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

作者：Iurascu Marius Ionut; Belaunzanar Osiris Marroquin; Cozma Claudia; Petrausch Ulf; Renner Christoph^*; Przybylski Michael^*

来源：Journal of the American Society for Mass Spectrometry, 2016, 27(6): 1105-1112.

DOI：10.1007/s13361-016-1361-9

• Affinity mass spectrometry
• Proteolytic epitope excision
• HLA B27
• Ankylosing Spondylitis
• Mixed-disulfide linked epitope
• Affinity quantification

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 beta 2-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 K-D towards the antibodies were determined using a surface acoustic wave (SAW) biosensor, and showed the highest affinity with a K-D of approximately 40 nM to the HD6 antibody for the (203-219)-SS-(257-273) mixed disulfide epitope.

• 出版日期2016-6