed
as a potential therapeutic approach."(8) However, enzymes studied
earlier
were not able to degrade gluten inside the stomach (before it reaches
the small
intestine), because they were "irreversibly inactivated by pepsin and
acidic pH, both present in the stomach."(8)
Nevertheless, over the past
years, researchers have discovered an enzyme--a newly-identified prolyl
endoprotease--from Aspergillus niger, that was
observed to "work optimally at 4-5pH and remains stable at 2pH"(8).
Perhaps this enzyme will lead us to an alternative treatment for this
disorder(8). Studies have shown that prolyl endoprotease from A.niger
is able to "degrade gluten in vitro and under conditions
similar
to the ones present in the gastrointestinal tract." (8) ; However,
due
to
licensing restrictions, we have opted not to work with this protein.
Recently (year 2007), a study
has suggested an alternative approach by
combining a glutamine-specific endoprotease (EP-B2 from barley) and a
prolyl
endopeptidase (SC PEP from Sphingomonas
capsulata) with gastric
activity and
complementary substrate specificity(12). Using this method, there is a
possibility of
increasing the
safe threshold of ingested gluten(12). One of the advantages of this
“combination product is that both enzymes are active and stable in
stomach and
can therefore be administered as lyophilized powders or simple capsules
or
tablets”(12).
Our approach:
Gliadin induces an immune
system response when it is absorbed into
the blood stream through the small intestine. We plan to take advantage
of this by breaking down gliadin in the stomach before it reaches the
small intestine. We have developed a secretion system to release an
enzyme that can break down gliadin. Also, in order to prevent our
delivery system from taking residence anywhere outside of the stomach,
we plan to incorporate the difference in pH between the stomach and
small intestine with a pH-inducible apoptosis system. By adopting these
two systems, we will create a pH-moderated secretion system.
Unlike other suggested treatments
requiring the consumption of capsules or tablets, our method of using E.
coli as our delivery system is more cost- and time-efficient.
Our project is
split into two parts:
1. Induced Secretion
- Adding secretion
- Gene sequence for secretion system
- Assay for measuring protein localization and activity
