Molecular characterization of covalent complexes between tissue transglutaminase and gliadin peptides

Burkhard Fleckenstein, Shuo-Wang Qiao, Martin Røssel Larsen, Günther Jung, Peter Roepstorff, Ludvig M Sollid

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Tissue transglutaminase (TG2) modifies proteins and peptides by transamidation or deamidation of specific glutamine residues. TG2 also has a central role in the pathogenesis of celiac disease. The enzyme is both the target of disease-specific autoantibodies and generates deamidated gliadin peptides recognized by intestinal T cells from patients. Incubation of TG2 with gliadin peptides also results in the formation of covalent TG2-peptide complexes. Here we report the characterization of complexes between TG2 and two immunodominant gliadin peptides. Two types of covalent complexes were found; the peptides are either linked via a thioester bond to the active site cysteine of TG2 or via isopeptide bonds to particular lysine residues of the enzyme. We quantified the number of gliadin peptides bound to TG2 under different conditions. After 30 min of incubation of TG2 at 1 microm with an equimolar ratio of peptides to TG2, approximately equal amounts of peptides were bound by thioester and isopeptide linkage. At higher peptide to TG2 ratios, more than one peptide was linked to TG2, and isopeptide bond formation dominated. The lysine residues in TG2 that act as acyl acceptors were identified by matrix assisted laser desorption ionization and nanoelectrospray mass spectrometry and tandem mass spectrometry analysis of proteolytic digests of the TG2-peptide complexes. At a high molar excess of gliadin peptides to TG2 altogether six lysine residues of TG2 were found to participate in isopeptide bond formation. The results are relevant to the understanding of how antibodies to TG2 are formed in celiac disease.
Original languageEnglish
JournalJournal of Biological Chemistry
Volume279
Issue number17
Pages (from-to)17607-16
Number of pages10
ISSN0021-9258
DOIs
Publication statusPublished - 23. Apr 2004

Fingerprint

Gliadin
Peptides
Lysine
Celiac Disease
Tandem Mass Spectrometry
transglutaminase 2
Mass spectrometry
T-cells
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Enzymes
Glutamine
Autoantibodies
Ionization
Cysteine

Keywords

  • Amino Acid Sequence
  • Biotinylation
  • Blotting, Western
  • Dose-Response Relationship, Drug
  • Electrophoresis, Capillary
  • Electrophoresis, Polyacrylamide Gel
  • GTP-Binding Proteins
  • Gliadin
  • Glutathione Transferase
  • Humans
  • Lysine
  • Mass Spectrometry
  • Models, Molecular
  • Molecular Sequence Data
  • Peptides
  • Protein Binding
  • Protein Isoforms
  • Recombinant Fusion Proteins
  • Sequence Homology, Amino Acid
  • Spectrometry, Mass, Electrospray Ionization
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Time Factors
  • Transglutaminases

Cite this

Fleckenstein, Burkhard ; Qiao, Shuo-Wang ; Larsen, Martin Røssel ; Jung, Günther ; Roepstorff, Peter ; Sollid, Ludvig M. / Molecular characterization of covalent complexes between tissue transglutaminase and gliadin peptides. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 17. pp. 17607-16.
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abstract = "Tissue transglutaminase (TG2) modifies proteins and peptides by transamidation or deamidation of specific glutamine residues. TG2 also has a central role in the pathogenesis of celiac disease. The enzyme is both the target of disease-specific autoantibodies and generates deamidated gliadin peptides recognized by intestinal T cells from patients. Incubation of TG2 with gliadin peptides also results in the formation of covalent TG2-peptide complexes. Here we report the characterization of complexes between TG2 and two immunodominant gliadin peptides. Two types of covalent complexes were found; the peptides are either linked via a thioester bond to the active site cysteine of TG2 or via isopeptide bonds to particular lysine residues of the enzyme. We quantified the number of gliadin peptides bound to TG2 under different conditions. After 30 min of incubation of TG2 at 1 microm with an equimolar ratio of peptides to TG2, approximately equal amounts of peptides were bound by thioester and isopeptide linkage. At higher peptide to TG2 ratios, more than one peptide was linked to TG2, and isopeptide bond formation dominated. The lysine residues in TG2 that act as acyl acceptors were identified by matrix assisted laser desorption ionization and nanoelectrospray mass spectrometry and tandem mass spectrometry analysis of proteolytic digests of the TG2-peptide complexes. At a high molar excess of gliadin peptides to TG2 altogether six lysine residues of TG2 were found to participate in isopeptide bond formation. The results are relevant to the understanding of how antibodies to TG2 are formed in celiac disease.",
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author = "Burkhard Fleckenstein and Shuo-Wang Qiao and Larsen, {Martin R{\o}ssel} and G{\"u}nther Jung and Peter Roepstorff and Sollid, {Ludvig M}",
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Molecular characterization of covalent complexes between tissue transglutaminase and gliadin peptides. / Fleckenstein, Burkhard; Qiao, Shuo-Wang; Larsen, Martin Røssel; Jung, Günther; Roepstorff, Peter; Sollid, Ludvig M.

In: Journal of Biological Chemistry, Vol. 279, No. 17, 23.04.2004, p. 17607-16.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Molecular characterization of covalent complexes between tissue transglutaminase and gliadin peptides

AU - Fleckenstein, Burkhard

AU - Qiao, Shuo-Wang

AU - Larsen, Martin Røssel

AU - Jung, Günther

AU - Roepstorff, Peter

AU - Sollid, Ludvig M

PY - 2004/4/23

Y1 - 2004/4/23

N2 - Tissue transglutaminase (TG2) modifies proteins and peptides by transamidation or deamidation of specific glutamine residues. TG2 also has a central role in the pathogenesis of celiac disease. The enzyme is both the target of disease-specific autoantibodies and generates deamidated gliadin peptides recognized by intestinal T cells from patients. Incubation of TG2 with gliadin peptides also results in the formation of covalent TG2-peptide complexes. Here we report the characterization of complexes between TG2 and two immunodominant gliadin peptides. Two types of covalent complexes were found; the peptides are either linked via a thioester bond to the active site cysteine of TG2 or via isopeptide bonds to particular lysine residues of the enzyme. We quantified the number of gliadin peptides bound to TG2 under different conditions. After 30 min of incubation of TG2 at 1 microm with an equimolar ratio of peptides to TG2, approximately equal amounts of peptides were bound by thioester and isopeptide linkage. At higher peptide to TG2 ratios, more than one peptide was linked to TG2, and isopeptide bond formation dominated. The lysine residues in TG2 that act as acyl acceptors were identified by matrix assisted laser desorption ionization and nanoelectrospray mass spectrometry and tandem mass spectrometry analysis of proteolytic digests of the TG2-peptide complexes. At a high molar excess of gliadin peptides to TG2 altogether six lysine residues of TG2 were found to participate in isopeptide bond formation. The results are relevant to the understanding of how antibodies to TG2 are formed in celiac disease.

AB - Tissue transglutaminase (TG2) modifies proteins and peptides by transamidation or deamidation of specific glutamine residues. TG2 also has a central role in the pathogenesis of celiac disease. The enzyme is both the target of disease-specific autoantibodies and generates deamidated gliadin peptides recognized by intestinal T cells from patients. Incubation of TG2 with gliadin peptides also results in the formation of covalent TG2-peptide complexes. Here we report the characterization of complexes between TG2 and two immunodominant gliadin peptides. Two types of covalent complexes were found; the peptides are either linked via a thioester bond to the active site cysteine of TG2 or via isopeptide bonds to particular lysine residues of the enzyme. We quantified the number of gliadin peptides bound to TG2 under different conditions. After 30 min of incubation of TG2 at 1 microm with an equimolar ratio of peptides to TG2, approximately equal amounts of peptides were bound by thioester and isopeptide linkage. At higher peptide to TG2 ratios, more than one peptide was linked to TG2, and isopeptide bond formation dominated. The lysine residues in TG2 that act as acyl acceptors were identified by matrix assisted laser desorption ionization and nanoelectrospray mass spectrometry and tandem mass spectrometry analysis of proteolytic digests of the TG2-peptide complexes. At a high molar excess of gliadin peptides to TG2 altogether six lysine residues of TG2 were found to participate in isopeptide bond formation. The results are relevant to the understanding of how antibodies to TG2 are formed in celiac disease.

KW - Amino Acid Sequence

KW - Biotinylation

KW - Blotting, Western

KW - Dose-Response Relationship, Drug

KW - Electrophoresis, Capillary

KW - Electrophoresis, Polyacrylamide Gel

KW - GTP-Binding Proteins

KW - Gliadin

KW - Glutathione Transferase

KW - Humans

KW - Lysine

KW - Mass Spectrometry

KW - Models, Molecular

KW - Molecular Sequence Data

KW - Peptides

KW - Protein Binding

KW - Protein Isoforms

KW - Recombinant Fusion Proteins

KW - Sequence Homology, Amino Acid

KW - Spectrometry, Mass, Electrospray Ionization

KW - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

KW - Time Factors

KW - Transglutaminases

U2 - 10.1074/jbc.M310198200

DO - 10.1074/jbc.M310198200

M3 - Journal article

VL - 279

SP - 17607

EP - 17616

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 17

ER -