Molecular Basis of Celiac Flashcards
HLA-DQ2 - DQ8 aminoacid preference: - Gliadin
Negative charge on 4th, 6th 7th places on DQ2
1st and 8th on DQ8
Gluten itself doesn’t have E (glutamic acid), D = which brings negative charge.
It has a lot of proline and glutamine (P) (Q) immunogenic. 1/3 of gluten is glutamine
Gliadin is a cleavage product of pepsin = and pepsin is the only enzyme in stomach that can cleave proteins.
Gliadin is DQ8 restricted
Sequence still doesn’t have E-D negative charge.
posttranslational modification is required.
Glutamine glutamic acid conversion
Glutamine is really similar to
glutamic acid, you can turn it.
It happens when you lower the ph= if you lower it to 2, some glutamine will turn to glutamic acid.
2nd = enzymatic modification. Tissue transglutaminase!
When converted, first peptide in DQ8 becomes E, T cell recognition increases 100 fold. Same happens when you change the 9th position.
Gluten itself isn’t so immunogenic = but its modified gluten that causes huge problems.
Celiac disease patients make autoAbs for tissue Transglutaminase!
Steps of celiac disease (BEST ONE STUDY THIS)
1) Eat gluten, it’s degraded in stomach into gluten peptides by pepsin
2) Some of the peptides can bind to HLA DQ2-8 = very low affinity. But it happens if there’s a lot of gluten.
3) T cell responds to gluten = this leads to tissue damage
4) In tissue damage, transglutaminase is released for tissue repair.
5) People eat a lot of gluten, and the gliadin (gluten component) can also bind to transglutaminase = when it happens, you get deamidation, Q becomes an E (-) charge, and now they can bind HLA DQ 2-8 perfectly.
6) Amplification of damage, more transglutaminase, more binding to T cells = never ending cycle. Unless you stop eating gluten. = treatment.
Which glutamines are targeted in the gluten from tissue transglutaminase?
If a glutamine is followed by proline = no modification
- Glutamine followed by 1 random aminoacid, then proline = always modified
- Glutamine followed by 2 random aminoacid, then proline = not modified
- Glutamine + X + Proline + Y or F = modified.
But 1/3 of Gluten is glutamine, 1/5 of Gluten is Proline = so it’s modified anytime. These sequences are very common.
When looking for immunogenic sequences in Gluten = look for sequences that turn Q to E. Then they can bind to HLA DQ2-8.
Wheat - barley - rye - oat study + it’s relation with T cell memory response
300 possible gluten sequences exist. Every gluten protein is around 30 kb, 100 aminoacids. = 30.000 potential peptides. Searched for peptides that will bind to HLA-DQ2.
Somehow gluten in wheat/barley and rye are immunogenic, but the ones in oat don’t bind to HLA-DQ2. They can eat oat. Now they found a lot of peptides that can trigger this HLA response though, not a single peptide is responsible from Celiac’s disease.
Now scenario changed: When modified, gluten fragment can bind to the HLA-DQ2 – but there’s multiple of those fragments. And they all can bind to HLA DQ 2-8 = and trigger multiple T cells!
So in Celiac patients = there’s multiple T cell phenotypes with affinity to multiple peptide types! And obv there’s memory T cells occurring, and they will stay with you forever. That’s the reason why they will never eat gluten again = because every time they eat it back, a memory T cell is triggered and this response starts all over again.
What initiates the disease, only %1 people have it? - TcR study
Everyone is able to generate 10^15 of TcRs with VDJ = huge! Let’s clone every TcR of patients and check them.
Everyone has alpha-26 Beta 9 receptor (TRAV), and either one of them can be identical with other people. There’s apparently 1 specific TcR that can recognize gluten really good. = 1st box.
There’s 2nd one = 2nd box. 2 different T cells from 2 different patients are completely identical! The chance that this occurs is almost 0 if it was random. So something is pressuring them to get these receptors, and you don’t find these receptors in healthy people.
It’s always an R sequence in recognition site = and it doesn’t happen in germline, it only occurs via rearrangement! There’s not only a selection for specific receptor, but also for Arg.
DQ8 - TcR- gliadin relation - CryoEM results
DQ2 - TcR- glia alpha 2 results
TcR makes contact with both HLA and gluten. So receptor distinguishes/selects for both HLA and gluten.
If you remove Tyr, Leu or Arg = recognition doesn’t work.
Multiple patients overlapped = same. Celiac disease patients all recognize gluten in same fashion.
Arginine conserved - DQ2
Why generate TcRs for a harmless protein?
Molecular mimicry - cross reactivity between microbial antigens and gluten epitopes?
Glia alpha 1 epitope = Very immunogenic gluten peptide recognized by all patients that have HLA-DQ2.
Another gluten peptide, alpha2 = also very immunogenic, recognized by HLA-DQ2 again.
Both have Pseudomonas/Bordetella sequences that are very similar.
They themselves are also continuous epitopes. 2 extra on ends of each, rest is overlapping. Freakingly, in Pseudomonas, you see the exact same thing! Those 2 epitopes that is very similar with Alpha 1 and alpha 2 in pseudomonas are like that too.
So Pseudomonas really has very strong homolog peptides with gluten peptides.
Would T cells in Celiac patients also recognize Pseudomonas proteins? Yes. So if they can recognize alpha 1-2, they recognize Pseudomonas too. Same with Bordetella. So driving recognition of such epitopes can be driving Celiac disease by crossreactivity.
The epitope found in Pseudomonas are also seen in opportunistic versions of Pseudomonas that lives in gut/very common in vegetable, in contact with it. They can settle in intestine, and this disease might develop.
Gluten epitopes / Pseudomonas similarity in Cryo-EM
DQ2 - glia-alpha1
Pseudo
Complete overlap! Except in places that there’s 2 aa difference. Does it matter in recognition of TcR? It doesn’t matter in recognition.
Conserved ELPYP is what’s really recognized by TcRs. Recognised sequences in both Bordetella and glia are identical. And there’s additional salt bridge in bordetella, which makes the interaction even more. (marked) T cells might be triggered by infection rather than gluten.
So if you interact a peptide, and accidentally recognize a wrong epitope = you might get Celiac’s. As long as you don’t have TcRs, you won’t get the disease. And also like there’s a lot of events that have to come together in order to have it. = really unlucky lmao.
Mass cytometry
Flow cyt without florochromes but metal labeled Abs.
Good point is there’s no overlap in isotope signals like in florochromes – and you can recognize up to 50 markers at the same time. = You can visualize much more heterogeneity.
Useful for biopsies since you have limited material = you get all information all together.
tSNE
Too much marker = too much data = hard to analyse
Dimentionality reduction technique. Groups the similar cells.
Reduce it more: Do dual t-SNE: Every dot is an immunological sample.
example: PBMCs from intestine.
Celiac patients in blue are completely separated from black sample patients.
You can do a completely unbiased analysis with cells = also useful.
Imaging Mass Cytometry
IHC with 36 markers at the same time, normally you can do 3.
You can distinguish every T cells.
