Autoantibodies (aAb) associated with Alzheimer’s disease (AD) have not been sufficiently characterized and their exact involvement is undefined. The use of information technology and computerized analysis with phage display technology was used, in the present research, to map the epitope of putative self-antigens in AD patients. A 12-mer random peptide library, displayed on M13 phages, was screened using IgG from AD patients with two repetitions. Seventy-one peptides were isolated; however, only 10 were positive using the Elisa assay technique (Elisa Index > 1). The results showed that the epitope regions of the immunoreactive peptides, identified by phage display analysis, were on the exposed surfaces of the proteins. The putative antigens MAST1, Enah, MAO-A, X11/MINT1, HGF, SNX14, ARHGAP 11A, APC, and CENTG3, which have been associated with AD or have functions in neural tissue, may indicate possible therapeutic targets.
Alzheimer’s disease (AD) is the most important cause of dementia. Its prevalence increases with age and, together with increasing life expectancy, has created the expectation of an increase in the number of cases, especially in developed countries [
Following the original description of AD in 1906, the presence of
Soon after it was observed that SP and NFT are accompanied by an inflammatory process in the immune system, this system began to be investigated regarding its role in AD pathogenesis [
Several studies have discovered an abundant presence of antibodies directed at targets in brain neural tissue, cerebrospinal fluid, and the serum of patients with AD. Antibodies against neurotransmitter receptors (glutamate, dopamine, serotonin, and acetylcholine), enzymes (ATP synthase and aldolase), cytoskeletal proteins, and microglia have been described [
Phage display (Ph.D.) technology is useful for the identification of peptides or antibodies on the surface of the filamentous M13 bacteriophage capsid. This capsule permits exposure to an extensive diversity of peptides that can bind to various targets and be identified using peptide library techniques. This methodology has been proven useful not only for the selection of peptides that mimic proteins but also for the identification and description of epitopes recognized by antibodies [
Phage display findings can be analyzed with different bioinformatic tools: the identification of consensus motifs among selected sequences, the identification of possible targets by linear and conformational (3D structure) comparison with protein databanks, and assessments of their putative epitopes with their degree of antigenicity. This information can be extremely useful for planning experiments, designing drugs, and other applications [
The present study identified mimetic peptides of target antigens in the circulating IgG present in the serum of patients with AD. Our use of the phage display technique, together with bioinformatic tools, may represent one of the first evidences of the presence of autoantibodies and their putative epitope mapping, in AD.
Serum samples from AD patients and healthy controls, matched by sex and age, were obtained from the University Hospital of Uberlandia. For the diagnosis of dementia, the DSM-IV TR criteria were used [
The phage selection was performed using a pool of sera from AD patients and healthy (control) individuals. Immunoglobulin G (IgG) was secured using magnetic beads coupled to protein G Dynabeads (Invitrogen). For subtraction of nonspecific peptides, 10
Selected phages were amplified, purified, and titrated according to the Ph.D. Phage Display Libraries Instruction Manual (New England Biolabs).
After three rounds of selection, 96 blue colonies were randomly selected and their phage single strand DNA was isolated using iodide buffer extraction procedures [
The selected peptide-phage clones were used in the bead-ELISA assay against IgG from controls and AD patients to evaluate their reactivity and specificity.
Fifty microliters of phage supernatant was incubated with IgG coupled in magnetic beads (Invitrogen) for one hour with stirring, at room temperature. Using a magnetic apparatus, the microspheres were precipitated, washed six times with TBS-T 0.1%, and incubated with monoclonal anti-M13 peroxidase conjugate (GE Healthcare) diluted 1 : 5000 in TBS-T 0.1% and 5% BSA for one hour with stirring, at room temperature. Microspheres were again precipitated and washed six times and the reaction was observed with buffer orthophenylenediamine (OPD) to 1 mg/mL plus 3% hydrogen peroxide (H2O2). The results were expressed as an arbitrary ELISA Index (EI) and calculated as follows: EI = Abs of serum sample/cut-off, where the cut-off was determined as the mean absorbance of the negative control sera plus two standard deviations. Values of EI > 1.0 were considered positive.
The vector sequences were removed and the deductions of peptide sequences were performed using the ExPASy Translate Tool (
Bioinformatics workflow.
For a more detailed analysis, the sequence of positive peptides selected by the ELISA assay (EI > 1) was subjected to alignment using the BLAST tool (
The proteins indicated in alignment were selected for the next step of the analysis. We excluded unnamed sequences which had only been predicted or that were from unknown proteins. Those sequences with low
The program PEPSURF (
Statistical analysis was performed using the GraphPad Prism version 5.00 (GraphPad Software Inc.).
In this study 100 patients who had registered cognitive disorders were evaluated. Only 10 of these patients had complete AD diagnosis with laboratory tests, imaging, and assessment of cognitive function by neuropsychological tests. As paired healthy control (HC), we used 10 cognitively healthy individuals.
Phage display selection of a 12-mer random peptide library generated 75 peptides, of which 71 were distinct sequences. A phage ELISA assay was performed with these clones using a pool of serum from the patients and from the controls. The result showed that of the 71 peptides, only 10 were highly reactive mimotopes when compared with the controls (IE > 1). This suggested that circulating IgG from AD patients recognizes these specific peptides (Figure
Detection of IgG antimimotope in serum from patients with Alzheimer’s disease by Elisa using peptide-phage selected by phage display. Values of EI > 1.0 were considered positive (Student’s
Those peptides with distinct sequences were subsequently chosen for in-depth characterization through bioinformatics. The data are presented in Table
Peptide sequence and position of alignment in putative Alzheimer’s disease self-antigens.
Clone | Peptide sequence | Alignment region | Putative protein matched | PDB | Accession number |
---|---|---|---|---|---|
ALZ01 | TSISINPPRRPS | 672–683 | MAST1 | 2M9X | AAH27985.2 |
ALZ02 | SRPRPLIRNRRP | 341–350 | Enah | 2XQN | AAH65238.1 |
ALZ03 | MTIRRHRHRPKI | 128–131 | MAO-A | 2Z5Y | P21397.1 |
ALZ04 | SRRRIPRINRPQ | 431–438 | X11/MINT1 | 1X11 | Q02410.3 |
ALZ05 | KRRNTILINLPN | 4–9 | HGF | 2HGF | P14210.2 |
ALZ06 | TPIKKMIRRLPH | — | — | — | — |
ALZ07 | LPTKRIIKRMRR | 502–508 | SNX14 | 4BGJ | Q9Y5W7.3 |
ALZ08 | MSLNLRMRPMRI | 449–453 | ARHGAP 11A | 3EAP | Q6P4F7.2 |
ALZ09 | KMTRRTHINQIS | 111–115 | APC | 1AUT | 1AUT_C |
ALZ10 | RSIPRIHINTTN | 235–246 | CENTG3 | 3IHW | 3IHW_A |
After the initial identification of targets for alignment and prediction of linear and structural epitopes, the three-dimensional alignment, using the PepSurf program, was performed. This result demonstrated that peptide sequences from phage display were mapped in exposed regions (external surfaces) of target proteins and could be accessible to antibodies (Figure
Three dimensional epitope prediction using the PepSurf program. The peptide alignment regions are shown in red. All of the peptides align with external regions. (a) MAST1; (b) Enah; (c) MAO-A; (d) X11/MINT1; (e) HGF; (f) SNX14; (g) ARHGAP 11A; (h) APC; (i) CENTG3. Source: Martz E. FirstGlance in Jmol (
Phage display technology can be considered a subtractive proteomic strategy for the selection of specific molecules without known targets. This is due to its combinatorial nature, favoring the random binding to several molecules. It is, for this reason, an important tool for the identification of biomolecules because it exposes a large variety of ligands to many targets at the same time and requires only minimal knowledge of the starting proteome/immunome target [
Since there was the possibility of the phage binding on components of the screening system such as plastic, magnetic bead, protein G [
Our selection and analysis strategy resulted in the identification of ten potential mimotopes recognized by the IgG present in the serum of patients with AD. It was possible to select peptides by phage display and prevalidate them as potential new products for specific diagnosis of thyroid cancer [
Autoantibodies are important for AD progression. Patients with AD have a low titer of serum levels of the anti-beta-amyloid antibodies (A
The putative epitopes of the self-antigens, using the mimotopes, were mapped and are presented in Table
Identity of the self-antigens mapped by mimotopes.
Database ID | Description | Protein |
---|---|---|
AAH27985.2 | Microtubule associated serine/threonine kinase 1 | MAST1 |
AAH65238.1 | Enabled homolog (Drosophila) | ENAH |
P21397.1 | Monoamine oxidase A | MAOA |
Q02410.3 | Amyloid beta (A4) precursor protein-binding, family A, member 1 | APBA1 |
P14210.2 | Hepatocyte growth factor (hepapoietin A; scatter factor) | HGF |
Q9Y5W7.3 | Sorting nexin 14 | SNX14 |
Q6P4F7.2 | Rho GTPase activating protein 11A | ARHGAP11A |
NM_000312 | Protein C (inactivator of coagulation factors Va and VIIIa) | PROC |
AF413079.1 | Homo sapiens centaurin gamma 3 mRNA | CENTG3 |
The ALZ01 peptide is aligned with the MAST1 sequence. MAST1 is a member of the microtubule associated serine/threonine kinase family [
The Enah or Mena proteins, with which peptide ALZ02 is aligned, are a component of the neural growth cone [
Alz03 is a putative mimotope of MAO-A, an important enzyme of the catecholamine pathway. Some studies have shown changes of the catecholamine in AD. NE levels are decreased in the hippocampus of patients with AD [
The ALZ04 peptide sequence is aligned with the X11 protein family. These proteins, also known as Mints or APBA (APP binding family A), are multidomain adaptor proteins [
Another interesting mimotope of a putative self-antigen identified in the present research was the HGF. This polypeptide is a growth factor that acts like a semaphorin in the neural development [
The SNX14 protein, also mapped by the peptides from phage display, is an important element for endocytosis and endosomal signaling [
The APC protein, in mice, was found to reduce the production of Ab. The mechanism involved appears to be a stimulation of the alpha secretase activity [
The CENTG3 antibody, also known as AGAP3 and mapped by our mimotopes, is important for AMPA receptor traffic to the neural membrane during long term potentiation, which strengthens the synapse [
The involvement of aAb in neurodegenerative diseases can be varied and uncertain. Antibodies can act as receptor agonists or antagonists, coagonists, activate the complement proteins, or lead to internalization of receptors [
The role of aAb in AD has not been determined despite frequent descriptions of its presence in the serum and cerebrospinal fluid of AD patients. Some aAb, such as that generated against A
Nagele et al. [
The use of mimic peptide as a diagnostic, rather than full protein, may yield increases in the specificity of the
The combination of
The authors declare that there is no conflict of interests regarding the publication of this paper.
This research was supported by Grants from the following Brazilian research agencies: Fundação de Amparo a Pesquisa de Minas Gerais (FAPEMIG-APQ CBB-APQ-01952-13), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, 445679/2014-0), and Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES).