GLUTEN SENSITIVITY

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GLUTEN SENSITIVITY MEETING POINT FOR GENETICS, PROTEIN CHEMISTRY AND IMMUNOLOGY Western societies: 1% of the population Coeliac disease : caused by a genetically determined, specific immune response to antigens present in wheat gluten, focused on a limited region of the α-gliadin. The antigenic 33-mer peptide generated by digestion with intestinal enzymes produce a highly stimulatory antigen for CD4+ T cells. Moreover, this peptide is resistant to further digestion by intestinal brush border enzymes, because of it’s high proline and glutamine content. The epitopes’ recognition by CD4+ T cells previously requires the deamination of the glutamine residues by the tissue Transglutaminase (TTG).

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GLUTEN SENSITIVITY. MEETING POINT FOR GENETICS , PROTEIN CHEMISTRY AND IMMUNOLOGY  Western societies: 1% of the population Coeliac disease : caused by a genetically determined, specific immune response to antigens present in wheat gluten, focused on a limited region of the α-gliadin . - PowerPoint PPT Presentation

Transcript of GLUTEN SENSITIVITY

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GLUTEN SENSITIVITY

MEETING POINT FOR GENETICS, PROTEIN CHEMISTRY AND IMMUNOLOGY

Western societies: 1% of the population

Coeliac disease : caused by a genetically determined, specific immune response to antigens present in wheat gluten, focused on a limited region of the α-gliadin. The antigenic 33-mer peptide generated by digestion with intestinal enzymes produce a highly stimulatory antigen for CD4+ T cells. Moreover, this peptide is resistant to further digestion by intestinal brush border enzymes, because of it’s high proline and glutamine content.

The epitopes’ recognition by CD4+ T cells previously requires the deamination of the glutamine residues by the tissue Transglutaminase (TTG).

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THE PROJECT A GLOBAL DESCRIPTION

Inspiring from probiotic yogurts, we would chose an oral supplementation of these probiotic bacteria, choosing the Lactobacillus Acidophilus. This bacteria support the acidic conditions of our stomach As well as it support the higher pH of the intestine Naturally present in our flora, so that it won’t induce an immune response BUT: for the pH sensing, introducing such a network (PAC sensing

pathway) : not sure if it is possible to completely clone it and the secretion is also a problem

Escherichia coli is one of the many species of bacteria present in our gut, and this would represent many advantages: Well understood genetics, manipulations quite easier For this organism, we can use known pH sensor

nhaA encodes an Na1/H1 antiporter in E. coli which is essential for adaptation to high salinity and alkaline pH in the presence of Na.

MOLECULAR PHYSIOLOGY OF THE Na+/H+ ANTIPORTER IN E. COLI

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THE PROJECT A GLOBAL DESCRIPTION

This bacteria should so produce an enzyme that will degrade these resistant α-gliadin: we will use the prolyl endoprotease of the Aspergillus niger (a fungi) for this purpose This enzyme works optimally at pH 4-5 and remain stable at

pH 2, the pH of the stomach where it would be optimal to begin with the proteins degradation

Moreover, this enzymes is completely resistant to digestion with pepsin, produced naturally by the chief cells in the stomach

Another advantages, it has been shown that this enzyme is capable of degrading all T cell stimulatory peptides as well as intact gluten molecules, a very good point for us, because this degradation will go on in the intestine.

The rapidity of degradation of this enzyme is 60 times faster than a prolyl oligopeptidase found in our body

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THE PROJECT A GLOBAL DESCRIPTION

After the oral ingestion of the yogurt, the bacteria will remain for a while in the stomach. Under these low pH conditions, the genetic network will be induced and it will begin the production of the enzyme for a first degradation of the dietary food taken. cf inducible genetic network

We think that it is important that the secretion of the enzyme can be inducible, in fact that it only begins in the stomach, and thus for different reasons: conservation and stability of the product, survival of the bacteria

The enzyme should then be produced all along the intestinal tract to ensure a sufficiently good degradation of all gluten proteins present

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THE PROJECT A GLOBAL DESCRIPTION

It would be another good idea to induce the bacteria to commit “suicide” when the “work” is finished, but this is only optional and it would probably occur naturally in the gut.

The bacteria has to live long enough to degrade with a certain efficiency all proteins found, and particularly the gluten’s one, so the rate of degradation is important, and the life time is something we have to check: so a population dynamics analysis would also be a good thing for this project

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THE PROJECT A TECHNICAL

DESCRIPTION

Genetically modified BACTERIA: L. Acidophillus DNA cloning strategies:

Genes of interest: put restriction sites by polymerase chain reaction PCR Use 2 enzymes to check the differences:

Prolyl oligopeptidase from F. Meningosepticum FM-POP Prolyl endoprotease from A. Niger AN-PEP

Controls: Analysis of PCR products by agarose gel electrophoresis,

DNA ligation, transformation of plasmid DNA into bacteria

Inoculation of bacterial cultures quantitation and analysis of DNA by UV spectrophotometry Analysis of plasmid DNA by restriction enzyme digestion and

DNA sequence analysis Protein quantification by spectrophotometric assay, SDS-

Polyacrylamide gel electrophoresis

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THE PROJECT A TECHNICAL

DESCRIPTION

Determine the pH optimum:Using Z1-Gly-Pro-Z2 as a substrate and different pH values:

measure amount of released Z2

Check the stability at low pH + resistance to pepsin degradation our secreted enzyme:Mixing with pepsin in a first step, neutralizing it’s activity

with inhibitor, pepstatin: measure left enzymatic activity

Make an activity assay in solutions that mimic stomach and intestinal conditions, in terms of pH, enzymatic contents etc..Using a fluorogenic substrate Z1-Gly-Pro-Z2

(Spectrophotometry)

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THE PROJECT A TECHNICAL

DESCRIPTION

Real Enzymatic digestions + Degradation rate measurement: Same type of experiment but with synthetic

peptides dissolved, all containing the this 33-mer resistant peptide: LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF

To do that: produce recombinant gliadins (such α and γ-gliadins) : take the gene, use a plasmid to transform into E. coli

Have to mimic real digesting conditions: Make a brush border enzyme preparation using (if

possible) rat small intestine (jejunum)

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Genetic Network, E.Coli K12Expression of en enzyme to process gluten

FM-POP AN-PEP

Induction of expression (in response to pH decrease) Na1-induced transcription of nhaA, which encodes an Na1/H1

antiporter in Escherichia coli, is positively regulated by nhaR and affected by hnsNYMU-Taipei, iGEM 2008

Burst of the bacteria to release the enzymes Lysis cassette including λ phage lysis genes. Lysis occurs 40-

45 minutes after inductionCaltech, iGEM 2008

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Induction in E.ColipH activates nhaA promoter sequences

CRE-recombinase

nhaA

CRE-Lox recognition sites

CRE-lox mechanism is used as a trigger to ensure expression of the protein will be maximum before burst

AN-PEPSTOP

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Bacteria lysisFrom lambda phage: holin/anti-holin, endolysin, and rz / rz1 genes

Lysis cassette

nhaA

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Need to investigate

Induction working in a strain of E.Coli compatible with conditions in gut.

Is lysis using genes from lambda phage working in the same strain?

What pH induces the cascade?Delay before lysis

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Help of bioinformatics

Tune a model to link the amount of enzymes needed to digest gluten in a normal life. Diffusion model: the enzyme has to cut efficiently most of the

gluten present in a low concentration inside a high volume. We have to know how many bacteria can produce these enzymes What concentration of gluten can be reached after degradation?

Try to quantify the number of bacteria triggered at pH from gut (or any induction system)

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Lactobacillus acidophilusAdvantages :

Gram positive (easier for secretion)Grow at low pHOccur naturally in the gastrointestinal tract

Culture :Anaerobic conditionsGrow on MRSWork with a limited range of plasmidA protocol exists for plasmid pNZ123

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L. Acidophilus (2)MIT worked in 2008 with L. bulgaricusL. bulgaricus would also be a possible

bacteriaThe y wrote the protocols for electroporation

for L. bulgaricus and L. acidophilusDifficulties because these bacteria aren’t

much used.