Comparison of Antigenic Regions Identified on IgG1Fc Using Bioinformatics vs Pepscan Analysis

Abstract

Epitope mapping allowed the location of antigenic determinants on a protein macromolecule to be identifi ed. In particular, pepscan techniques that utilize a series of overlapping peptides, help detect key amino acid residues that are important in antibody recognition and binding. In a previous study, we employed 15-mer peptides spanning the entire length of IgG1Fc to ascertain successfully the target epitopes of isotypic/allotypic monoclonal reagents.As an extension to this work we have used these peptides to evaluate the location of epitope targets of fi ve IgM rheumatoid factor antibodies (RFAbs). Overall, 2 antibodies, RFAb TS2 and TS1, detected a similar epitope within the CH3 domain (360-KNQVSLTCLVKGFYP-374), whilst 1 (RFAb SJ1) recognised an epitope in the CH2 domain (294-EQYNSTYRVVSVLTV-308). In contrast, 2 RFAbs, PRSJ2 and PRTS1 detected four and fi ve epitopes respectively within the Fc region. RFAb PRSJ2 recognised epitopes detected by RFAB TS2 and TS1 but also further epitopes in the CH2 domain (256-TPEVTCVVVDVSHED-270) and CH3 domain (418-QQGNVFSCSVMHEAL-432). Similarly, RFAb PRTS1 detected all four epitopes plus a fi fth in the CH3 domain (382-ESNGQPENNYKTTPP-396). In essence there was a consensus of target epitopes identifi ed by these rheumatoid factor antibodies. Interestingly, two epitopes (256-270, CH2 domain and 360-374, CH3 domain) were novel in that they had not been identifi ed in previous pepscan studies. The other epitopes recognised, either overlapped or were immediately adjacent to previous epitopes detected by poly/monoclonal rheumatoid factor antibodies.Molecular modelling (PCImdad) of IgG1Fc showed that all fi ve epitopes were exposed and surface accessible for antibody interaction. In addition, a bioinformatics analysis of the Fc region using ExPASy was employed to identify key antigenic determinants. This 'in silico' approach may provide a means of determining key regions without the need to develop overlapping peptides spanning the entire length of a macromolecule.

Rheumatoid Arthritis is Associated with IgG Antibodies to Human Endogenous Retrovirus Gag Matrix: A Potential Pathogenic Mechanism of Disease?

“…This proposes an unwanted immune reaction to host protein(s) while mounting an immune response to virus. Interestingly, a preliminary alignment search of HERV-K10 matrix peptide MAG1 highlights an amino acid sequence (GKELK) similar to IgG1Fc (GKEYK), a key target for RF 16,34 . The finding of a unique epitope shared by virus and host protein could provide a novel molecular mimic that has the potential to trigger and/or augment an immune response in RA 35,36 .…”

“…Development of HERV-K10 Gag matrix peptide. Bioinformatic analysis was performed on HERV-K10 using established algorithms 16 . In brief, the sequence of HERV-K10 (accession no.…”

“…We have previously described the use of bioinformatic algorithms 16 to predict antigenic peptides of proteins and viruses that can be tested in ELISA 17 . In our study, we have determined a highly antigenic peptide on the matrix protein of HERV-K10 and assessed the mean IgG antibody response to this peptide in patients with RA, inflammatory bowel disease (IBD), osteoarthritis (OA), and healthy individuals.…”