A peptide mimic blocks the cross-reaction of anti-DNA antibodies with glomerular antigens

Abstract

Anti-DNA antibodies play a pivotal role in the pathogenesis of lupus nephritis by cross-reacting with renal antigens. Previously, we demonstrated that the binding affinity of anti-DNA antibodies to self-antigens is isotype-dependent. Furthermore, significant variability in renal pathogenicity was seen among a panel of anti-DNA isotypes [derived from a single murine immunoglobulin (Ig)G3 monoclonal antibody, PL9-11] that share identical variable regions. In this study, we sought to select peptide mimics that effectively inhibit the binding of all murine and human anti-DNA IgG isotypes to glomerular antigens. The PL9-11 panel of IgG anti-DNA antibodies (IgG1, IgG2a, IgG2b and IgG3) was used for screening a 12-mer phage display library. Binding affinity was determined by surface plasmon resonance. Enzyme-linked immunosorbent assay (ELISA), flow cytometry and glomerular binding assays were used for the assessment of peptide inhibition of antibody binding to nuclear and kidney antigens. We identified a 12 amino acid peptide (ALWPPNLHAWVP, or 'ALW') which binds to all PL9-11 IgG isotypes. Preincubation with the ALW peptide reduced the binding of the PL9-11 anti-DNA antibodies to DNA, laminin, mesangial cells and isolated glomeruli significantly. Furthermore, we confirmed the specificity of the amino acid sequence in the binding of ALW to anti-DNA antibodies by alanine scanning. Finally, ALW inhibited the binding of murine and human lupus sera to dsDNA and glomeruli significantly. In conclusion, by inhibiting the binding of polyclonal anti-DNA antibodies to autoantigens in vivo, the ALW peptide (or its derivatives) may potentially be a useful approach to block anti-DNA antibody binding to renal tissue.

Keywords: SLE; anti-DNA antibodies; lupus nephritis; phage display library.

Figure 1

Anti‐DNA antibodies bind to the ALWPPNLHAWVP (ALW) peptide. (a) Representative graph of surface plasmon resonance (SPR) analysis for ALW peptide (0–250 nM) binding to PL9‐11 immunoglobulin (Ig)G3. (b) Representative graph of SPR analysis for PL9‐11 isotypes binding to the ALW peptide (120 nM). (c) PL9‐11 antibodies bind to biotin‐labelled ALW in order of IgG2b > IgG2a > IgG3 > IgG1. Polyclonal mouse IgG was used as control. The difference in binding affinity observed between the panels in this figure reflects methodological variations in both the peptide antigen preparation and binding assay, with the peptide immobilized on the chip and the binding measured by SPR in (a) and (b), versus biotin labelling and enzyme‐linked immunosorbent assay (ELISA) in (c), respectively. Data were from three independent experiments.

Figure 2

The ALWPPNLHAWVP (ALW) peptide inhibits the binding of PL9‐11 antibodies to dsDNA and laminin. (a) The ALW peptide (0·16–80 µg/ml) inhibits the binding of PL9‐11 isotypes (2 µg/ml) to dsDNA in the order of immunoglobulin (Ig)G2b > IgG2a > IgG1 > IgG3. (b) The graph for one site binding curve analysis is shown. (c) ALW inhibits the binding of PL9‐11 antibodies (2 µg/ml) to laminin in the order of IgG2b > IgG3 > IgG1 > IgG2a. The inhibitory capacity of the unmodified ALW in PL9‐11 binding to dsDNA and laminin was higher than that of the scrambled peptide (data not shown). (a,c) Representative graphs from three independent experiments are shown.

Figure 3

The ALWPPNLHAWVP (ALW) peptide inhibits PL9‐11 antibody binding to DNase I‐treated mesangial cells and glomeruli. (a) ALW inhibits the binding of PL9‐11 immunoglobulins (Ig)Gs to mesangial cells. Serial dilutions from an initial peptide concentration of 80 µg/ml were tested in this experiment. (b,c) Flow cytometry shows a significant decrease in both the percentage of positive cells (b) and mean fluorescence intensity (MFI) (c) after PL9‐11 IgG2a was preincubated with the ALW peptide before binding to the mesangial cells. The other PL9‐11 isotypes showed similar results (not shown). (a–c) Data are representative of three independent experiments. (d) ALW, but not the control peptides (P1, PLP), inhibit the binding of PL9‐11 IgG3 to isolated glomeruli. There were no significant differences in the inhibition between the three control groups. The bottom part of the panel depicts immunoglobulin binding to a single glomerulus fixed to the glass slide. (e) Binding of PL9‐11 IgG2a and IgG2b to glomeruli was inhibited similarly by ALW. (d,e) Ten glomeruli in each group were stained, and representative images are shown. Scale bar = 25 µm. *P < 0·05, compared to the no ALW (or control peptide) group accordingly.

Figure 4

Alanine scanning of the ALWPPNLHAWVP (ALW) peptide. (a) Alanine replacement at the third or eighth amino acid residue decreases immunoglobulin (Ig)G3 binding to ALW. The other PL9‐11 IgG isotypes exhibited similar results (not shown). (b) In both direct and inhibition enzyme‐linked immunosorbent assays (ELISAs), reduction of peptide binding was most seen frequently when either the third (W) or eighth (H) amino acid residue was replaced by alanine. The optical density (OD) values were normalized to the original ALW peptide, and the degree of binding or inhibition is indicated by the shade of colour.

Figure 5

Identification of isotype specific peptide sequences. (a) Four phage clones were used for phage inhibition enzyme‐linked immunosorbent assays (ELISAs), each of them selected exclusively by a single PL9‐11 isotype during library screening. ++ = Strong binding; + = moderate binding; – = weak or no binding. (b,c) The immunoglobulin (Ig)G3‐selected VHLQAGELMNPK (VHL) phage only inhibited PL9‐11 IgG3 but not the binding of the other isotypes to dsDNA (b) or laminin (c). (d) The binding of PL9‐11 IgG2a to dsDNA was inhibited exclusively by the phage (TPM) selected singularly by that particular isotype. (e) Similar inhibition specificity was also seen with PL9‐11 IgG3 selected phage. Three independent experiments were performed, and representative graphs are shown in panels (b) to (e).

Figure 6

ALWPPNLHAWVP (ALW) inhibits lupus sera binding to self‐antigens. (a,b) Sera from Murphy Roths Large lymphoproliferation (MRL)lpr) mice (n = 6) binding to dsDNA (a) or laminin (b) were inhibited by ALW. (c,d) ALW inhibition of the binding of Sle1.3 mice sera (n = 8) to dsDNA (c) and laminin (d). (e,f) ALW inhibition of the binding to dsDNA (e) or laminin (f) by sera from lupus patients (n = 10). Each individual line in the graphs from (a) to (f) represents the binding of an individual serum sample. (g) Preincubation with ALW decreased the binding of human and mouse lupus sera (n = 3 of each group) to isolated glomeruli (n = 4 for each serum sample). (h) Representative images of the glomerular binding assay results are shown. Scale bar = 25 µm. *P < 0·05, comparing binding intensity with and without ALW.