Isolation, characterization, and molecular modeling of a rheumatoid factor from a Hepatitis C virus infected patient with Sjögren's syndrome

Abstract

We have previously isolated several IgG rheumatoid factors (RFs) from patients with both rheumatoid arthritis and idiopathic thrombocytopenia purpura using phage display system. To study IgG RFs in patients with other autoimmune diseases, phage display antibody libraries from a hepatitis C virus infected patient with Sjögren's syndrome were constructed. After panning, a specific clone RFL11 was isolated for characterization in advance. The binding activity and specificity of RFL11 to IgG Fc fragment were comparable to those of RFs previously isolated. The analysis with existed RF-Fc complex structures indicated the homology model of RFL11 is similar to IgM RF61 complex with high binding affinity of about 6 × 10⁻⁸ M. This effect resulted from longer complementarity-determining region (CDR) combining key somatic mutations. In the RFL11-Fc interfaces, the CDR-H3 loop forms a finger-like structure extending into the bottom of Fc pocket and resulting in strong ion and cation-pi interactions. Moreover, a process of antigen-driven maturation was proven by somatically mutated VH residues on H2 and H3 CDR loops in the interfaces. Taken together, these results suggested that high affinity IgG RFs can be generated in patients with Sjögren's syndrome and may play an important role in the pathogenesis of this autoimmune disease.

Figure 1

(a) Eluted phage titers after each round of panning. (b) Western blotting analysis of IPTG-induced Fab antibody expression under nonreducing condition. The protein patterns were visualized using goat anti-human λ light chain antibodies. (c) Randomly selected clones from 4th panned library were examined for their binding to human Fc fragment by ELISA. GG3 and GG48 denoted previously isolated clones expressing recombinant Fab molecules with Fc-binding activity. BL12 and LPSL are negative controls of irrelevant Fab molecules expressed in E. coli. Bound Fab was detected using goat anti-human λ light chain antibodies.

Figure 2

The Fc-binding reactivity of RFL11 clone using Western blot analysis. Panel (a) showed human Fc fragment on a Coomassie blue-stained polyacrylamide gel. After transferred onto the nitrocellulose membrane, the Fc fragments were detected by serum of HCV-infected patient with Sjögren's syndrome (b) and RFL11 (c) but not an irreverent Fab molecule in cellular lysate (d).

Figure 3

The binding specificities of the RFL11-Fc binder. Fab molecules were analyzed comparatively against Fc fragment and a panel of five unrelated antigens, including collagen, KLH, LPS, ssDNA, and thyroglobulin. GG3 and BL12 denoted positive and negative Fab controls, respectively.

Figure 4

Competitive inhibition assay of RFL11 Fab antibody against the Fc fragment. The amount of bound Fab in the presence of free Fc inhibitor was measured and expressed as a percentage of the binding of Fab in the absence of an inhibitor. B and B0 denoted the amount of bound Fab in the presence and absence of the inhibitor, respectively.

Figure 5

Sequence analysis of variable regions of light chain (VL) in the panel (a) and heavy chain (VH) in the panel (b) of RFL11 Fab antibody. The sequences of putative germline counterpart were included for comparison. Sequence gaps were introduced to maximize alignment as indicated by blank spaces. The boundaries of framework region (FR) and complementarity-determining region (CDR) were indicated above each germline gene sequences.

Figure 6

Multiple sequence alignment of variable domains of the light and heavy chains of RFL11 with 2J6E and 1ADQ antibodies. The background of the residues is colored according to sequence similarity. Deep blue color shows conserved residues in all sequences. The color scheme is from deep blue and light blue to cyan corresponding to identify highly and low conserved residues, respectively. The residue backgrounds colored white are not similar. Residues of somatic mutation of RFL11 have a red background. Residues of the CDRs are indicated by bold letters in red.

Figure 7

Comparison of RFL11 and RF61 (PDB id: 2J6E) complexes with human IgG Fc. The orientation of CDRs in both RFs was colored identically, except H2 and H3 loops. The affinity improvement by slight difference in the interactions was discussed particularly in advance.

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.