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Read Article: Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease by Molberg et al.

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1. Read a Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease by Molberg et al. found on Blackboard.

2. Answer the following questions:

1. What is the hypothesis of the paper?

2. Is this a correlative or causative study? Justify

3. How do the authors support their hypothesis?

4. Do you believe the author’s data presented in the paper (and any supplements) support their hypothesis. If not, why not?

5. Are there other experiments or experimental modification that should have been performed by the researchers? 

6. Does the discussion carefully lay out an argument that supports the hypothesis? If not, what do you believe was missing?

7. Does the statistical analysis strengthen or weaken the conclusions of the paper.

8. What are the conclusions of the paper? Do you agree with the conclusions?

9. Does the author overstate their findings or perhaps not enough?


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Attachment 1

© 1998 Nature Publishing Group http://www.nature.com/naturemedicine• ....................................................................................................................................................................................................................... ARTICLES Tissue transglutaminase selectively modifies gliadin peptides

that are recognized by gut-derived T cells in celiac disease




'Institute of Transplantation Immunology and 'LIIPA T, Institute of Pathology,

Rikshospitalet, University of Oslo, N-0027 Oslo, Norway 2Department of Molecular Biology, Odense University, DK-5230 Odense M, Denmark

'Department of Clinical Immunology, Aarhus University Hospital, Skeiby Sygehus DK-8200 Aarhus N,Denmark 4Deptartment of Clinical Immunology, Rigshospitalet, University of Copenhagen,

6Department of Medical Biocehmistry and Genetics, Biochemistry Laboratory C, The Panum Institute,

University of Copenhagen, DK-2200 Copenhagen N, Denmark

Correspondence should be addressed to K.E.A.L. present address: Department of Medicine, Ullevaal Hospital, Oslo, Norway

The action of tissue Transglutaminase1 (TGase) on specific pro- tein-bound glutamine residues plays a critical role in numerous biological processes2-s. Here we provide evidence for a new role of this enzyme in the common, HLA-DQ2 (and DQ8) associated6

enteropathy, celiac disease (CD). The intestinal inflammation in CD is precipitated by exposure to wheat gliadin in the diet' and is associated with increased mucosa! activity of TGase8 • This en- zyme has also been identified as the main target for CD-associ- ated anti-endomysium autoantlbodies•, and is known to accept gliadin as one of its few substrates 10. We have examined the pos-

sibility that TGase could be involved in modulating the reactivity of gliadln specific T cells. This could establish a link between pre- vious reports of the role of TGase in CD and the prevailing view of CD as a T-cell mediated disorder1•11•12• We found a specific ef- fect of TGase on T-cell recognition of gliadin. This effect was lim- ited to gliadin-specific T cells isolated from intestinal CD lesions. We demonstrate that TGase mediates its effect through an or- dered and specific deamidation of gliadins. This deamidation creates an epitope that binds efficiently to DQ2 and is recog- nized by gut-derived T cells. Generation of epitopes by enzy- matic modification is a new mechanism that may be relevant for breaking of tolerance and initiation of autoimmune disease.

Immunofluorescence staining of small-intestine biopsy speci- mens from control subjects and untreated CD patients demon- strated expression of TGase mainly in the subepithelial region of the mucosa. The CD patients had a higher expression level than control subjects as well as some TGase expression in the brush border. Notably, in the untreated CD patients, the subepithelial region staining positive for TGase is heavily infiltrated with T cells carrying the CD3 antigen and large cells expressing DQ molecules (data not shown).

We tested a panel of T-cell lines and clones (from eight differ- ent CD patients) isolated from duodenal biopsies challenged ex vivo with pepsin -trypsin digested gliadin (PT-gliadin, an antigen known to provoke disease in vivo13) and we found that addition ofTGase to proliferative assays enhanced reactivity and sensitiv- ity to PT-gliadin for all these gut-derived T cells (Fig. 1, left panel and data not shown). None of the gut-derived T cells were reac- tive to TGase alone. The effect of TGase was not dependent on


type of antigen presenting cells (APC); both B lymphoblastoid cell lines (B-LCL) and T-cell-depleted peripheral blood mononu- clear cells (PBMC) had enhanced responses (10-fold and 30-fold respectively). Further, the effect of TGase on T-cell recognition appeared to be specific for gliadin. Two DR2 restricted, gut-de- rived T-cell lines specific for astrovirus (one from an untreated CD patient) and two DR3 restricted, PBMC-derived T-cell clones specific for PPD (Purified Protein Derivative from Mycobacterium tuberculosis) were not sensitive to TGase-mediated antigen modi- fication (data not shown).

TGase catalyzes protein crosslinking through the formation of isopeptide bonds between Gin and Lys residues1•3• Gliadins are rich in Gin residues, but have very few Lys residues14• Nonetheless, gliadin- gliadin complexes are known to form in the presence of TGase10• Since particulate antigens are more effi- ciently taken up by APC15, TGase could enhance gut-derived T- eel! recognition of gliadin by catalyzing formation of gliadin complexes. However, with the exception of one single DQ2 re- stricted T-cell clone, no increase in PT-gliadin-specific prolifera- tion of PBMC-derived T cells from six CD patients16 was detected after addition of TGase (Fig. 1, right panel). This indicates that increased uptake of gliadin mediated by TGase cannot account for the enhanced proliferation of gut-derived T cells. These data also indicate that gliadin-specific T cells that reside in peripheral blood and gut mucosa may recognize distinct epitopes and, most importantly, that TGase could be instrumental in the creation of gliadin epitopes recognized by gut-derived T cells.

The PT-gliadin recognized by gut-derived T cells has been sub- jected to digestion at low pH, a condition known to facilitate non- enzymatic deamidation of Gin residues 11 . Interestingly, TGase can catalyze substrates to react with water to be deamidated when amine acceptors are in deficit' . Indeed, such Gln deamidations have been demonstrated with gliadin as a substrate10• We tested whether TGase-mediated deamidation of gliadin was important for its recognition by gut-derived T cells by analyzing the effect of TGase on gut-derived T-cell recognition of a water-soluble, chy- motrypsin-digested gliadin. Digestion with chyrnotrypsin was done at neutral pH, thereby avoiding spontaneous non-enzymatic deamidation. Chymotrypsin-digested gliadin only weakly stimu- lated gut-derived T cells from six CD patients (four DQZ+ and two


© 1998 Nature Publishing Group http://www.nature.com/naturemedicine

ARTICLES • Small Peripheral

HLA- intestine

HLA- blood

TCC restriction TCC restriction

412 R-2 002 -TGasea -TGasea + TGasel!lil RNB.14 DR3 + TGase II

412 R-3 002 AK35 DR3

412 R-5 002 OHR 17 DR3

387 R-3 002 DHR41 DR3

387 R-16 002 DHR23 DR4

411 R-1 002 DHR26 DR4

380 E-27 002 AK16 002

282 1.12 DOB GVL 13 002

282 2.8 DOB AK44 002

282 2.27 DOB GVL40 008

282 2.35 DOB AK35 DP1

20 40 60 BO 100 20 60 100 140 180

cpmx 10·1 cpm x 10·'

Fig. 1 Comparison of the effect of TGase on PT-gliadin-specific T-cell clones derived from the small intestine or from peripheral blood of celiac disease patients. 100 µg/ml PT-gliadin was incubated for 2 h at 37 °C with or without 100 µg/ml TGase and then incubated with APC (irradiated allo- genic HLA homozygous B-LCL) for 16 h. Recognition of antigens by the T

cells was measured as 3H-thymidine incorporation 48-72 h after antigenic stimulation. Data given as (cpm; Proliferation of [T cells + APC incubated with antigen] - [T cells + APC incubated without gliadin]. Data for each T-

eel\ clone are from one representative experiment (of two or three).

DQ8·). However, it could be converted to an efficient antigen by incubating it in acid (pH 1.8) for 1 hour at 98 °C. Notably, TGase treatment of the chymotrypsin-digested gliadin generated a more potent antigen for all of the gut-derived T cells than the acid/heat treatment (Fig. 2 and data not shown).

For a detailed molecular analysis of the actions of TGase on gliadin, we focused on a recently characterized gliadin epitope recognized by gut-derived DQ2 restricted T cells from three dif- ferent CD patients (H.S. et al., manuscript submitted). This epi- tope was originally identified within a 20 amino acid fragment isolated from a purified y-type gliadin. In its unmodified form this fragment were determined to have the sequence QQLPQPQQPQQSFPQQQRPF, a sequence that matched residues 134-153 of GDB2_WHEAT (SWISSPROT accession number P08453). Acid/heat treatment of this fragment resulted in a com- plex mixture of peptides with varying numbers of deamidated Gin residues. Of these, the Glu at position 148 was essential for recognition by gut-derived T cells, as without this deamidation the 134-153 peptide was barely stimulatory. The minimal T-cell epitope was mapped to residues 141-150 (H.S. et al, manuscript submitted).

T-cell lines from two patients (CD370 and CD412), as well as a T-cell transfectant (60.6) expressing a chimeric T-cell receptor containing variable regions cloned from the gliadin peptide-spe- cific, gut-derived T-cell clone 4.32 (H.S. et al, manuscript submit- ted), recognized the acid/heat treated peptide 134- 153. These T cells were used to demonstrate that TGase treatment trans- formed peptide 134-153 from a non-stimulatory to a potent T- cell stimulatory peptide.

Ion exchange chromatography of peptide 134-153, treated with TGase showed two distinct and dominant peaks (Fig. 3a). Fractions collected from the chromatography were separated into aliquotes. One aliquot was tested for recognition by the T-cell


tranfectant 60.6 and showed that the T-cell stimulatory capacity of the peptide was retained in the peak corresponding to fraction 28. Fractions 15 and 28, corresponding to the two peaks were sub- jected to methyl-esterification of acid residues (C-terminus and Glu) followed by Matrix-Assisted Laser Desorption/Ionization- Time-Of-Flight (MALDI-TOF) and ElectroSpray Ionization (ESI) ion trap mass spectrometry to determine the number and posi- tions of the deamidated glutamine residues. Both fractions had one dominant peptide, with two and three deamidations respec- tively (Fig. 3b). In the non-stimulatory fraction 15, Ql40 and Q150 were identified as the deamidated glutamine residues. In the T-cell stimulatory fraction 28 these residues were also deami- dated, as well as a third residue, predominantly at Ql48.

Incubation of synthetic 1251-labeled peptides encoding T-cell epi- topes (that is, peptides 134-153;£140 and 134-153;£140 E148, E150) with TGase and subsequent analysis by SDS-PAGE indicated that a fraction of each peptide became covalently coupled to the enzyme (data not shown). This demonstrates that TGase and gliadin peptides may form hapten-carrier-like complexes that can be involved in production of TGase autoantibodies9 •

The proposed peptide-binding motif for DQ2 predicts a prefer- ence for negatively charged residues in relative position 7 (refs. 18, 19) Testing of synthetic peptides in a cell-free DQ2 binding assay demonstrated a IO-fold increase in binding affinity of pep- tides with an E148 substitution (Fig. 4a and H.S. et al., manu- script submitted), indicating that position 148 could be in DQ2 pocket 7. Although the binding affinity to DQ2 of acid/heat treated peptide 138- 152; £148 the latter was a more potent anti- gen for the T-cell transfectant (Fig. 4). This indicates that al- though non-enzymatic deamidation generates a complex mixture of deamidated variants, of which only a minority are ad- vantageous for T-cell recognition, enzymatic deamidation by TGase preferentially acts on residues favorable for T-cell recogni- tion and binding to DQ2.

Our data indicate that the repertoire of antigen-specific HLA class II restricted CD4·T cells can be shaped by modified antigens and that organ-specific T cells in an inflammatory disease can recognize epitopes generated by a specific enzyme activated by the inflammation. This may go unreported as the standard use of synthetic peptides for the characterization of T-cell epitopes means that most in vivo modified epitopes would escape detec- tion. Our data indicate that the fine specificity of peripheral

Fig. 2 Recognition of chymotrypsin-digested gliadin by a DQ2-re- stricted T-cell line from the gut of an untreated celiac disease patient (CD4 l 2). Gliadin was in- cubated as described in Fig. 1. The acid heat- treated gliadin was incu- bated at pH 1.8, 98 °C for 60 min, freeze-dried and resuspended at pH 7.4. Allogenic, HLA- matched, T-cell-depleted PBMC (irradiated 25 Gy) were used as APC. T cell proliferation was mea-



b X 25 E a. V




-1:r- : Gladin alone + : Gliadin incubated with TGase -0-; Acid/heat-treated glladin

-+-: Acidlheat-lreated glladin incubated with TGase

10 0.1 0,01

Chymotrypsin-digested gliadin (mg/ml)

sured as in Fig. 1. Data are from one representative experiment (of three).

Similar data were obtained with T-cell lines from the small intestines of five

other CD patients.


© 1998 Nature Publishing Group http://www.nature.com/naturemedicine• ······················································································································································································································· ARTICLES a 5 15 28 38


0.3 OD 210

0.2 nm


180 min

15 28 46

b Fraction number

134· · . .. . . ... . ········· · --·· ·153 QQLPQPQQPQQSfPQQQRPF

frac tion

15 ·· .... .. E· ·" •• •· .... · ·E ••• 28 .. . .. . . . E .. ..... ... E ... E ...

blood T cells and organ-specific T cells recognizing the same complex antigen is different, emphasizes the danger of sampling PBMC when studying the immunology of other organs.

The spatial relationship (revealed by immunohistochemistry), between abundantly expressed extracellular TGase and DQ ex- pressing cells just under the mucosa! epithelium of CD patients suggests that TGase might be involved in processing gliadin be- fore it binds to DQ2 molecules. There is a infiltrate of T cells in the same region and ex vivo studies of gliadin-challenged biop- sies have shown that some of these T cells are gliadin-specific20 • Thus, the subepithelial region seems to be a microenvironment for TGase-mediated modification, DQ2 binding and T cell recog- nition of gliadin.

Detailed analysis of one T cell epitope (within residues 134-153 of a y-gliadin) recognized by DQ2 restricted gut-derived T cells from three CD patients showed that TGase mediated an ordered and specific deamidation of Gln residues which was im- portant for DQ2 binding and critical for T cell recognition . Our data indicate that peptide 134- 153 is not the only gliadin pep- tide that can be transformed into an efficient T-cell epitope through TGase modification. All the gut-derived T-cell clones displayed in figure 2 showed enhanced reactivity to TGase mod- ified gliadin, but none recognized TGase-treated peptide 134-153. Furthermore, analyses with different purified gliadins have demonstrated that these T-cell clones are heterogeneous with respect to fine-specificity (0.M., unpublished observa- tions), which suggests that TGase-mediated deamidation poten- tates several different gliadin epitopes.

Fig. 4 a, T cell recognition and DQ2 binding of the synthetic gliadin pep-

tide 138-152 and variants with Gin to Glu amino acid substitutions. Peptide

binding to DQ2 was measured in a cell-free assay with purified HLA-mole-

cules and binding affinity is given as IC,0, i.e. the amount of gliadin peptide

necessary for 50% inhibition of binding of a labeled indicator peptide. The

synthetic peptides were made with Gin to Glu substitutions in the same po-

sitions (140, 148 and 150) that are deamidated upon incubation of gliadin

peptide with TGase (Fig. 3b). Recognition of peptides by the DQ2 restricted

T-cell transfectant 60.6 was measured by IL-2 release in response to peptide

stimulation. b, Effect of acid/heat treatment of peptide 138-152; El 40, El 48, El 50 on T cell recognition and DQ2 binding. Acid/heat treatment of

the peptide was done at pH 1.8, 98 °C for 60 min .


Fig. 3 a, Kinetic analysis of TGase-mediated deamidation of gliadin pep-

tide 134-153. The peptide was incubated with TGase for various time pe-

riods and then analyzed by ion exchange chromatography. Depicted are

the chromatographs of the peptide after incubation with TGase for 0, 30,

60 and 180 min. The fractions of the 180 min chromatography were di-

vided. One aliquot was tested for recognition by the DQ2-restricted T-cell

transfectant 60.6. Fractions 15 and 28 from the other aliquot were methyl-

esterified and sequenced by ESI MS/ MS to determine the number and po-

sitions of deamidated glutamine residues. The peak corresponding to

fractions 38-46 in the chromatographs contains mainly impurities in the

Tris-HCI buffer. Recognition of each of the fractions by DQ2-restricted T-

cell transfectant 60.6 was measured by quantification of IL-2 release.

b, The upper lines show the amino acid sequence of gliadin peptide

134-153 prior to TGase treatment; lower lines, the number and positions

of deamidated glutamine residues in chromatography fractions 15 and 28

of peptide 134-153 after 180 min TGase treatment. The number and posi-

tions of deamidated glutamine residues in each fraction was assessed by

ESI MS/MS analysis.

The DQ2 binding epitopes recognized in CD are created by an enzyme which is the main focus of the autoantibody response in this disease9 • We have proposed that a prerequisite for produc- tion of these disease-associated autoantibodies is that hapten- carrier like complexes of gliadin and TGase are formed in vivo, which would provide (gliadin-specific) T-cell help to TGase-spe- cific B cells21 • Jndeed, our data indicate that, in addition to gen- erating gliadin fragments recognized by gut-derived T cells, TGase can also catalyze formation of complexes between gliadin

T-cell epitopes and TGase. Notably, in this model autoantibody production to TGase does not involve breaking of T-cell toler- ance to the abundantly expressed TGase protein.

lntroduction of negative charges at defined positions in pep- tides probably affects the repertoire of HLA molecules able to bind them. We observed an effect of TGase on both DQ2 and DQ8 restricted T-cell epitopes. Notably, both DQ2 and DQ8 have been predicted to preferentially bind peptides with negatively charged residues' 8· ' 9•22 • The skewing towards DQ2 and DQ8 re- striction of gliadin-specific gut-derived T cells may therefore re- flect that TGase deamidation favors selective binding of gliadin to these HLA molecules.

Although this report describes the enzymatic deamidation of a foreign antigen, self antigens could also be substrates for similar types of modifications. Mass spectrometric analysis of peptide fragments eluted from HLA molecules have identified post-trans- lationally-modified self proteins23 • Notably, autoreactive human CDS· T-cells antigen specifically recognizing modified peptides

a Binding to T cell transfectant DQ2 (IC50) (µM)




Py.PfQPQQSFP.!;Q.!;RP 1.8 50 " -0 3

b X

250 ~

Unmodified Py.P.!;QPQQSFP.!;Q.!;RP 1.8 150

Acid/heat-treated Py.P.!;,QPQQSFP.!;Q.!;RP 1.2 50

0,4 2 10 50

Peptide.cone. (µM)


© 1998 Nature Publishing Group http://www.nature.com/naturemedicine• ARTICLES have been described23•24. In a mouse arthritis model it has also been demonstrated that modified self-peptides are recognized by the class JI-restricted autoreactive T cells2s. Future work should determine whether enzymatic modification of antigens is a gen- eral mechanism for the immune system to define epitopes or if it is a process invariably associated with the kind of pathological immune activation described in this study.

Methods lmmunohistochemistry. Cryostat sections from healthy controls and un- treated CD patients were stained using a indirect three-color immunofluo- rescence technique20• Images were digitally analyzed using a Eclipse E800 microscope (Nikon) and a C5810 imaging system (Hamatsu, Shizouka, Japan). The following mouse monoclonal antibodies were used; anti-TGase (TG l 00, lgGl; Neomarkers, Fremont, California), anti-DQ (SPV-L3, lgG2a; gift H. Spits, DNAX Research Institute, Palo Alto, California) and anti-CD3 (RIV9, lgG3; Sanbio, Am Uden, The Netherlands). Secondary goat anti mouse antibodies (Southern Biotechnology, Birmingham, Alabama) were biotinylated anti-lgG3, FITC-conjugated anti-lgG2a and cy.3-conjugated anti-lgGl. The biotinylated anti-lgG3 was visualized by an AMCA conju- gated streptavidin (Vector, Burlingame, California).

Preparation of gliadin antigens. Digestion of crude gliadin (Sigma; G- 3375) with pepsin (Sigma; P-7012) and trypsin (Sigma; T-7418) was done as described". a-chymotrypsin (Sigma; C-3142) digestion of gliadin from the wheat strain Kadett was performed at 200:l (w/w) in 0.1 M NH,HC03 with 2 M urea at 37 •c for 24 hand stopped by incubating in boiling water for 5 min. Synthetic gliadin peptide quality-controlled by HPLC (> 80% pu- rity) and MS were purchased from Research Genetics (Huntsville, Alabama).

Generation of antigen specific T cells. T-cell lines derived from gut and specific for PT-gliadin were established from small intestinal biopsies as de- scribed", except that isolation of some T cells from the gliadin-challenged biopsy material was done without enrichment for CD25-expressing cells (0.M. et al., manuscript submitted). Gut derived T cell lines of three DQ2 + CD patients (CD370, CD380 and CD387) and two DQ8 + patients (CD282 and CD360) on a gluten-free diet and three untreated DQ2 + patients (CD4 l 0, CD4 l l and CD4 l 2) were tested. In addition, DQ2 restricted T-cell clones (370.E-l 4; 380.E-3, E-11 and E-27; 387.R-3,R-12 and R-16; 411 .R-1; 412.R-2, R-3 and R-5) (0.M. et al., manuscript submitted) made from these T-cell lines were used. Generation of gut-derived astrovirus-specific T-cell lines (ref. 26 and 0 .M . et al., manuscript submitted), gut-derived DQ8 re- stricted T-cell clones", PPD-reactive T-cell clones (RN4.26 and 5.28 from PBMC; E.A.L. et al., unpublished) and P/T-gliadin-specific T-cell clones from PBMC'6 have been described elsewhere.

Generation of a DQ2 restricted T-cell transfectant specific for gliadin. Va and VI} gene segments from the T-cell clone 4.32 derived from the gut" were amplified from genomic DNA and cloned into the T-cell receptor ex- pression vectors, p T acass and p T(}cass, respectively". These vectors allow for functional expression of murine or human variable region T-cell receptor spliced to murine constant regions (L.M., LS. and l.F., unpublished obser- vations). Transfection of these plasmids with CD4:pBs and a Neo selection marker into the T-cell hybridoma BW 58a-/(}- deficient for T-cell receptors29

allowed for the selection and cloning of a stable transfectant (60.6) express- ing human CD4 and chimeric T-cell receptor.

T-cell assays. T cells (5 x 10') were added to 5 x 104 APC (either HLA- matched allogenic B-LCL transformed by Epstein Barr virus and irradiated with 80 Gy or HLA-matched allogenic PBMC irradiated with 25 Gy) which had been incubated for 16-20 h with complex antigen or four h with pep- tides in a volume of l O µI RPMI 1640 (Life Technologies) supplemented with 15% pooled human serum. Assays were performed in 96-well U-bot- tom plates (Nunc) and T-cell proliferation was measured as 3H-thymidine incorporation 48-72 h after antigenic stimulation. Activation of the 60.6 T- cell transfectant was measured as IL2 release following an 18 h incubation of 2.5 x 1 o• transfectant cells with 5 x 1 o• DQ2 + B-LCL as APC. The con- centration of IL2 was quantified by time-resolved fluorometry, using Delfia reagents (Wallac, Finland) and murine anti-lL2 antibodies (JES6-1 A 12 and


JES6-5H4, PharMingen, California). Parallel assays using the original T-cell clone and the transfectant confirmed that the chimeric T-cell receptor ex- pressed by the transfectant maintained its specificity for MHC restriction and peptide (data not shown).

Tissue transglutaminase assays. PT-gliadin (1-1000 µg/ml), chy- motrypsin-digested gliadin (0.1-1000 µg/ ml) or gliadin peptides (0.2-100 µM) were incubated with 100 µg/ml guinea pig TGase (Sigma; T-5398) at 37 °c for 2 h in PBS with 0.8mM CaCI,. These concentrations of TGase and CaCl,were found to give optimal T-cell stimulation and are comparable to that described in other systems'·10• After 2 h incubation, gliadin/TGase mix- tures were incubated with APC and tested in T-cell assays as above.

DQ2 peptide binding assay. A competitive peptide binding assay using affinity purified HLA-DQ(al *0501,(}*0201) molecules, a 1251 -labeled indica- tor peptide KPLLIIAEDVEGEY (Mycobacterium Bovis 65 kDa heat shock pro- tein 243-255Y) and various unlabeled gliadin peptides was performed as described'".

SOS-PAGE of gliadin peptides incubated with TGase. Peptides 134-153; 134-153, El 48; and 134-153, El 40; E148, El 50 were synthesized with an extra N-terminal Y and radiolabeled with 1251 using the chloramine T method'". TGase was 125I-labeled as control. Trace amounts of '"I-peptides (20,000 cpm) were mixed with their corresponding unlabeled peptides in various concentrations and incubated with 1 OOµg/ml unlabeled TGase at 37 °c for 2 h in PBS with 0.8 mM CaCl2• The samples were boiled in Laemmli buffer with 2-mercaptoethanol for 5 min and separated by 15% SOS-PAGE.

Ion exchange chromatography. Mono-Q chromatography in the SMART system (Pharmacia) was run in 5 mM Tris-HCI buffer purified by treatment with DAEA-Sephadex A50 (buffer A, pH 6.5). The column was eluted by a gradient with a final concentration of 0.05 M NaCl in buffer A. Synthetic peptide 134-153 (1 mg/ml) was dissolved in buffer A supplemented with 0.8 mM CaCI, and incubated with 1 mg/ml TGase.

Mass spectrometry. Fractions collected from ion exchange chromatogra- phy of TGase treated peptides were subjected to methyl-esterification'0 of acid residues (C-terminus and Glu) followed by MALDI-TOF mass spectrom- etry" to determine the number of Glu residues. ESl32-ion trap" tandem mass spectrometry30 was used to determine the position of Glu residues. Peptide ions were isolated in the ion trap and fragmented by collisions with He gas creating a series of N- and C-terminal fragments with differing pep- tide chain lengths. By comparing the fragmentation patterns of the native peptide with the TGase treated fractions, mass increments of 15 Da were as- signed to methyl-esterified Glu residues in these fragment ions. MALDI-TOF mass spectra were acquired on a Reflex mass spectrometer (Bruker-Franzen Analytik GmbH, Bremen, Germany). ESI tandem mass spectra were recorded on an ESQUIRE ion trap mass spectrometer (Bruker-Franzen Analytik).


We thank E. Thorsby for review of the manuscript, E. Brundtland, T. Jensen and

D. Anthonsen for technical assistance, H. Gjertsen for providing T-ce/1 clones

and K. Kett for providing biopsy specimens. This work was supported with

grants from the Research Council of Norway, Medinnova, the Danish Medical

Research Council, the Danish Cancer Society, the Novo Nordisk Foundation

and the Lundbeck Foundation. 0. N. and H. S. are members of the Danish Biomembrane Research Centre.


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  • Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease
    • Methods
    • Acknowledgements