Isolation, Characterization, and Molecular Modeling of a Rheumatoid Factor from a Hepatitis C Virus Infected Patient with Sjögren's Syndrome

Lee, Yu-Ching; Tsai, Keng-Chang; Leu, Sy-Jye; Wang, Tuan-Jen; Liu, Chia-Yu; Yang, Yi-Yuan

From the results of competitive inhibition assay, a high binding affinity of RFL11 Fab having anti-human IgG Fc capability was confirmed. RFL11 shares high similarity with the L chain amino acid sequence of RF61 autoantibody, which has been reported, but is different from another autoantibody (PDB id: 1ADQ, lower binding affinity ∼ 6 × 10 −5 M) already published [12] . In addition, CDRs H2 and H3 on the H chain of RFL11 have two and three more amino acids than those of RF61, respectively, but the amino acid sequences on the frameworks are highly similar. Despite the fact that RFL11 and RF61 come from different immunoglobulin isotypes (IgG and IgM types, resp.), their antigens are both IgG Fc; accordingly, it can be reasonably inferred that the epitopes recognized by RFL11 and RF61 have a high similarity, and the docking simulation of RFL11 antibody and IgG Fc antigen by using zDOCK program [23] also revealed an epitope highly similar to that corresponding to RF61. In order to establish a reliable structure model of RFL11 antibody-Fc complex, we used structural alignment and CDR loop minimization to replace docking. From this approach, the dominating interactions of H2 and H3 loops between RFL11 and IgG Fc can be more clearly observed to further investigate why RFL11 demonstrates a superior binding affinity to RF61, as indicated from the experiments. Table 1 illustrates the epitope residues on IgG Fc. Gln438 and Lys439 on Fc individually form H-bonding and cationpi interactions with the CDR-H2 loop, so we can infer that Gln438 and Lys439 are key residues for both RFL11 and RF61 on the IgG Fc epitope. However, from the paratope of the CDR-H2 loop, Asn52 and Phe48 on the RFL11 CDR-H2 loop, generated by somatic mutation, respectively, form H-bonding and anion-pi interactions with respect to Gln438 and Asp356, indicating the importance of somatic mutation to the CDR-H2 loop. Compared with the RF61 CDR-H2 loop, the two additional amino acids Ser51 and Asn52 on the RFL11 CDR-H2 loop both interact with Gln438 on Fc with a strong Hbonding, showing that the RFL11 CDR-H2 loop has a binding affinity stronger than the RF61 CDR-H2 loop to IgG Fc.