An HLA-B27 Homodimer Specific Antibody Recognizes a Discontinuous Mixed-Disulfide Epitope as Identified by Affinity-Mass Spectrometry

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2016
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Iuraşcu, Marius-Ionuţ
Marroquin Belaunzanar, Osiris
Petrausch, Ulf
Renner, Christoph
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HLA-B27 homodimer formation is believed to be a hallmark of HLA-B27 associated spondyloarthritides. Recently, we have generated a homodimer-specific monoclonal antibody (HD6) and have demonstrated that HLA-B27 homodimer complexes are present on monocytes of healthy HLA-B27 gene carriers at low levels, with significantly increased levels at active disease. The capability of the HD6 antibody to discriminate between correctly formed HLA-B27 heterotrimers and pathology-associated homodimers is striking and cannot be explained by the primary structure of HLA-B27. We hypothesized that HD6 accesses a unique epitope and used affinity-mass spectrometry for its identification. The HD6 antibody was immobilized on an activated sepharose affinity column, and HLA-B27 homodimer characterized for affinity. The epitope was identified by proteolytic epitope excision and MALDI mass spectrometry, and shown to comprise a discontinuous Cys-203- 257-Cys mixed-disulfide peptide structure that is not accessible in HLA-B27 heterotrimers due to protection by noncovalently linked β2-microglobulin. The epitope peptides were synthesized by solid phase peptide synthesis, and the two monomeric peptide components, HLA-B27(203-219) and HLA-B27(257-273), as well as the homo- and hetero-dimeric disulfide linked combinations prepared. The affinity binding constants KD towards the antibodies were determined using a surface acoustic wave (SAW) biosensor, and showed the highest affinity with a KD of approximately 40 nM to the HD6 antibody for the (203-219)-SS-(257-273) mixed disulfide epitope. Graphical Abstract ᅟ.

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ISO 690IURAŞCU, Marius-Ionuţ, Osiris MARROQUIN BELAUNZANAR, Claudia COZMA, Ulf PETRAUSCH, Christoph RENNER, Michael PRZYBYLSKI, 2016. An HLA-B27 Homodimer Specific Antibody Recognizes a Discontinuous Mixed-Disulfide Epitope as Identified by Affinity-Mass Spectrometry. In: Journal of The American Society for Mass Spectrometry. 2016, 27(6), pp. 1105-1112. ISSN 1044-0305. eISSN 1879-1123. Available under: doi: 10.1007/s13361-016-1361-9
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@article{Iurascu2016HLAB2-34898,
  year={2016},
  doi={10.1007/s13361-016-1361-9},
  title={An HLA-B27 Homodimer Specific Antibody Recognizes a Discontinuous Mixed-Disulfide Epitope as Identified by Affinity-Mass Spectrometry},
  number={6},
  volume={27},
  issn={1044-0305},
  journal={Journal of The American Society for Mass Spectrometry},
  pages={1105--1112},
  author={Iuraşcu, Marius-Ionuţ and Marroquin Belaunzanar, Osiris and Cozma, Claudia and Petrausch, Ulf and Renner, Christoph and Przybylski, Michael}
}
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