Application of phage display peptide library to autoimmune diabetes: identification of IA-2/ICA512bdc dominant autoantigenic epitopes.
Autoantigenic epitope mapping represents a critical issue in autoimmune diseases. The islet tyrosine phosphatase-like protein IA-2/ICA512bdc is a major autoantigen in type 1 diabetes (IDDM), but the epitopes responsible for autoantibody binding have been only partially defined. The aim of our study was to identify ICA512bdc epitopes, and in particular mini-epitopes, utilizing a novel strategy for autoimmune diseases. The study was performed in three sequential steps: (1) construction of a lambda-phage surface-displayed ICA512bdc cDNA library with the methodology of tagged random priming with peptides displayed as a fusion to the C terminus of the capsid protein D; (2) affinity selection of the resulting library, followed by immunoscreening, enzyme-linked immunosorbent assay and sequence analysis of positive clones, and (3) radioimmunoprecipitation to detect autoantibodies to the selected clones. This strategy resulted in the identification of two epitopes (IA-2 residues 761 - 964 and 929 - 979), which were recognized by 100 % and 62.9 % ICA512bdc-positive IDDM patients, respectively. Interestingly, the larger clone was detected also by a proportion (16.7 %) of new onset ICA512bdc-negative patients, thus suggesting that this region contains not only the main autoantigenic repertoire of ICA512bdc molecule, but is able to detect IA-2 autoantibodies in even higher percentages of patients. In addition, this study showed the existence of multiple epitopes located in the C-terminal domain of the IA-2 protein, one of which is formed by the 50 C-terminal amino acids, and provided evidence that the strategy used represents a valid tool for identification of epitopes within autoantigenic molecules.
Farilla
Loredana
L
Department of Medical Sciences, Endocrinology, University La Sapienza, Rome, Italy.
Tiberti
Claudio
C
Luzzago
Alessandra
A
Yu
Liping
L
Eisenbarth
George S
GS
Cortese
Riccardo
R
Dotta
Francesco
F
Di Mario
Umberto
U
D000293:Adolescent; D001323:Autoantibodies / Q000276:immunology; D001324:Autoantigens / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D001667:Binding, Competitive; D002648:Child; D018076:DNA, Complementary / Q000235:genetics; D003922:Diabetes Mellitus, Type 1 / Q000235:genetics / Q000276:immunology*; D005260:Female; D006801:Humans; D016056:Immunodominant Epitopes / Q000235:genetics; D066298:In Vitro Techniques; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology*; D019151:Peptide Library; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000276:immunology*; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8
D001323:Autoantibodies; D001324:Autoantigens; D018076:DNA, Complementary; C096322:ICA512 autoantibody; D016056:Immunodominant Epitopes; D008565:Membrane Proteins; D019151:Peptide Library; C516862:PTPRN protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8