Toward a better understanding ofceliac diseaseThe kinetics of an enzyme reactionmay help researchers figure out howceliac disease (CD) develops. SiriDørum, Burkhard Fleckenstein, andcolleagues at the University of Osloand Rikshospitalet University Hospital(Norway) report in JPR (DOI 10.1021/pr800960n) that they have applied aquantitative MS methodand observed a positivecorrelation between thedegree of deamidationand the frequency withwhich various epitopesare recognized by T cellsof CD patients.

CD is a common di-gestive disease in whichnutrients from food arenot absorbed well.People with the diseasecannot tolerate gluten,which is a mixture ofproteins found in wheat,barley, and rye. When apatient consumes gluten,an immune response oc-curs. Villisfingerlike pro-jections in the small intestine that areresponsible for nutrientabsorptionsdisappear. Currently, theonly treatment is a change in diet toavoid proteins that trigger the immuneresponse. In most cases, a gluten-freediet reverses the intestinal damage.

So, how does gluten cause the im-mune system to run amok? Some de-tails of this process are still unknown,but a strong genetic association exists.Most CD patients express the humanleukocyte antigen (HLA) moleculesDQ2 or DQ8, which are receptors onantigen-presenting cells. When HLADQ molecules bind to foreign antigensor self-antigens, these antigens are pre-sented to T cells, which either promoteor suppress an immune response. Aftergluten has been digested into peptides,the enzyme transglutaminase 2 (TG2)deamidates some of these peptides andconverts certain glutamine residues toglutamic acid. This process introducesnegative charges, which increase theaffinities of these peptides for DQ2 and

DQ8, and an immune response istriggered.

Most of the peptides, or epitopes, thatT cells recognize in CD patients are de-rived from R- and γ-gliadins, which aregluten proteins. Some epitopes are bet-ter triggers of an immune response thanothers. For example, almost all patientshave T cells that recognize R-gliadinepitopes, but few have T cells that recog-

nize γ-gliadin epitopes. Many gliadinpeptides can be deamidated, so why aresome peptides recognized more fre-quently by T cells than others? “Whatwas not known was how fast theepitopes were deamidated by TG2, andwe were wondering what implicationsthis would have for the T cell responsesin celiac disease,” explains Dørum.

To see whether the rate of TG2 de-amidation correlates with T cell recog-nition of gluten peptide epitopes inpatients, the researchers turned to MS.Fleckenstein points out that the con-ventional method of assaying deamida-tion is to couple the transglutaminaseactivity to another enzymatic reaction,which releases ammonium. But thisapproach is indirect. “If there are sev-eral peptides in a mixture, which maybe of high complexity, one can onlymeasure the sum of ammonium pro-duction,” says Fleckenstein. However,with MS, the modifications on eachpeptide can be detected, and the loca-tions of those modifications can be

pinpointed within each peptide. Quan-tification was achieved by calculatingthe shift of the centroid masses of thepeptides’ isotopic envelopes after TG2treatment from the values obtainedbefore treatment.

The sequence context of glutamineresidues was known to influencewhether the residues would be deami-dated by TG2. However, the research-

ers discovered that thepeptide length is also akey factor. The longer agliadin peptide is, themore likely it is to havedeamidated glutamines.

Various gluten peptideswith known epitopeswere studied individuallyand in a mixture to de-termine the degree ofdeamidation. A 33-mer,shorter R-gliadin pep-tides, and one peptidefrom γ-gliadin weredeamidated quickly. Therest of the peptides wereonly partially deami-dated, even after a longincubation. “The results

seem to correlate with the observedfrequency of the T cell response in ce-liac disease patients,” says Dørum. The33-mer and the R-gliadin peptide weregood TG2 substrates and were recog-nized by T cells from almost all pa-tients. In comparison, the glutaminesof most γ-gliadin peptides were deami-dated less often and were recognizedless frequently by patient T cells. How-ever, one γ-gliadin peptide was an ex-ception. “The γ-II epitope is a verygood substrate for TG2 but is not oftenrecognized by T cells,” Dørum pointsout. She explains that proteolytic sta-bility may be an additional factor. “It isknown that the γ-II epitope is part of agluten fragment that is less stable com-pared with the 33-mer,” she states.

Although the scientists are not sur-prised by their findings, they say thatthese results fill in a large knowledgegap. “This adds one more piece to thepuzzle on epitope selection,” notesDørum.

—Katie Cottingham

Skip the bread. Researchers analyzed gluten peptides by MS to figure outwhether the rate of glutamine deamidation by TG2 plays a role in the recognitionof these peptides by the immune systems of CD patients.

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1620 Journal of Proteome Research • Vol. 8, No. 4, 2009 10.1021/pr900075y © 2009 American Chemical Society