2. RESULTS AND DISCUSSION
2.2. Epitope elucidation of Aß-specific human antibodies
2.2.3. Identification of the epitope recognized by Aß-autoantibodies isolated
The Aß-specific autoantibodies present in IVIG (OCTAGAM) were purified on affinity matrix containing immobilized Cys-Aß(1-40). The integrity of the purified Aß-autoantibodies was assessed by separation of the components present in the sample on a 1D-gel performed under denaturing and reducing conditions. The 1-D gel separation of the Aß-autoantibody sample is shown in Figure 27. The intense spots of the heavy and light chain indicate that the antibody contained >90 % intact antibody chains.
The identification of the epitope recognized by the Aß-reactive antibody from IVIG was performed using an affinity column containing 100 µg of immobilized antibody (column prepared according to the procedure described in the Experimental Section). The reactivity of the affinity column to Aß and the optimal amount of buffer for the removal of non-specifically bound Aß were tested in a first experiment. 25 µg of intact Aß was dissolved in PBS to a final concentration of 0.1 µg/µl and allowed to react with the antibody for 2 hours. The supernatant containing excess peptide was then removed and the column was washed with 30 ml of PBS. The last volume of the washing buffer was collected for mass spectrometric analysis in order to investigate whether non-specifically bound peptide fragments were present. The affinity bound peptides were then dissociated
Figure 27: 1-D gel of human Aß-autoantibody purified from IVIG and visualized by colloidal Coomassie.
67
45 36 29
24 20 14
Marker Proteins KDa
Aß-autoantibody
Heavy chain
Light chain 67
45 36 29
24 20 14
Marker Proteins KDa
Aß-autoantibody
Heavy chain
Light chain
by treatment with 0.1% trifluoroacetic acid. Figure 28 shows the MALDI-MS analyses of the supernatant, washing and elution fractions. Molecular ions corresponding to Aß(1-40) ([M+H]+calc. of 4328.88) were identified in the supernatant and elution fraction. This experiment confirmed that the column containing immobilized Aß-antibody was able to react with Aß(1-40) and the non-specifically bound fragments can be removed by washing with 30 ml of PBS.
a)
b)
c)
For the mass spectrometric identification of the epitope, a series of proteolytic cleavages were performed to remove the Aß-residues that were unprotected by the autoantibody binding. Endoproteinase GluC from Staphylococcus aureus strain V8 is able to cleave peptide bonds at the carboxyl side of glutamyl residues
Figure 28: MALDI-TOF mass spectra of the Aß affinity experiment with the immobilized Aß-autoantibodies. a) Supernatant, b) Wash test, c) Elution fraction.
[M+H]+calc.= 4329.8 Aß(1-40)
Aß(1-40)
[M+2H]2+
[M+H]+calc.= 4329.8 Aß(1-40)
Aß(1-40)
[M+2H]2+
if the reaction is carried out in ammonium bicarbonate (pH 7.8) and provides cleavage at the carboxyl side of either glutamyl and aspartyl residues if the reaction is carried out in phosphate buffer (pH 7.8). Due to the presence of 3 aspartyl residues and 3 glutamyl residues within the Aß(1-40) sequence the proteolytic cleavage was performed in phosphate buffer. Digestion of Aß(1-40) by GluC for 4 hours resulted in cleavage at the Glu-3 and Glu-11 residues. The peptide mixture produced by enzymatic proteolysis using GluC was allowed to react with the affinity column for 2 hours. MALDI-MS analysis of the unbound peptide fragments revealed the fragments [4-11], [1-11] and [12-40] whereas the mass spectrum of the elution fraction contained an abundant ion of m/z 3022.2 that corresponded in mass to the amino acid sequence [12-40]. In order to verify whether a shorter sequence than [12-40] is able to display binding to the antibody, Aß(1-40) was digested in solution for 20 hrs and the resulting peptide mixture was exposed to the affinity column. The supernatant contained peptide fragments resulting from cleavage at Glu-3, Asp-7, Glu-11, Glu-22 and Asp-23. The peptide fragments identified in the supernatant and elution fraction are shown in the Figure 29 a and b and summarized in Table 16 (par. 3.6.3.).
Although longer N-terminal peptide fragments such as [1-11], [12-22] and [1-23], were produced at extended digestion time (20 hrs) with GluC, the elution fraction contained only the Aß(12-40). This result showed that the epitope is not contained in the N-terminal sequence [1-11] and in the sequence [12-22]. However, the carboxy-terminal fragments [23-40] and [24-40] present in the supernatant did not show affinity to the autoantibody. These preliminary data were interpreted to suggest that the autoantibody might recognize a middle domain of Aß. Consistent with the results provided by epitope extraction, the predominant peptides identified in the mass spectrum of the elution fraction upon epitope excision performed by digestion of immobilized Aß(1-40) with GluC for 20 hrs, were [12-40] and intact Aß(1-40) (see Figure 29c).
a)
b)
A second enzyme employed for proteolytic excision was trypsin. Aß(1-40) contains 3 possible cleavage sites of trypsin: Arg-5, Lys-16 and Lys-28. Considering the sequence Aß(12-40) identified by proteolytic digestion with endoproteinase GluC, the proteolytic reaction at the Lys-17 and Lys-28 residues could provide additional information. Upon cleavage by trypsin of Aß(1-40) in complex with the antibody, the mass spectrum of the elution fraction contained an intense ion at m/z 3707.9 due to the tryptic peptide [6-40] [M+H]+calc.= 3711.2 and with lower abundance an ion at m/z 2391.5 corresponding to the fragment [17-40] [M+H]+calc.= 2392.8 (see
Figure 29: MALDI-TOF mass spectra of the epitope identification using GluC a) the supernatant of the epitope extraction b) the elution fraction after epitope extraction c) elution fraction obtained by epitope excision with endoproteinase GluC The insert in the upper right corner of the figure contains the Aß(1-40) sequence. The possible cleavage sites of GluC are indicated by a square and the cleavage sites observed by the identification of the proteolytic peptides present in the supernatant are indicated by arrows.
Figure 30). The ions at m/z 637.5 and 1335.4 observed in the supernatant were identified as singly charged ions of the fragments Aß(1-5) [M+H]+calc.= 637.6 and Aß(6-16) [M+H]+calc.= 1337.3. The low intensity of the molecular ion corresponding to the peptide Aß(1-17) might indicate that the cleavage at Lys16 residue by trypsin occurs with a lower rate, and possibly suggest that the sequence around this residue might be involved in the binding to the antibody.
a)
b)
In order to probe more possible cleavage sites within the sequence, the epitope excision experiment was also carried out using α-chymotrypsin as protease which catalyzes the hydrolysis of peptide bonds at the C-terminal side of Phe, Tyr, Trp and Leu peptide bonds. Hydrolysis of peptide bond at slow rates also occurs at
Figure 30: MALDI-TOF mass spectra showing the ions observed in the supernatant a) and elution fraction b) produced by epitope excision with trypsin. The insert shows the sequence of Aß(1-40). The possible cleavage sites are indicated with squares and the cleavage sites determined upon identification of the tryptic fragments present in the supernatant and elution fractions are indicated with arrows.
Aß(1-5) Aß(6-16)
Matrix
Aß(1-40) Aß(6-40)
Aß(17-40)
Aß(1-5) Aß(6-16)
Matrix
Aß(1-5) Aß(6-16)
Matrix
Aß(1-40) Aß(6-40)
Aß(17-40) Aß(1-40)
Aß(6-40)
Aß(17-40)
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV
1 40
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV
1 40
Met, Ile, Ser, Thr, Val, His, Gly and Ala. The mass spectrum (Figure 31 a) acquired for the peptide mixture resulting from the digestion of Aß(1-40) by alpha-chymotrypsin for 20 hrs revealed several ions that are summarized in Table 18 (par. 3.6.4.) The digestion led to proteolytic cleavage at all the primary cleavage sites, Tyr-10, Phe-4, Phe-19, Phe-20 and Leu-17 and one of the secondary cleavage sites Met-35, whereas the digestion of Aß(1-40) in complex with the antibody led to cleavage only at the Phe-4 residue. Thus, the mass spectrometric analysis of the elution fraction provided the identification of fragment Aß(5-40) while the supernatant contained no signal.
a)
b)
To examine if the amino acid sequence of the epitope can be further reduced, epitope excision was carried out using pronase. In contrast to the proteases
Aß(5-40) Aß(5-10)
Aß(28-35)
Aß(18-27) Aß(11-17)
Aß(21-35) Aß(5-17)
Aß(5-40) Aß(5-40) Aß(5-10)
Aß(28-35)
Aß(18-27) Aß(11-17)
Aß(21-35) Aß(5-17)
Aß(5-10) Aß(28-35)
Aß(18-27) Aß(11-17)
Aß(21-35) Aß(5-17)
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV
1 40
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV
1 40
Figure 31: MALDI-TOF mass spectra of the peptide mixture produced by proteolytic digestion of Aß(1-40) by alpha-chymotrypsin in solution a) and the elution fraction upon epitope excision using alpha-chymotrypsin b). The insert shows the Aß(1-40) sequence. The proteolytic cleavage sites identified by digestion of Aß(1-40) in solution are indicated by arrows. The peptide eluted after epitope excision using alpha-chymotrypsin is highlighted in grey.
employed in the previous experiments, pronase is a non-specific protease able to cleave proteins down into individual amino acids and exerts its proteolytic activity on denatured as well as on native proteins. This property is attributed to the composition of the preparation, comprising several types of endoproteinases (serine and metaloproteases) and exoproteinases (carboxypeptidases and aminopeptidases). The optimum pH value is 7.0-8.0 and the optimum temperature is in the range 40-60ºC. Aß after complexing with the antibody was treated with Pronase (0.5 µg/µl) for 2 hrs at 40ºC, and compared to the digestion in solution.
No molecular ion could be identified in the mass spectrum acquired for the digestion mixture of Aß in solution after 2 hours, which indicated pronase degraded free Aß to the individual amino acids (see Figure 32).
a)
b)
The mass spectrum of the elution fraction collected upon epitope excision with pronase provided the identification of several Aß(1-40) fragments as summarized in the Table 2. The proteolytic cleavage by pronase of Aß(1-40) in complex with the autoantibodies occurred predominately at the N-teminal part of the peptide. All the fragments resulted were truncated at the N-terminal part and one fragment,
Figure 32: MALDI-TOF mass spectra of a) Aß(1-40) digested in solution with Pronase for 2 hours and b) Aß(1-40) peptide fragments eluted after epitope excision using Pronase.
Aß(21-40)
Aß(14-40) Aß(7-40) Aß(11-37)
Aß(6-40)
Aß(5-40)
Aß(1-40) Aß(21-40)
Aß(14-40) Aß(7-40) Aß(11-37)
Aß(6-40)
Aß(5-40)
Aß(1-40) Aß(21-40)
Aß(14-40) Aß(7-40) Aß(11-37)
Aß(6-40)
Aß(5-40)
Aß(1-40)
Aß(11-37) was identified upon cleavage at both N-and C-terminal ends of the Aß-sequence. The cleavages at the amino-terminal side occurred after amino acid residues Phe-4, Arg-5, His-6, Tyr-10, His-14 and Phe-20. The only C-terminal cleavage occurred after Val-36. Considering these results, the minimal sequence that preserves the affinity to the antibody was [21-37]. The presence of an additional molecular ion containing methionine sulphoxide for each of the sequences [11-37], [7-40], [6-40], [5-40] and [1-40] confirmed the correct identification as the Aß-sequence contains only one methionine residue located in the C-terminal part (Met-35). The ions containing oxidized methionine are indicated in the mass spectrum by an arrow.
Table 2: Peptide sequence assignment of the molecular ions present in the elution fraction after digestion of the immune complex with pronase
[M+H]+exp [M+H]+calc. ∆m Da
Proteolytic fragment
Sequence 1885.5 1886.22 0.72 [21-40] 21AEDVGSNKGAIIGLMVGGVV40
2786.3 2786.31 0.01 [14-40] 14HQKLVFFAEDVGSNKGAIIGLMVGGVV40 2895.3 2897.36 2.07 [11-37] 12EVHHQKLVFFAEDVGSNKGAIIGLMVG37
3572.4 3574.09 1.70 [7-40] 7DSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV40 3709.8 3711.23 1.44 [6-40] 6HDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV40 3866.5 3867.42 0.99 [5-40] 5RHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV40
The various proteolytic enzymes provided detailed information about the sequence recognized by the Aß-autoantibodies (see Figure 33). The minimal sequence [21-37] was determined by epitope excision using pronase. However, the information provided by the specific enzymes (trypsin, GluC and α-chymotrypsin) significantly contributes to the unequivocal identification of the epitope. Proteolytic cleavages of Lys-28 by trypsin, Glu-22 and Asp-23 by GluC were not observed confirming that these residues are shielded in the immune complex. Noteworthy, the Lys-17 residue is cleaved at a very low rate indicating that the activity of trypsin at this site might be sterically hindered. Similarly, the epitope excision with alpha-chymotrypsin reveals that the enzyme is able to hydrolyze only the peptide bond following Phe-4, and cleavage after Tyr-10, Leu-17, Phe-19 and Phe-20 are hindered when Aß is bound to the antibody while in the free form the enzyme is able to hydrolyze these peptide bonds.
These results ascertained that the Aß-antibody isolated from the IVIG preparation recognizes an epitope located in the carboxy-terminal part of Aß within the amino acid residues [21-37]. Presumably due to the polyclonality of the antibody amino acid residues located in the sequence [12-20] might also contribute to an increased reactivity of the Aß to the autoantibody.
In contrast to the IVIG the human IgG preparation from Calbiochem is a lyophilized solid prepared only for research use. In a further part of study it was investigated whether this product contains anti-Aß autoantibodies and the epitope specificity was compared with the Aß-autoantibodies isolated from IVIG. 250 mg of the solid powder containing IgG were reconstituted in PBS and incubated with the Cys-Aß(1-40) affinity column according to the procedure detailed in the Experimental Part. Based on the amount of specific Aß-autoantibody quantified in the eluate the Aß-antibodies represented 0.1-0.2% of the IgG molecules in the Calbiochem product. In order to identify the epitope recognized, an affinity column containing immobilized Aß-antibodies was prepared. 150 µg of the Aß-antibody quantified in the elution fractions of the antigen column were lyophilized and reconstituted in coupling buffer to a final concentration of 0.5 µg/µl. The solution was applied to Sepharose matrix and the antibody was covalently immobilized according to the procedure described in the Experimental Part. To ensure minimal non-specific interaction of the antigen to the column material, the amount of antigen applied to the antibody column was reduced to 5 µg of intact or digested Aß(1-40). Due to the identification in the previous epitope identification experiments of the sequence [12-40] and [21-37], the enzyme of choice was GluC protease, at a digestion time of 20 hours.
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG11 EDVGSNKGAIIGLMVGGVV GluC Pronase Trypsin
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG11 EDVGSNKGAIIGLMVGGVV
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG11 EDVGSNKGAIIGLMVGGVV GluC Pronase Trypsin GluC
Pronase Pronase Trypsin Trypsin
Figure 33: Summary of the results leading to the identification of the Aß(21-37) epitope. The cleavages that occur when the antigen-autantibody complex is subjected to enzymatic proteolysis are indicated by continuous arrow lines and the shielded cleavage sites by dashed arrow lines.
The MALDI-TOF mass spectra of the supernatant and elution fractions from the epitope extraction are shown in the Appendix 3, Figure 83. In the mass spectrum of the supernatant, ions corresponding to the N-terminal peptide [4-11], to the middle domain [12-22], to fragments containing missed cleavage sites [12-40], [4-40] and intact [1-[4-40] were identified. The elution fraction contained only N-terminal truncated peptides with the most intense ion signal corresponding to the sequence [12- 40]. In the case of the epitope excision, a low amount of Aß(1-40) remained unbound and was identified in the mass spectrum of the non-binding fraction. The supernatant, collected after incubation for 20 hrs with GluC contained Aß(1-40) and the fragment [12-22] (see Figure 84). After washing the column with 30 ml PBS the last volume of the PBS was collected to check by mass spectrometry for background ions prior to elution. The mass spectrum of the washing fraction yielded no contaminant ion while the mass spectrum of the elution fraction contained molecular ions corresponding to the fagments [12-40] and [4-40] with low intensity and the intact Aß(1-40) with high intensity. The difference in the intensities of the fragment [12-40] and the intact Aß(1-40) that can be observed in the elution fractions of the epitope extraction and epitope excision suggest a lower rate of the proteolytic cleavage of Aß(1-40) by GluC in the complexed state.
In a further set of experiments an affinity column containing unfractioned IgG was prepared using identical experimental conditions as for the immobilisation of the Aß-antibody. Considering that the concentration of Aß-autoantibody is approximately 0.1-0.2% of the total IgG, 150 µg of IgG should contain maximum 0.3 µg of Aß-autoantibody which is negligible for showing a specific interaction with Aß. Therefore the column containing 150 µg of immobilized IgG represents a suitable control to examine the unspecific affinity that might appear during the epitope identification due to the interaction of intact Aß(1-40) and of the proteolytic fragments with the matrix, the column or with domains of the IgG that are not part of the CDRs. The epitope extraction using the unfractionated IgG control column was carried out in parallel with the experiments performed on the Aß-antibody column. The mass spectra of the supernantant collected after exposing the proteolytic mixture resulting from digestion of Aß with GluC to the column showed molecular ions corresponding to the fragments [4-11], [12-22] and [12-40]. The elution fraction from the epitope extraction on the control column showed the
absence of any molecular ion (Figure 85). The epitope excision on the unfractioned IgG column was carried out in the same manner in comparison to the epitope excision on the Aß-autoantibody column. The non-binding fraction collected after 2 hrs of incubation with the IgG column contained unbound Aß(1-40) (Figure 86). The enzyme was added to the column and incubated for 20 hrs followed by removal of the supernatant fraction. The mass spectrometric analysis revealed the presence of Aß(1-40) at a very low extent. The elution fraction contained a molecular ion corresponding to Aß(1-40).
The epitope identification carried out for the Aß-autoantibody purified from the immune globulin preparation purchased from Calbiochem showed that the epitope resides within the sequence [12-40] and is consistent with the epitope recognized by the Aß-autoantibodies purified from IVIG. The control experiments carried out for the examination of the non-specific binding of the Aß(1-40) peptides indicated that intact Aß(1-40) has nonspecific binding to the affinity column, however the molecular ion pattern identified in the elution fraction of the epitope identification using the autoantibody affinity column was not observed at the control experiments.
2.2.4. Clonal diversity and sequence analysis of human serum Aß