Production of a panel of recombinant gliadins for the characterisation of T cell reactivity in coeliac disease.
OBJECTIVE
Coeliac disease is a chronic intestinal disorder most probably caused by an abnormal immune reaction to wheat gliadin. The identification of the HLA-DQ2 and HLA-DQ8 as the molecules responsible for the HLA association in coeliac disease strongly implicates a role for CD4 T cells in disease pathogenesis. Indeed, CD4 T cells specific for gliadin have been isolated from the small intestine of patients with coeliac disease. However, identification of T cell epitopes within gliadin has been hampered by the heterogeneous nature of the gliadin antigen. To aid the characterisation of gliadin T cell epitopes, multiple recombinant gliadins have been produced from a commercial Nordic wheat cultivar.


METHODS
The alpha-gliadin and gamma-gliadin genes were amplified by polymerase chain reaction from cDNA and genomic DNA, cloned into a pET expression vector, and sequenced. Genes encoding mature gliadins were expressed in Escherichia coli and tested for recognition by T cells.


RESULTS
In total, 16 alpha-gliadin genes with complete open reading frames were sequenced. These genes encoded 11 distinct gliadin proteins, only one of which was found in the Swiss-Prot database. Expression of these gliadin genes produced a panel of recombinant alpha-gliadin proteins of purity suitable for use as an antigen for T cell stimulation.


CONCLUSIONS
This study provides an insight into the complexity of the gliadin antigen present in a wheat strain and has defined a panel of pure gliadin antigens that should prove invaluable for the future mapping of epitopes recognised by intestinal T cells in coeliac disease.
Arentz-Hansen
E H
EH
Institute of Immunology, Rikshospitalet, University of Oslo, Norway.
McAdam
S N
SN
Molberg
O
O
Kristiansen
C
C
Sollid
L M
LM
D000595:Amino Acid Sequence; D002446:Celiac Disease / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D018604:Epitope Mapping / Q000379:methods; D005903:Gliadin / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D006801:Humans; D007421:Intestine, Small / Q000276:immunology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis; D016176:T-Lymphocyte Subsets / Q000276:immunology*
D011994:Recombinant Proteins; D005903:Gliadin