Employing a Recombinant HLA-DR3 Expression System to Dissect Major Histocompatibility Complex II-Thyroglobulin Peptide Dynamism: A GENETIC, BIOCHEMICAL, AND REVERSE IMMUNOLOGICAL PERSPECTIVE

• Published in: The Journal of biological chemistry 2009-12, Vol.284 (49), p.34231-34243
• Main Authors: Jacobson, Eric M, Yang, Heyi, Menconi, Francesca, Wang, Rong, Osman, Roman, Skrabanek, Luce, Li, Cheuk Wun, Fadlalla, Mohammed, Gandhi, Alisha, Chaturvedi, Vijaya, Smith, Eric P, Schwemberger, Sandy, Osterburg, Andrew, Babcock, George F, Tomer, Yaron
• Format: Article
• Language: English
• Subjects: Algorithms; Animals; Autoimmune Diseases; Cathepsins - chemistry; Cell Line; Gene Expression Regulation; HeLa Cells; Histocompatibility Antigens Class II; HLA-DR3 Antigen - metabolism; Humans; Peptides - chemistry; Protein Structure and Folding; Rats; Recombinant Proteins - chemistry; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thyroglobulin - chemistry; Thyroid Diseases - immunology; Thyroid Gland - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109.041574

Previously, we have shown that statistical synergism between amino acid variants in thyroglobulin (Tg) and specific HLA-DR3 pocket sequence signatures conferred a high risk for autoimmune thyroid disease (AITD). Therefore, we hypothesized that this statistical synergism mirrors a biochemical interaction between Tg peptides and HLA-DR3, which is key to the pathoetiology of AITD. To test this hypothesis, we designed a recombinant HLA-DR3 expression system that was used to express HLA-DR molecules harboring either AITD susceptibility or resistance DR pocket sequences. Next, we biochemically generated the potential Tg peptidic repertoire available to HLA-DR3 by separately treating 20 purified human thyroglobulin samples with cathepsins B, D, or L, lysosomal proteases that are involved in antigen processing and thyroid biology. Sequences of the cathepsin-generated peptides were then determined by matrix-assisted laser desorption ionization time-of-flight-mass spectroscopy, and algorithmic means were employed to identify putative AITD-susceptible HLA-DR3 binders. From four predicted peptides, we identified two novel peptides that bound strongly and specifically to both recombinant AITD-susceptible HLA-DR3 protein and HLA-DR3 molecules expressed on stably transfected cells. Intriguingly, the HLA-DR3-binding peptides we identified had a marked preference for the AITD-susceptibility DR signatures and not to those signatures that were AITD-protective. Structural analyses demonstrated the profound influence that the pocket signatures have on the interaction of HLA-DR molecules with Tg peptides. Our study suggests that interactions between Tg and discrete HLA-DR pocket signatures contribute to the initiation of AITD.