2 Results and discussion
2.5 Interaction studies of autoantibodies against Rheumatoid Arthritis with nuclear
2.5.2 Binding affinity of RA33 epitope peptides to autoantibodies
RA33 (hnRNP A2/B1) was determined to be a one major autoantigen in RA patients and anti-RA33 autoantibodies were found to appear shortly after onset of RA.
Therefore, identification of autoantibody-specific RA33 epitope(s) is of high interest.
El-Kased et al. have identified several epitopes using a recombinant RA33 protein in interaction studies with a monoclonal anti-RA33 antibody (peptide 1, Table 11) and the RA33 autoantibodies (peptides 2, 3, 4 & 5) isolated from the sera of Rheumatoid
Table 11: Amino acid sequences of epitopes identified by El-Kased et al. using a recombinant RA33 protein in interaction with monoclonal anti-RA33 antibody (peptide 1) and RA33 autoantibodies (peptides 2, 3, 4 & 5) isolated from the sera of Rheumatoid Arthritis patients.
Peptide
code Peptide sequence Epitope Ref.
1 78MAARPHSIDGRVVEP92 epitope [212]
2 245GYGGG249 epitope [213]
3 59RSRGFGF65 cryptic [213]
4 111KKLFVG116 cryptic [213]
5 266NQQPSNYG273 epitope [213]
The epitope sequence, 78MAARPHSIDGRVVEP92 (peptide 1) was identified using recombinant RA33 protein in interaction with a monoclonal anti-RA33 antibody [212], by subjecting the recombinant RA33 protein to BrCN cleavage and separating the resulting fragments by SDS-PAGE. Western blotting of the SDS-PAGE separation showed a positive response of the monoclonal anti RA33 antibody for several bands.
The BrCN-derived RA33 fragments were also separated by RP-HPLC and analyzed by dot blotting for antibody specificity. The region identified on the dot blot was compared with data obtained by peptide chip analysis using 15-meric synthetic peptides attached to a glass surface. The results from these analyses demonstrated that the epitope is located within the region of amino acids [84-92].
Figure 77 shows the sequence of the recombinant RA33 protein used by El-Kased et al. for epitope identification. The sequence resembles the A2 isoform of RA33 with an additional His-tag at the N-terminus, but lacks the 40 amino acids following residue 250 found in the A2 isoform. The identified epitope (residues 84-92) consists of two β-sheet motifs linked with a hairpin [212].
1MSHHHHHHHH10SMEREKEQFRK LFIGGLSFET TEESLRNYYE QWGKLTDCVV MRDPASKRSR GFGFVTFSSM AEVDAAMAAR PHSIDGRVVE PKRAVAREES GKPGAHVTVK KLFVGGIKED TEEHHLRDYF EEYGKIDTIE IITDRQSGKK RGFGFVTFDD HDPVDKIVLQ KYHTINGHNA EVRKALSRQE MQEVQSSRSG RGGNFGFGDS RGGGGNFGPG PGSNFRGGSD GYGSGRGFGD GYNGYGGGPG GGNFGGSPGY GGGRGGYGGG GPGYGNQGGG YGGGYDNYGG GNYGSGNYND FGNYNQQPSN YGPMKSGNFG GSRNMGGPYG GGNYGPGGSG GSGGYGGRSR YLE314
a
b
Figure 77: (a) Amino acids sequence of recombinant RA33. The sequence resembles the A2 isoform of RA33 with an additional His-tag at the N-terminus (italic). The recombinant RA33 lacks 40 amino acids after residue 250 compared to the A2 isoform. The identified epitope (residues 84-92) is printed in purple and is underlined. The black underlined residues 78-84 were added to the epitope sequence and synthesised as epitope peptide 1 for present studies. (b) Ribbon structure of hnRNP-A1 (PDB 1HA1). The epitope is highlighted in purple [212].
Residues [78-84] were added to the epitope sequence and the resulting peptide synthesised (epitope peptide RA 1) for SAW interaction studies with the monoclonal anti-RA33 antibody. A second peptide comprisiong residues [55-71] and designed as peptide 6 showed no interaction with the mouse anti-RA33 antibody by peptide chip ananlysis. Peptide sequence 6, [57-719], H-57SKRSRGFGFVTFSSM71-NH2 was synthesised as a negative control. The peptides were synthesised using the SPPS/Fmoc
strategy, further purified and characterised by mass spectrometry. As an example, the HPLC chromatogram and MS spectrum of peptide 1 are shown in Figure 78.
1 : H-78MAARPHSIDGRVVEP92-NH2
Time (min) m/z
a b
Figure 78: RP-HPLC chromatogram (a) and ESI-mass spectrum (b) of peptide 1.
Further analysis of the affinity binding of monoclonal anti-RA33 antibody with peptides 1 and 6 was performed with the SAW biosensor. After activation of the SAM-COOH surface with EDC/NHS, peptide 1 was covalently linked to channels 1 and 2 and the control peptide 6 to channels 4 and 5 using a 10 µM peptide solution for 40 min. Affinity binding experiments were performed after blocking the nonspecific by injection of a 100 nM anti-Lysozyme antibody solution. The affinity binding was monitored with injection of 200 nM m anti-RA33 antibody (Figure 79a). SAW binding curves and dot blot analysis of peptides 1 and 6 showed that only peptide 1 was recognised by the antibody (Figure 79).
-200 0 200 400 600 800 1000
H-57SKRSRGFGFVTFSSM71-NH2 6
H-78MAARPHSIDGRVVEP92-NH2 1
H-57SKRSRGFGFVTFSSM71-NH2 6
H-78MAARPHSIDGRVVEP92-NH2 1
Figure 79: (a) SAW binding curves and dot blot analysis of monoclonal anti-RA33 antibody to immobilised peptides 1 and 6. (b) Graphical evaluation of the affinity binding of the monoclonal anti-RA33 antibody. Peptide 1 was recognised by the monoclonal anti-RA33 antibody, whereas peptide 6 showed no binding.
The KD for peptide 1 was determined using a linear regression of the fitted kobs value versus the antibody concentration; KD value determined to be approximately 29 nM (Figure 80b). The antibody concentrations used in this analysis ranged from 8 to 128 nM and 0.1 M HCl was used as the regeneration solution.
-200 0 200 400 600 800 1000 1200 antibody with the immobilised peptide 1. (b) The kobs values, achieved by fitting the experimental data with the associated software, are linearly plotted against the concentration of the antibody dilutions. KD is determined by division of the intercept by the slope of the linear fit. KD value of approximately 29 nM was determined.
Since monoclonal antibodies normally show high affinity towards their antigen, as determined in the present KD in the low nanomolar range, this result is consistent with the assignement of peptide 1 being the epitope for the anti-RA33 antibody.
El-Kased et al. also identified the epitopes for autoantibodies from sera of rheumatoid arthritis patients [213]. Mass spectrometric epitope mapping, in combination with peptide chip analysis, identified four sequence motifs on RA33 protein (Table 11). The sequential epitope motif 245GYGGG249 has been found by El-Kased et al. at the C-terminal part of RA33 which matches the Western blot results using the full length protein. Therefore, this motif has been regarded as a disease associated epitope. Additonally, the N-terminal sequences of 59RSRGFGF65 and
111KKLFVG116 and the C-terminal sequence of 266NQQPSNYG273, have been identified as “cryptic epitopes” by peptide chip analysis. These motifs are illustrated in Figure 81 and correlates with the epitope-containing regions as determined by mass spectrometry (Figure 81a) [213].
a
b
Figure 81: Structural representations of recombinant RA33. The three-dimensional structure representation has been modeled using the “I-TASSER”
algorithm (http://zhang.bioinformatics.ku.edu/I-TASSER). The molecular surface is indicated with van der Waals spheres. The His-tag region is displayed in black, domain I in yellow, domain II in blue, and domain III in green. (a) The epitope-containing regions as determined by mass spectrometry are shown in red. (b) The four epitope motifs as derived from the peptide chip analysis are displayed in red and are circled. Sequence ranges of the motifs are given [100].
Therefore the epitopes peptides 1-5 (Table 11) were used for further interaction studies with autoantibodies from four RA patient samples obtained from the Department of Immunology (Prof. Dr. Glocker, University of Rostock). All 4 patients sera were examined for the presence of anti-RA33 autoantibodies using Western blot
Western blot
Figure 82: Rheumatoid Arthritis patient samples and Western blot analysis of all four serum samples. Only two sera, 11 and 25, were identified as autoantibody positive [100].
Two strategies were applied in the interaction studies with autoantibodies from the RA patient samples. In the first strategy the peptides were immobilised offline, followed by binding of patient sera at a 1:100 dilution. Since this dilution showed a high background, it was optimised to a 1:200 dilution. In the second strategy, two channels were activated and deactivated followed by online immobilisation of 30 µM RA peptides. The patient sera were then exposed to the surface and binding affinity analysed. The data were processed by subtracting the reference channel and the ratio between the bound autoantibodies in the sera versus the peptides was determined.
a b c
a : peptide X (off-line)
b : capping by ethanolamine (off-line) c : peptide Y (off-line)
c
c: capping by ethanolamine (off-line) a: immobilization of the peptide X (on-line)
a
Strategy 1 Strategy 2 Data processing 1:100 1:200
• Extract the reference
• n (autoAb) : n (peptide)
Figure 83: Strategies applied in the interactions analysis of patient sera with the synthetic RA peptides. In the first strategy the peptides were immobilised offline, followed by binding of patient sera at a 1:100 dilution. Since this dilution showed a high background, it was optimised to a 1:200 dilution. In the second strategy, two channels were activated and deactivated followed by online immobilisation of 30 µM RA peptides. The patient sera were then exposed to the surface and binding affinity analysed. The data were processed by subtracting the reference channel and the ratio between the bound autoantibodies in the sera versus the peptides was determined.
The binding curve of the positive patient sample S11 (1:200) to immobilised peptide 1 is illustrated in Figure 84. The peptide was immobilised online and the empty surface was created by activating and deactivating the surface without immobilising the peptide. This empty channel demonstrates just how much non-specific binding to the chip surface is present. Therefore, was necessary to use the empty surface to reference away this nonspecific signal.
0 500 1000 0
10 20 30
empty
Phase [deg]
Time [s]
1 H-78MAARPHSIDGRVVEP92-NH2 1 H-78MAARPHSIDGRVVEP92-NH2 1
Phase [°]
Time [s]
Figure 84: Binding curves of sample 11 to immobilised peptide 1. The empty surface, used as a negatve control signal, was activated by EDC/NHS and then deactivated with ethanolamine without any peptide being coupled.
After subtracting the reference channels, the ratio between the bound autoantibodies and the peptides was determined and the results are summarised in Figure 85 and Table 12.
The SAW bioaffinity analyses showed high sensitivity for the detection of RA autoantibodies present in patient sera. Samples 11 and 25 showed a high level of autoantibodies. Peptides 2, 3, 4 and 5 showed only weak interactions with the autoantibodies present in the patient sera. Peptide 1 is the longest peptide from all peptides chosen in this study and the conformation of this peptide might play a role in the interaction with and detection of the autoantibodies.
0 0,05 0,1 0,15 0,2 0,25
1 2 3 4 5
RA peptide
n autoAb: n Peptide
S11 S25 S6 S7
Figure 85: Summary of the binding of patient sera to RA peptides. The ratio between the autoantibodies and the peptides is plotted versus all RA peptides and all patients samples used in this analysis. Sample 11 has a high level of autoantibody in comparision to the other patient samples.
Table 12: Summary of the peptide-sera interactions analysis.
Sample code Peptide code Peptide sequence
11 25 6 7
1 H-78MAARPHSIDGRVVEP92-NH2 + + + +
2 H-245GYGGG249-NH2 + + + -
3 H-59RSRGFGF65-NH2 + + + +
4 H-111KKLFVG116-NH2 + + + +
5 H-266NQQPSNYG273-NH2 + - - -
+, analysis was performed; -, analysis was not performed.
Further interaction studies are needed in order to understand the details of exact interaction between the epitope peptides and the autoantibodies present in the patient sera.