Mapping of myeloperoxidase epitopes recognized by MPO-ANCA using human-mouse MPO chimers (1)
Antineutrophil cytoplasmic autoantibodies (ANCA) directed against myeloperoxidase (MPO) or proteinase 3 are associated with pauci-immune necrotizing and crescentic glomerulonephritis, microscopic polyangiitis, and Wegener’s granulomatosis. Sera from 64 and 71% of patients bound to the carboxy-terminus of the heavy chain, in the regions of amino acids 517–667 or 668–745, respectively. No patient serum bound the MPO light chain or the aminoterminus of the heavy chain.
Despite an 85% amino-acid identity (90% homology) between the human and mouse MPO sequences, the majority of human MPO-ANCA do not bind mouse MPO.
The heavy chain of MPO was ‘divided’ into four segments, namely A, B, C, and D.
Human and mouse recombinant MPO and five of the six chimeric MPO molecules were successfully expressed in human embryonic kidney 293 cells and the purified recombinant proteins verified by Western blot analysis.
Based on the binding pattern of sera from our 14 patients, sera from 0 patients bound to region A (amino acids (a.a.) 1–108 of heavy chain), sera from six patients bound to region B (a.a. 109–237), sera from nine patients bound to region C (a.a. 238–388), and sera from 10 patients bound to region D (a.a. 389–469) at some point during the course of disease. Sera from six patients bound to one region, sera from six patients reacted to two regions, and sera from two patients to possibly three regions over time.
Sera from 71% of patients did not bind recombinant mouse MPO. Similarly, serum from only 1 of 36 (2.7%) patients with MPO-ANCA vasculitis bound rat MPO. MPO-ANCA recognize epitopes on human MPO that are absent on rat or mouse MPO. All the patient sera bound to the heavy chain, and none bound to the light chain. Based on the crystal structure of MPO, the three-dimensional model of MPO reveals that, in the dimer form, the light chain is largely ‘hidden’ in the groove between the two MPO monomers and is poorly ‘accessible’ to antibody binding.
