| Summary: | 碩士 === 國立中興大學 === 分子生物學研究所 === 101 === P64k protein composed of 594 amino acid residues is a lipoamide dehydrogenase, the E3 component of the meningococcal meningococcal pyruvate dehydrogenase complex. An unusual feature of P64K is that it also contains a lipoyl domain of the E2 component (aa1-81) that is linked to the E3 region by an Alanine riched linker (aa110-594). It was first identified as the minor outer membrane of Neisseria meningitidis and is currently used as an immunological carrier for weak immunogens due to its strong immunogenicity. P64K is the target antigen of the monoclonal antibody (mAb) 107-01 obtained from a mouse immunized with heat-inactivated meningococci. The epitope of 107-01 is exposed on the meningococcal surface because 107-01 binds to intact meningococcal cells. The minimal region possessing the antigenic reactivity to mAb 107-01 has previously been defined to the E5-V146 region and the 6th residue was suggested to be critical by replacing the Leu with Pro. To further delineate the effect of the 6th residue on the binding of mAb 107-01 to the P64K, mutated P64K(M1-V146) fragments with Ala, Ile, Gly Asp, and Lys as the 6th residue were produced. The half maximal inhibitory concentrations (IC50) for the wild type, L6A, L6I, L6G, L4K and L6D mutants were 4.703, 3.906, 4.355, 4.531, 8.727, and 72.76 μg/mL, respectively. The results indicate that an aliphatic amino acid at the 6th position is critical for the 107-01 epitope on the P64K. Notably, although fragments M1-E139, M1-K142, and P9-V146 were not recognized by 107-01 in Western blot analysis, the IC50 for the M1-E139 and M1-K142 were lower than that of L6D which showed weak reactivity in the Western blot. In addition, the anti- M1-E139, anti- M1-K142, and anti-M1-V146 all exhibited strongest reactivity towards the M1-V146 fragment. Collectively, these results suggest that 107-01 recognizes a conformational epitope and residues140-146 play a key role in the stabilization of the 107-01 epitope conformation. This assumption is supported by the molecular modeling. The 3D-structure of mutated P64K fragments and the wild type M1-V146 fragment generated by using the I-TASSER server with the available 3D-structures (aa1-81 and 110-594) as the templates. The results showed that the P64k M1-V146 is composed of β-strands which form a flattened beta barrel within M1-T81 and disordered structure and α-helix in the remaining region. The flattened β barrel was changed significantly when the 6th residue is not an aliphatic amino acid. The β barrel in the M1-E139 is relative intact but that of P9-V146 is completed destroyed. In conclusion, the results of this study strongly suggest that mAb107-01 recognizes a conformational epitope that requires both the N-terminal 9 amino acid residues and aa140-146 to maintain its optimal structure
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