Gliadin peptides induce tissue transglutaminase activation and ER-stress through Ca2+ mobilization in Caco-2 cells.
BACKGROUND
Celiac disease (CD) is an intestinal inflammatory condition that develops in genetically susceptible individuals after exposure to dietary wheat gliadin. The role of post-translational modifications of gliadin catalyzed by tissue transglutaminase (tTG) seems to play a crucial role in CD. However, it remains to be established how and where tTG is activated in vivo. We have investigated whether gliadin peptides modulate intracellular Ca(2+) homeostasis and tTG activity.


RESULTS
We studied Ca(2+) homeostasis in Caco-2 cells by single cell microfluorimetry. Under our conditions, A-gliadin peptides 31-43 and 57-68 rapidly mobilized Ca(2+) from intracellular stores. Specifically, peptide 31-43 mobilized Ca(2+) from the endoplasmic reticulum (ER) and mitochondria, whereas peptide 57-68 mobilized Ca(2+) only from mitochondria. We also found that gliadin peptide-induced Ca(2+) mobilization activates the enzymatic function of intracellular tTG as revealed by in situ tTG activity using the tTG substrate pentylamine-biotin. Moreover, we demonstrate that peptide 31-43, but not peptide 57-68, induces an increase of tTG expression. Finally, we monitored the expression of glucose-regulated protein-78 and of CCAAT/enhancer binding protein-homologous protein, which are two biochemical markers of ER-stress, by real-time RT-PCR and western blot. We found that chronic administration of peptide 31-43, but not of peptide 57-68, induces the expression of both genes.


CONCLUSIONS
By inducing Ca(2+) mobilization from the ER, peptide 31-43 could promote an ER-stress pathway that may be relevant in CD pathogenesis. Furthermore, peptides 31-43 and 57-68, by activating intracellular tTG, could alter inflammatory key regulators, and induce deamidation of immunogenic peptides and gliadin-tTG crosslinking in enterocytes and specialized antigen-presenting cells.
Department of Chemistry and Biology, University of Salerno, Fisciano, Salerno, Italy. icaputo@unisa.it
Ivana
I
Caputo
Agnese
A
Secondo
Marilena
M
Lepretti
Gaetana
G
Paolella
Salvatore
S
Auricchio
Maria Vittoria
MV
Barone
Carla
C
Esposito
D000595:Amino Acid Sequence; D001710:Biotin / Q000031:analogs & derivatives / Q000378:metabolism; D015153:Blotting, Western; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics / Q000378:metabolism; D018938:Caco-2 Cells; D002118:Calcium / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000187:drug effects* / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects*; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D005903:Gliadin / Q000737:chemistry / Q000494:pharmacology*; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000494:pharmacology*; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D059010:Single-Cell Analysis; D011503:Transglutaminases / Q000235:genetics / Q000378:metabolism*
D022762:CCAAT-Enhancer-Binding Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D010446:Peptide Fragments; D001710:Biotin; D005903:Gliadin; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins; D002118:Calcium