2. RESULTS AND DISCUSSION
2.1. Characterisation of Aß-plaque specific antibodies
2.1.4. Effect of zinc ions on the recognition of N-terminal ß-amyloid epitope
Zinc is an important catalytic and structural component of many proteins in human brain as well as a modulating agent in synaptic transmission, but is neurotoxic at high concentrations [112]. High levels of zinc ions have been detected within amyloid deposits [113, 114], which could result (i) from zinc-induced amyloid deposition [89, 110, 115], (ii) from its accumulation as a protective agent after amyloid deposition, or (iii) from a disruption of zinc homeostasis associated with AD. In support of the Zn2+ mediated stabilization of Aß amyloid fibrils, solubilisation of Aß from Alzheimer´s disease brain tissue has been found to be significantly enhanced by the presence of metal chelators such as EGTA, N,N,N´,N´-tetrakis(2-pyridyl-methyl) ethylene diamine and bathocuproine [116]. In recent studies the metal chelator clioquinol has been reported to inhibit the aggregation of Aß [87]
and currently there are studies that investigate the effects of clioquinol administered to transgenic mice developing amyloid plaques.
Different structural models propose that the Aß conformation within amyloid fibrils contains two parallel ß-strands formed by the residues 18-26 (ß1) and 31-42 (ß2), separated by a 180° bend formed by residues 27-30 [ 117]. The ß-strands form 2 layers of intermolecular, parallel ß-sheets. The BallView 1.1.1 program was used to depict the three-dimensional structure of the Aß fibril based on the structural data available at the Protein Data Bank with the accession number 2beg (Figure 19). By contrast, considerable NMR data obtained on Aß argue that the soluble monomeric peptide has an unordered conformation in aqueous solution and mainly adopts an α-helical structure in membrane-mimicking media [118]. The residues 1-17 that form the N-terminal region of Aß are disordered and are omitted from the structural model of the amyloid fibril. These residues constitute the outer wall of the fibril and are not involved in the interactions of Aß within the fibrils, thus remaining accessible to the interaction with partners critical for the pathology or with therapeutic agents. The structure model of the N-terminal region in the presence and absence of zinc ions will be discussed at the end of this section.
Figure 19: Structure model of amyloid fibril indicating the core structure of residues 17-42. The direction of the fibril axis is indicated by an arrow. In the ribbon diagram of the Aß(17-42) the ß-strands are indicated by blue arrows and the nonregular secondary structure is depicted by green spline curves. The hydrophobic, polar, negatively charged, positively charged amino acid side chains are shown in grey, green, red and blue. The ß-strands are indicated by blue arrows and the loop connecting the 2 ß-strands is depicted by green spline curve. A schematic view of the fibril is shown in the insert on the upper right side.
Based on the mass spectrometric fragmentation patterns of Aß(1-16)-Zn2+
complexes in the gas phase and the NMR data of the complexes in aqueous solution at pH 6.5, the Aß(1-16) sequence has been identified as the minimal zinc-binding domain. The residues His-6, His-13, His-14 and the Glu-11 carboxylate were identified as ligands that tetrahedrally coordinate the zinc ions [109, 110, 119-122].
As described in the Introduction, the N-terminal region of Aß represents an attractive therapeutic target for active and passive immunization approaches. Only the antibodies raised against the N-terminal part of Aß are able to reduce the plaque burden and restore cognitive deficits in mice models of AD. With the assumption that active anti-Aß-antibodies and zinc ions target a similar or identical domain of Aβ [16], the Aβ-epitope recognition by two different antibodies in the presence of zinc was investigated in the present work. The mAb anti(1-17) clone
L17
6E10 was chosen due to the previous identification of the Aß(4-10) epitope specificity by a combination of affinity chromatography, proteolytic digestion and mass spectrometric experiments performed in our laboratory. As a second antibody, mAb-anti(1-40) clone Bam-10 targeting an epitope residing within the amino acids Aß(1-12) was studied.
To investigate the effects of zinc ions on the binding of the antibodies to the N-terminal domain of Aß, a sandwich ELISA was designed. The Aß(1-16) peptide was synthesized with a pentaglycine spacer and a biotin attached at the amino-terminal end. As a negative control for the binding of the zinc ions, biotin-(G)5 -Aß(1-10) was employed. This peptide does not contain the minimal zinc binding domain but comprises the N-terminal epitope of the antibodies as previously described (s. par. 2.1.3.). An example of the HPLC profile and MALDI-FT-ICR mass spectrum of the pure Biotin-(G)5-Aß(1-16) peptide is depicted in Figure 20.
a) b)
A detailed description of the experimental conditions and a schematic representation of the sandwich ELISA are given in the Experimental Section. The peptides containing a biotin and a pentaglycine spacer at the amino-terminal end were incubated with the metal ions before addition to the microtiter plate. The monoclonal antibody specific for the N-terminal peptide was allowed to adsorb to the surface of the plate, followed by the addition of the peptide-metal ion complexes and the anti-biotin antibody conjugated with horseradish peroxidase. In comparison to the indirect ELISA in which the antigen was immobilized through
Figure 20: HPLC profile a) and MALDI-FT-ICR mass spectrum b) of Biotin-(G)5-Aß(1-16).
0 10 20 30 40 50 60
0,2 0,4 0,6 0,8 1,0 1,2 1,4
Abs (220 nm)
Time (min) 21.9
1000 1500 2000 2500 3000 3500 m /z
2465.0861
0 10 20 30 40 50 60
0,2 0,4 0,6 0,8 1,0 1,2 1,4
Abs (220 nm)
Time (min) 21.9
1000 1500 2000 2500 3000 3500 m /z
2465.0861
the biotin to the streptavidin coated plates, the sandwich ELISA provides the advantage of allowing the interaction of the antibody with the free peptide-metal ion complex thus minimizing the washing steps following addition of peptide-metal ion complex.
In a first set of experiments, biotin-G5-Aß(1-16) and biotin-G5-Aß(1-10) were tested for the binding to 6E10 and Bam-10. Both peptides reacted in a dose-dependent manner with the 6E10 and Bam-10 antibodies (Figure 21). However, biotin-G5 -Aß(1-10) gave identical responses with both antibodies, while in the case of biotin-G5-Aß(1-16) the affinity with the Bam-10 antibody was twofold higher than with 6E10.
a)
b)
Figure 21: Dose-response ELISA signal of biotin-G5-Aß(1-16) (squares) and biotin-G5-Aß(1-10) (triangles) in the absence of transition metal cations, as measured with a) 6E10 (filled symbols) and b) Bam-10 (open symbols).
0,0
In a further set of experiments the effect of different transition metal ions on Aß recognition was tested. Different peptide dilutions (0.01-60 µM) and a fixed concentration of metal ions (either 0 or 100 µM) (Figure 22) were employed in a first experiment. The presence of 100 µM Zn2+ ions caused a significant increase of the binding of biotin-G5-Aß(1-16) to both the 6E10 and Bam-10 antibody, which resulted in a 4- and 10-fold increase in the ELISA response for 60 µM of Aß-peptide respectively. By contrast, the presence of Zn2+ did not influence the ELISA response of biotin-G5-Aß(1-10). The cations Co2+ and Ni2+ had no effect on biotin-G5-Aß(1-16) recognition. The presence of Cu2+ ions did not influence the recognition of biotin-G5-Aß(1-16) by 6E10 mAb but resulted in a higher ELISA response with the Bam-10 antibody.
a)
b)
Peptide concentration (µM)
Figure 22: Dose-response ELISA signal of biotin-G5-Aß(1-16) in the absence (open squares) or in the presence of 100 µM Zn2+ (filled squares), Cu2+ (cross), Co2+ (filled triangles) or Ni2+
(filled lozenges) ions, as measured with 6E10 and Bam-10 mAbs.
0,0
a)
b)
A further set of experiments was performed at a fixed peptide concentration (60 µM) but at increasing the metal ion concentration from 0 to 1.2 mM (Figure 23).
These experiments also showed a significant increase of biotin-G5-Aß(1-16)
Figure 23: Effect of divalent ions concentration on the ELISA response of biotin-G5-Aß(1-16) (filled symbols) and biotin-G5-(1-10) (open squares),as measured with 6E10 and Bam-10 mAbs. 60 µM of peptide were introduced in the presence of the indicated concentrations of Zn2+ (squares), Cu2+ (cross), Co2+ (triangles) or Ni2+ (lozenges).
Biotin-G5-Aß(1-10) was tested only in the presence of Zn2+. 0,0
0,5 1,0 1,5 2,0
Absorbance (450nm)
0,0 0,5 1,0 1,5 2,0
0 100 200 300 400 500 600 700 800 900 1000 1100 1200 ion concentration (µM) Biotin-G5-Aß(1-10)
Biotin-G5-Aß(1-16) + ZnCl2 Biotin-G5-Aß(1-16) + CuCl2
Biotin-G5-Aß(1-16) + CoCl2 Biotin-G5-Aß(1-16) + NiCl2
0,0 0,5 1,0 1,5 2,0
Absorbance (450nm)
0,0 0,5 1,0 1,5 2,0
0 100 200 300 400 500 600 700 800 900 1000 1100 1200 ion concentration (µM) Biotin-G5-Aß(1-10)
Biotin-G5-Aß(1-16) + ZnCl2 Biotin-G5-Aß(1-16) + CuCl2
Biotin-G5-Aß(1-16) + CoCl2 Biotin-G5-Aß(1-16) + NiCl2
recognition by the 6E10 and Bam-10 antibodies in the presence of zinc ions, which was concentration dependant up to a plateau reached at 500 and 150 µM ZnCl2
for 6E10 and Bam10 respectively. As described above the presence of Zn2+ ions had no effect on the recognition of biotin-G5-Aß(1-10). Likewise, the presence of Co2+ or Ni2+ had no significant influence on the recognition of biotin-G5-Aß(1-16).
Consistent with the results obtained in the previous set of ELISA experiments, Cu2+ ions at low concentration (up to 100 µM) led to an increased recognition of biotin-G5-Aß(1-16) by the Bam 10 antibody, but had no effect on the recognition by the 6E10 antibody.
From these results, it can be concluded that the sequence Aß(1-16) undergoes a conformational change induced by the cation binding which results in an enhanced recognition of the epitope by both antibodies. This result is consistent with the three-dimensional structures of the Aß(1-16) (available at the pdb accession number 1ze7) and Aß(1-16)-Zn2+ complex (available at the pdb accession number 1ze9). The three-dimensional structures were determined from the NMR spectra recorded in aqueous solution, pH 6.5. According to these data, the first 6 amino acids are unstructured while the region 7 to 15 of the Aß(1-16) has a well defined structure but not canonical structure (see Figure 24). Upon zinc binding, the mainly affected region of Aß(1-16) is (9-15), which contains three of the zinc ligand residues Glu-11, His-13 and His-14.
Figure 24: Three-dimensional structure of Aß(1-16) (a and b) and Aß(1-16) in complex with Zn2+
(c and d). The α-helix is depicted in red and the nonregular secondary structure is indicated by green spline curves. The amino acids that are part of the zinc binding domain are highlighted in the figures a and c and the residues 4-10 that are targeted by the monoclonal antibodies are highlighted in the figures b and d.
In contrast, biotin-G5-Aß(1-10), which lacks the Glu-11, His-13 and His-14 residues does not undergo a conformational change upon addition of Zn2+ and thus no enhancement of the ELISA response is determined. Consequently this peptide may be considered a negative control for the binding to the antibody in the presence of divalent ions.
1