Employing a recombinant HLA-DR3 expression system to dissect major histocompatibility complex II-thyroglobulin peptide dynamism: a genetic, biochemical, and reverse immunological perspective.
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.
Division of Endocrinology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45220, USA. Eric.Jacobson@uc.edu
Eric M
EM
Jacobson
Heyi
H
Yang
Francesca
F
Menconi
Rong
R
Wang
Roman
R
Osman
Luce
L
Skrabanek
Cheuk Wun
CW
Li
Mohammed
M
Fadlalla
Alisha
A
Gandhi
Vijaya
V
Chaturvedi
Eric P
EP
Smith
Sandy
S
Schwemberger
Andrew
A
Osterburg
George F
GF
Babcock
Yaron
Y
Tomer
D000465:Algorithms; D000818:Animals; D001327:Autoimmune Diseases; D002403:Cathepsins / Q000737:chemistry; D002460:Cell Line; D005786:Gene Expression Regulation; D015803:HLA-DR3 Antigen / Q000378:metabolism*; D006367:HeLa Cells; D000949:Histocompatibility Antigens Class II; D006801:Humans; D010455:Peptides / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013954:Thyroglobulin / Q000737:chemistry; D013959:Thyroid Diseases / Q000276:immunology; D013961:Thyroid Gland / Q000378:metabolism
D015803:HLA-DR3 Antigen; D000949:Histocompatibility Antigens Class II; D010455:Peptides; D011994:Recombinant Proteins; D013954:Thyroglobulin; D002403:Cathepsins