Identification of functional amino acid residues within the ß-amyloid

The mass spectrometric approach to the elucidation of the epitope recognized by the polyclonal anti-Aß(1-42) antibodies lead to the identification of the N-terminal sequence Aß(4-10) (FRHDSGY). The same antigenic determinant was identified by mass spectrometric epitope indentification in the case of the monoclonal antibody anti-Aß(1-17). In order to identify the relative contributions of each of the individual amino acids comprised by the epitope to the interaction with the antibodies, the reactivities of the antibodies towards synthetic peptides containing alanine single-site mutations were investigated by ELISA.

ß-secretase

Figure 15: Proposed mechanism of action of the plaque-specific antibodies. N-terminal specific antibodies are able to bind amyloid fibrils due to exposed N-terminal domain of Aß. Plaque breakdown by microglia activated through Fc receptor mechanisms could explain the Aß plaque clearance effect of these antibodies.

Biotinylated Aß(1-10) peptides, both wild type and mutant constructs with a N-terminal pentaglycine spacer, were prepared by Solid Phase Peptide Synthesis (SPPS) according to the Fmoc strategy. The synthesis protocol was as follows: (i) DMF washing (3 x 1 min), (ii) deprotection with 20 % piperidine in DMF (15 min), (iii) DMF washing (6 x 1 min), (iv) coupling of 5 equivalents of Fmoc amino acid using PyBOP and NMM in DMF (50 min), (v) DMF washing (3 x 1 min). After completion of the syntheses and removal of the N-terminal Fmoc protecting group, the free amino group was biotinylated using 5 equivalents of D-(+)-biotin. Then the peptides were cleaved from the resin using a cleavage solution containing 95 % TFA as cleavage reagent and 2.5 % triethylsilan as scavengers. After cleavage, the solution containing the resin and free peptide was filtrated to remove the resin and washed twice with TFA. The peptide present in the filtrate was precipitated using 10 volumes of cold tert-butyl-methyl-ether over the volume of filtrate. The precipitate was filtrated, then the solid material was washed three times with diethylether (10 ml) and dissolved in 5 % acetic acid (aqueous solution) prior to freeze-drying. The crude products were purified by RP-HPLC and analysed by mass spectrometry.

Table1 shows the characteristics of the synthetic peptides used in the alanine scanning mutagenesis experiment. To allow a comparable extent of immobilization to the microtiter plates the peptides were synthesized with a biotin residue at the amino-terminal end. The binding of the peptides to the plates is mediated by the interaction of the biotin with the streptavidin coated to the plates. A pentaglycine spacer was placed between biotin and the Aß(1-10) sequence to ensure the accessibility of the epitope to the antibody. The molecular masses of the synthesized peptides were determined using an ESI-FTICR-MS. The measured masses exactly matched the predicted molecular weights indicating that the correct sequences were obtained in the synthesis of the peptides. The purity of the peptides was assessed by analytical RP-HPLC and mass spectrometry. For analytical RP-HPLC an analytical Nucleosil 300-7 C18 column (Macherey-Nagel, Düren, Germany) was used as stationary phase.

Table 1: Characteristics of mutant and wild type Aß-epitope peptides. Linear gradient elution (0min 0 %B; 5 min 0 % B; 50 min 90 % B) with eluent A (0.1 % TFA in water) and eluent B (0.1 % TFA in (80 % acetonitrile, 0.1 % TFA in water) was employed at a flow rate of 1 mL/min at ambient temperature. Peaks were detected at λ=220 nm. The samples were dissolved in eluent A.

Peptide

No. Mutation Sequence HPLC

Rt (min)

[M+H]⁺ calculated found 1 WT Biotin-GGGGGDAEFRHDSGY 23.63 1706.6976 1706.7367 2 F4A Biotin-GGGGGDAEARHDSGY 21.15 1630.6663 1630.6948 3 R5A Biotin-GGGGGDAEFAHDSGY 24.14 1621.6336 1621.6540 4 H6A Biotin-GGGGGDAEFRADSGY 24.04 1640.6758 1640.6977 5 D7A Biotin-GGGGGDAEFRHASGY 24.40 1662.7078 1662.7124 6 S8A Biotin-GGGGGDAEFRHDAGY 23.79 1690.7027 1690.7340 7 G9A Biotin-GGGGGDAEFRHDSAY 23.97 1720.7132 1720.7395 8 Y10A Biotin-GGGGGDAEFRHDSGA ^{22.69 1614.6714 1614.7321} 9 (F4-Y10)A Biotin-GGGGGDAEAAAAAAA 20.8/23.54 1341.5852 1341.5614

All mutant peptides were immobilized on microtiter plates at a fixed concentration (1 ng/µl). After blocking, the antibodies were added in 8 serial twofold dilutions using stock solutions of 1 µg/µl. The bound anti-amyloid antibody was detected by an alkaline phosphatase-conjugated antibody as described at the Experimental Section. In order to provide accurate background substraction, triplicate wells of each antibody dilution without antigen were used as shown in Figure 16.

As expected, the response curves were significantly different for the polyclonal and the monoclonal antibodies although the antibodies target the same epitope Aß(4-10). The wild type construct (depicted with filled squares) reacted in a dose-dependent manner with both antibodies Figure 16 a) and b).

a)

b)

Figure 16: Dose-response curves of the alanine scanning mutagenesis experiment: a) reactivity of wild type Biotin-(G)5-Aß(1-10) (filled square) and of the alanine mutated constructs towards polyclonal anti-Aß42 antibody; b) reactivity of wild type Biotin-(G)₅-Aß(1-10) and of the alanine mutated constructs towards monoclonal anti-Aß(1-17) antibody. The insert in the figure 16b shows a detailed view of the response shown by the alanine mutated constructs. Background signals from wells without antigen were substracted.

0

In the case of the polyclonal anti-Aß(1-42) antibody the ELISA fluorescence values of all the mutant peptides obtained for the antibody dilution 1:250 were normalized to the wild-type construct. Therefore, the binding ability of the mutant peptides is depicted in Figure 17 as percentage of the binding of Biotin-(G)5-Aß(1-10).

According to the experimental results the alanine mutants can be sorted into 4 groups. A first group contains the mutants D7A and S8A which display binding properties to the polyclonal anti-Aß(1-42) antibody close to that of the wild type peptide. The Y10A can be considered as a particular example because it possesses a 2.8 times lower binding ability of that of the wild type without abolishing the binding. Mutations F4A, R5A and H6A led to complete abrogation of antibody binding. In contrast, the replacement of glycine with alanine increased slightly the binding ability of the mutant peptide.

As depicted in Figure 18 the results show a substantial difference between the functional amino acid residues recognized by the antibodies. In contrast to the polyclonal antibody, the reactivity of the epitope [4-10] towards the monoclonal antibody was abolished by all single site alanine mutations introduced in the

WT F4A R5A H6A D7A S8A G9A Y10A

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120%

Biotinylated peptides

binding ability (% of WT)

F R H D S G Y

Biotin-(G)₅- - COOH

WT F4A R5A H6A D7A S8A G9A Y10A

0%

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Biotinylated peptides

binding ability (% of WT)

F R H D S G Y

Biotin-(G)₅- - COOH

Figure 17: ELISA-detected reactivity of alanine mutants to the polyclonal anti-Aß(1-42) antibody.

The relative immunoreactivities of the wild type and mutant peptides to the polyclonal antibody were assessed by an indirect solid-phase ELISA as described in the Experimental Section. The panel depicts the binding capability (in percent of that of the wild type peptide) for all the mutants at an antibody concentration of 1 µg/ml.

Error bars represent the S.D. of triplicate determinations.

sequence. Structural data were from [110] and the ribbon representation of the structure was prepared with BALLWiew v1.1.1. .

The ELISA was effective to discriminate among the mutants of the Aß(4-10) epitope showing that the residues Phe4, His5 and Arg6 are crucial for the binding to the polyclonal anti-Aß(1-42) antibody, while the other four residues are less important. This antibody was obtained by active immunization of transgenic AD mice with Aß(1-42) oligomers and was shown to disaggregate preformed Aß-fibrils.

Interestingly, the 3-6 amino acid sequence located in the N-terminal region of Aß (Glu-Phe-Arg-His) has been found previously as the epitope of 2 different anti-aggregating antibodies [111].

F R H D S G

Figure 18: Location of residues affecting the epitope binding specificity to the antibodies.

Schematic comparison of the critical amino acid residues (dark grey) within the Aß(4-10) epitope involved in the interaction with a) the polyclonal anti-Aß(1-42) and b) the monoclonal anti-Aß(1-17).

2.1.4. Effect of zinc ions on the recognition of N-terminal ß-amyloid epitope