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

To further study and characterize these pathogenic RFs, it is important to comprehend the interaction between RF and Fc as well as to realize the influence of somatic mutations on antibody genes. After a comparison of the heavy chain and light chain variable gene sequence with germline counterpart, RFL11 was identified by ten probable somatic mutations, two in frameworks (Tyr88Phe (VL) and Gln16His (VH)) and eight in CDRs (Asp93Gln and Ser95Asn (L3); Tyr54Phe and Lys58Asn (H2); Asn108Thr, Tyr109Thr, Tyr110Asp, and Tyr111Phe (H3)) ( Figure 6 ). Unlike the antibody gene variations isolated in our previous works, where a substantial amount of somatic mutations occurs in CDRs or even in the framework regions in order to fight against exogenous pathogens, these pathogenic RFs caused by immune system disorders, however, possess a sequence similar to the endogenous germline sequence on the genetic level, except that critical mutations occur on the CDRs to provide a binding-specific ability to the IgG Fc domain. This implies that the generation of RF autoimmune antibodies is attained by critical changes which convert physiological RFs into pathogenic RFs. In general, output phage value after enrichment implies the success of the panning process if greater than 50-fold [27] . At lower enrichment levels, nonspecific and low affinity The Scientific World Journal 9 L2 H3 L1 H1 L3 H2 CDR color RFL11 scFv-Fc complex model Figure 8 : The interface structure of the RFL11-Fc was focused on the CDR-H2 (colored in blue) and CDR-H3 (colored in magenta) loops in this panel. Human IgG Fc antigen was colored in cyan. The colors of the six CDRs of RFL11 were the same as that in Figure 6 . The format of residues of Fc antigen and H2/H3 loops is abbreviated as three words and residues codes for distinguishing between antigen and antibody, respectively. The green and red dotted lines in the above panel represent H-bonding and anion-cation interactions of RFL11 with Fc, respectively. In the below panel, the green, red, and yellow dotted lines show H-bonding, cation-pi, and hydrophobic interactions, respectively. binders could cause outgrowth in the output phage after amplification. Moreover, in the panning process, although eluted phage titer with mild increasing only raised one order of magnitude after final round of panning, we still enriched and isolated specific phage binders from the original library successfully (Figure 1(a) ). It is interesting to show that the output phage numbers in our panning process had instant increment after the third cycle, although it dropped slightly in the second round (Figure 1(a) ). This result agrees with other studies [28, 29] . After sequencing of isolated clone following the final panning, almost all clones were identified to be similar. It is another verification to convince an efficient panning process resulting in high affinity clone to become dominant in the final library pool.