ScientificWorldJournal. 2013 Dec 30;2013:516516.

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

Lee YC, Tsai KC, Leu SJ, Wang TJ, Liu CY, Yang YY.

1The Institute for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei 110, Taiwan ; Antibody and Hybridoma Core Facility, Taipei Medical University, Taipei 110, Taiwan.

 

ABSTRACT:

We have previously isolated several IgG rheumatoid factors (RFs) from both patients with rheumatoid arthritis and idiopathic thrombocytopenia purpura using phage display system. To study IgG RFs in patients with other autoimmune disease, 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 about 6 x 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 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.

Key words: rheumatoid factor, phage display, Sjögren’s syndrome, homology model

PMID: 24489505

 

Acknowledgments

We thank the National Science Council for financial support (NSC 99-2628-B-038-002-MY3). We are grateful to the National Center for High-performance Computing in Taiwan for computer time, Discovery Studio software and facilities.

Contact information

Yi-Yuan Yang, Ph.D.,
School of Medical Laboratory Science and Biotechnology,
College of Medical Science and Technology,
Taipei Medical University,
#250 Wu-Hsing Street, Taipei, Taiwan 110
Tel: +886-2-27361661 ext.3325
Fax: +886-2-27324510
E-mail: yangyuan@tmu.edu.tw

Fig. 1Figure 1. (A) Eluted phage titers after each round of panning. (B) Western blotting analysis of IPTG-induced Fab antibody expression under non-reducing condition. The protein patterns were visualized using goat anti-human l 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 l light chain antibodies.

 

Fig. 2Figure 2. 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, 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.

Fig. 3Figure 3. 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 as the same color as the Figure 6. The format of residues of Fc antigen and H2/H3 loops are 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.

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