Diagnosis and therapy of multiple sclerosis
11505592 · 2022-11-22
Assignee
Inventors
- Roberto Paterno (Naples, IT)
- Mariarosaria Santillo (Naples, IT)
- Vittorio Enrico Avvedimento (Caserta, IT)
- Luigi Michele Pavone (Naples, IT)
Cpc classification
C07K14/4713
CHEMISTRY; METALLURGY
G01N2333/90209
PHYSICS
C07K14/705
CHEMISTRY; METALLURGY
C07K14/723
CHEMISTRY; METALLURGY
A61K39/00
HUMAN NECESSITIES
G01N2333/70571
PHYSICS
G01N2800/52
PHYSICS
C07K14/70571
CHEMISTRY; METALLURGY
C07K16/286
CHEMISTRY; METALLURGY
G01N33/564
PHYSICS
A61K39/0008
HUMAN NECESSITIES
International classification
C07K14/705
CHEMISTRY; METALLURGY
G01N33/564
PHYSICS
C07K16/28
CHEMISTRY; METALLURGY
A61K39/00
HUMAN NECESSITIES
G01N33/94
PHYSICS
Abstract
The serotonin receptor 5HT2A (5HT2aR) and membrane NADPH oxidases (NOX enzymes) are found to be a target of autoantibodies present in Multiple Sclerosis patients. The present invention refers to peptides comprised in the extracellular regions of the human 5HT2aR and/or NOXs for diagnosis and therapy of Multiple Sclerosis.
Claims
1. A peptide consisting of an amino acid sequence that is 100% identical to: (a) LYGYRWPLPSKL (SEQ ID NO: 158); (b) YRWPLPSKL (SEQ ID NO: 14); (c) RWPLPSKL (residues 2-9 of SEQ ID NO: 14); or (d) RWP (residues 2-4 of SEQ ID NO: 14); wherein the N-terminal amino group of the peptide is acylated with an N-acyl group selected from the group consisting of acetyl, lauroyl, myristoyl, palmitoyl, steroyl, oleoyl, or lineoyl; and wherein the peptide is able to bind multiple sclerosis auto-antibodies.
2. The peptide according to claim 1 wherein the sequence consists of YRWPLPSKL (SEQ ID NO: 14).
3. The peptide according to claim 1 wherein the sequence consists of RWPLPSKL (residues 2-9 of SEQ ID NO: 14).
4. The peptide according to claim 1, wherein the sequence consists of RWP (residues 2-4 of SEQ ID NO: 14).
5. The peptide according to claim 1, in linear or conformational form.
6. A pharmaceutical composition comprising the peptide according to claim 1, and pharmaceutically acceptable excipients.
7. The pharmaceutical composition according to claim 6, further comprising a therapeutic agent, selected from the group consisting of vitamins, nootropics, neuroprotective agents, racetams, isoflavones, choline, amphetamines, xanthines, adrenergics, cholinergics, serotonigergic, dopaminergics, eugeroics, GABA blockers, AMPAkines, PDE4 inhibitors, glutamate antagonists, statins, antioxidants, caspase inhibitors, neurotrophic factors, antiapoptotic agents, and anti-pain medications.
8. The pharmaceutical composition according to claim 6, further comprising a therapeutic agent selected from the group consisting of beta-interferon, methylphenidate, vitamin B, vitamin C, vitamin D, vitamin E, choline, 170-Estradiol, ginsenoside Rd, progesterone, nicotine, caffeine, and natalizumab.
9. The pharmaceutical composition according to claim 7, further comprising a peptide that is 100% identical to the amino acid sequence as set forth in SEQ ID NO: 157, or a fragment thereof.
10. The pharmaceutical composition according to claim 9, wherein the fragment consists of the sequence SKVFKEGS (residues 3-10 of SEQ ID NO: 157).
11. The pharmaceutical composition according to claim 9, wherein the fragment consists of FKE (residues 6-8 of SEQ ID NO: 157).
12. A method for detecting multiple sclerosis auto-antibodies in a patient, the method comprising: (a) obtaining a biological sample isolated from the patient; and (b) detecting whether multiple sclerosis auto-antibodies are present in the biological sample by contacting the biological sample with a peptide, and detecting binding between the multiple sclerosis auto-antibodies and the peptide; wherein the peptide comprises a peptide according to claim 1 or a fragment thereof consisting of the sequence SKL (residues 7-9 of SEQ ID NO.14).
13. The method according to claim 12, further comprising: (c) detecting whether multiple sclerosis auto-antibodies are present in the biological sample by contacting the biological sample with a peptide that is 100% identical to the amino acid sequence as set forth in SEQ ID NO: 157, or a fragment thereof, and detecting binding between multiple sclerosis auto-antibodies and said peptide or fragment thereof.
14. The method according to claim 13 wherein the fragment of the peptide of SEQ ID NO: 157 consists of the sequence SKVFKEGS (residues 3-10 of SEQ ID NO: 157).
15. The method according to claim 13 wherein the fragment of the peptide of SEQ ID NO: 157 has the sequence FKE (residues 6-8 of SEQ ID NO: 157).
16. A method for detecting multiple sclerosis auto-antibodies in a patient according to claim 13, further comprising using the detection of binding between multiple sclerosis auto-antibodies and the peptide of SEQ ID NO: 157 or fragment thereof wherein said fragment has the sequence SKVFKEGS (residues 3-10 of SEQ ID NO: 157) or FKE (residues 6-8 of SEQ ID NO: 157) for diagnosing or monitoring the progression of multiple sclerosis, identifying a therapy for multiple sclerosis, or monitoring a therapy for multiple sclerosis.
17. A method for detecting multiple sclerosis auto-antibodies in a patient according to claim 12, further comprising using the detection of binding between multiple sclerosis auto-antibodies and the peptide for diagnosing or monitoring the progression of multiple sclerosis, identifying a therapy for multiple sclerosis, or monitoring a therapy for multiple sclerosis.
18. A kit for diagnosing or monitoring the progression of multiple sclerosis, or for identifying or monitoring a therapy for multiple sclerosis, comprising the peptide according to claim 1.
19. A kit according to claim 18, further comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 157 or fragment thereof.
Description
(1) The present invention will be illustrated by means of non-limiting examples in reference to the following figures.
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(5) Then cells were incubated for 30 min with 200 μg of serum IgG from MS (IgGMS) or neurological (other neurological disorders affected patients, IgGCtr), and stained for 30 min with PE-conjugated goat anti human IgG. Cells were washed and resuspended in 200 μL of PBS for flow cytometric analysis. EGFP-positive cells, corresponding to the R2 region shown in the FSC/SSC dot blot panel, were 43.8%. The value reported inside each FSC/FL-2 dot blot, represent the percent of FL-2 positive cells inside the R2 region (GFP-positive cells). The upper panel shows the binding of serum Ig (MS or neurological (N) patients) to mock transfected cells. In each panel, control cells incubated with secondary antibody alone, are shown.
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Specific Embodiments Of The Invention
(21) 1. A peptide consisting of an amino acid sequence that is 100% identical to: (a) LYGYRWPLPSKL (SEQ ID NO: 158); (b) YRWPLPSKL (SEQ ID NO: 14); (c) RWPLPSKL (residues 2-9 of SEQ ID NO: 14); (d) RWP (residues 2-4 of SEQ ID NO: 14); or (e) SKL (residues 7-9 of SEQ ID NO: 14);
wherein the peptide is able to bind multiple sclerosis auto-antibodies.
(22) 2. The peptide according to embodiment 1 wherein the sequence consists of YRWPLPSKL (SEQ ID NO: 14).
(23) 3. The peptide according to embodiment 1 wherein the sequence consists of RWPLPSKL (residues 2-9 of SEQ ID NO: 14).
(24) 4. The peptide according to embodiment 1, wherein the sequence consists of RWP (residues 2-4 of SEQ ID NO: 14).
(25) 5. The peptide according to embodiment 1, wherein the sequence consists of SKL (residues 7-9 of SEQ ID NO: 14).
(26) 6. The peptide according to embodiment 1, in linear or conformational form.
(27) 7. A pharmaceutical composition comprising the peptide according to embodiment 1, and pharmaceutically acceptable excipients.
(28) 8. The pharmaceutical composition according to embodiment 7, further comprising a therapeutic agent, selected from the group consisting of vitamins, nootropics, neuroprotective agents, anti-pain medication, racetams, isoflavones, vitamins, choline, amphetamines, xanthines, adrenergics, cholinergics, serotonigergic, dopaminergics, eugeroics, GABA blockers, AMPAkines, PDE4 inhibitors, glutamate antagonists, statins, antioxidants, caspase inhibitors, neurotrophic factors, antiapoptotic agents, and anti-pain medications.
(29) 9. A method for detecting multiple sclerosis auto-antibodies in a patient, the method comprising: (a) obtaining a biological sample isolated from the patient; and (b) detecting whether multiple sclerosis auto-antibodies are present in the biological sample by contacting the biological sample with a peptide, and detecting binding between multiple sclerosis auto-antibodies and the peptide ; wherein the peptide comprises a peptide according to embodiment 1.
(30) 10. A kit for diagnosing or monitoring the progression of multiple sclerosis, or for identifying or monitoring a therapy for multiple sclerosis, comprising the peptide according to embodiment 1.
(31) 11. A nucleic acid molecule encoding a peptide consisting of an amino acid sequence that is 100% identical to: LYGYRWPLPSKL (SEQ ID NO: 158), wherein the peptide is able to bind multiple sclerosis auto-antibodies.
(32) 12. The pharmaceutical composition according to embodiment 7, further comprising a therapeutic agent selected from the group consisting of b-interferon, methylphenidate, vitamin B, vitamin C, vitamin D, vitamin E, choline, 17β-Estradiol, ginsenoside Rd, progesterone, nicotine, caffeine, and natalizumab.
(33) 13. The pharmaceutical composition according to embodiment 7, further comprising a peptide or fragment thereof consisting of an amino acid sequence that is 100% identical to SEQ ID NO: 157.
(34) 14. The pharmaceutical composition according to embodiment 13, wherein the fragment consists of the sequence SKVFKEGS (residues 3-10 of SEQ ID NO: 157).
(35) 15. The pharmaceutical composition according to embodiment 13, wherein the fragment consists of FKE (residues 6-8 of SEQ ID NO: 157).
(36) 16. The method according to embodiment 9, further comprising: (c) detecting whether multiple sclerosis auto-antibodies are present in the biological sample by contacting the biological sample with the peptide or fragment thereof consisting of the amino acid sequence of SEQ ID NO: 157, and detecting binding between multiple sclerosis auto-antibodies and the peptide or fragment thereof comprising the amino acid sequence of SEQ ID NO: 157.
(37) 17. A kit according to embodiment 10, further comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 157 or fragment thereof.
(38) 18. A method for detecting multiple sclerosis auto-antibodies in a patient according to embodiment 9, further comprising using the detection of binding between multiple sclerosis auto-antibodies and the peptide for diagnosing or monitoring the progression of multiple sclerosis, identifying a therapy for multiple sclerosis, or monitoring a therapy for multiple sclerosis.
(39) 20. The method according to embodiment 16 wherein the fragment of the peptide of SEQ ID NO: 157 consists of the sequence SKVFKEGS (residues 3-10 of SEQ ID NO: 157).
(40) 21. The method according to embodiment 16 wherein the fragment of the peptide of SEQ ID NO: 157 has the sequence FKE (residues 6-8 of SEQ ID NO: 157).
(41) 22. A method for detecting multiple sclerosis auto-antibodies in a patient according to embodiment 16, further comprising using the detection of binding between multiple sclerosis auto-antibodies and the peptide of SEQ ID NO: 157 or fragment thereof wherein said fragment has the sequence SKVFKEGS or FKE for diagnosing or monitoring the progression of multiple sclerosis, identifying a therapy for multiple sclerosis, or monitoring a therapy for multiple sclerosis.
DETAILED DESCRIPTION OF THE INVENTION
(42) Materials and Methods
(43) Patients
(44) In the study MS group comprises men and women between 15 and 50 years of age who meet all the following criteria: diagnosis of relapsing/remitting MS, according to McDonald criteria; an Expanded Disability Scale Score (EDSS) between O and 5.0; lesions detected by MRI compatible with the diagnosis of multiple sclerosis; at least one acute episode in the last 12 months.
(45) Control samples include other neurological disorders affected patients (including inflammatory, degenerative diseases not involving direct or indirect de-myelinization, i. e.: cerebral cancers, stroke, vasculitis, etc) that need differential diagnosis with multiple sclerosis. Control patients were selected by sex and age to be similar to multiple sclerosis patients. Blood serum was collected, from each patients. From the blood serum, to perform the experiments on cell culture, the IgG fractions were purified.
(46) Patients gave written informed consent before any study-related procedures was performed.
(47) Purification of Immunoglobulins
(48) The purification of IgG fractions from serum of MS and control Neurological subjects has been carried out by affinity chromatography on A/G Sepharose columns (Pierce, Rockford, Ill.). The protein concentration of immunoglobulin fractions has been assessed spectrophotometrically.
(49) Cell Cultures
(50) MO3-13 Cells
(51) The MO3-13 cells are an immortal human-human hybrid cell line with the phenotypic characteristics of primary oligodendrocytes (OLs), derived from the fusion of a 6-thioguanine-resistant mutant of a human rhabdomyosarcoma with OLs obtained from adult human brain (CELLution Biosystem Inc., Canada). They were grown in Dulbecco's Modified Eagles Medium (DMEM; GIBCO Invitrogen), containing 4.5 g/L glucose (GIBCO, Auckland, New
(52) Zealand), supplemented with 10% Foetal Bovine Serum, 100 U/ml penicillin and 100 μg/ml streptomycin (FBS; Sigma S. Louis, USA).
(53) HEK293 Cells
(54) HEK293 is a cell line derived from human embryonic kidney cells (American Type Culture Collection, ATCC, USA). They were grown in Dulbecco's Modified Eagles Medium (DMEM; GIBCO Invitrogen), containing 4.5 g/L glucose (GIBCO, Auckland, New Zealand), supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml (Sigma S. Louis, USA).
(55) Hela and SH-SY5Y Cells
(56) The human cervical adenocarcinoma Hela cells and the human neuroblastoma SH-SY5Y cell lines (American Type Culture Collection, ATCC, USA) were grown in DMEM-F12 medium (GIBCO Invitrogen) containing 4.5 g/L glucose (GIBCO, Auckland, New Zealand), supplemented with 10% FBS (Sigma S. Louis, USA), 100 U/ml penicillin and 100 μg/ml. The cells were kept in a 5% CO2 and 95% air atmosphere at 37° C.
(57) Flow Cytometric Assay of 5-Ht2AR
(58) MO3-13 cells were grown to semiconfluency in 60-mm culture dishes. After trypsin detachment, 5.Math.10.sup.5 cells were suspended in 1 mL of phosphate buffered saline (PBS) and fixed overnight with 1% formaldehyde at room temperature. Next, cells were permeabilized with 0.1% Triton X-100 for 40 min at 4° C., washed 4× with 2 mL of PBS containing 2% FBS, 0.01% NaN3, 0.1% Triton X-100 (buffer A), and incubated for 45 min at 4° C. with 1:50 dilution of Rabbit polyclonal to 5HT2aR antibody (Abcam ab81864). The cells were then washed twice with the same buffer and incubated for 45 min at 4° C. with Cy3-conjugated anti-(rabbit IgG) Ig (Amersham Pharmacia Biotech) at 1:50 dilution. Control cells were incubated with Cy3-conjugated anti-(rabbit IgG) IgG alone. After two washes in buffer A, cells were resuspended in PBS and analyzed by flow cytometry using FACSCAN (BD, Heidelberg, Germany) and WINMDI software.
(59) Flow Cytometric Assay of Serum IgG Binding to 5-HT2aR
(60) HEK293 cells were plated in 100 mm Petri dishes and grown to semiconfluence. After trypsinization and wash in PBS, the cells were resuspended in 200 μl PBS and then incubated with mouse serum for 30 min at 4° C., to block nonspecific binding; then, they were incubated for 30 min with 200 μg of serum IgG (MS or neurological), and stained for 30 min with PE-conjugated goat anti human IgG. Cells were washed and resuspended in 200 μL of PBS for flow cytometric analysis of phycoerythrin positive cells with a FACSscan apparatus (Becton-Dickinson). Data were analyzed using WinMDI software.
(61) Immunoprecipitation and Immunoblotting Experiments
(62) MO3-13 cells, grown to semiconfluence in 100 mm dishes, were incubated for 18 h in 0.2% FBS medium.
(63) The cells were washed twice with PBS and harvested in cold RIPA buffer containing 2.5 mM Na-pyrophosphate, 1 mM β-glycerophosphate, 1 mM NaVO4, 1 mM NaF, 0.5 mM PMSF, and the cocktail of protease inhibitors. The cells were kept for 15 min at 4° C. and disrupted by repeated aspiration through a 21-gauge needle. Cellular debris was pelleted by centrifugation at 11600 g for 15 min at 4° C. 300 μg of cellular lysates were immunoprecipitated with IgG from Neurological or MS patients at 1:10 dilution. Samples were rocked gently for 16 h; thereafter 20 μl of protein A/G PLUS-Agarose (Santa Cruz Biotechnology), resuspended in RIPA buffer, was added to immunoprecipitates. Samples were further rocked for 1 h, centrifuged at 3000 rpm. Supernatants were collected and the protein A/G PLUS-Agarose was added again, to obtained the immunodepleted samples. Then, the pellets were washed thrice in RIPA buffer and once with PBS before the addition of 20 μl Laemmli sample buffer.
(64) Immunoprecipitated/immunodepleted samples and 50 μg of total lysates in Laemmli buffer were boiled for 5 min and centrifuged for 1 min at 11600 g at room temperature (22° C.). The pellets were discarded and supernatants were resolved by 7.5% SDS-PAGE and transferred onto nitrocellulose membrane.
(65) Next, the membrane was blocked in 3% dry-fat milk in TBS-Tween20 (0.05%) and probed with a polyclonal anti-human anti NOX3 (Abcam ab81864) or 5HT-2a receptor (Abcam ab85496) antibodies at 1:1000 dilution. Then, the membrane was washed and incubated with a secondary horseradish peroxidase-linked antibody (Amersham Pharmacia Biotech) 1:2000 and was detected by ECL.
(66) Indirect ELISA
(67) Diluted peptide (20 μg/ml) was coated to the wells of a PVC microtiter plate and incubated at 4° C. overnight. After many wash, the remaining protein-binding sites was blocked with 3% BSA solution and incubated at 4° C. overnight. The patient extracted immunoglobulins was diluted in blocking buffer and incubated at 4° C. overnight. The plate was washed for four times with PBS. For the detection, we used a secondary antibody anti human (recognized constant region of the patient antibody conjugated with HRP and (3,3′,5,5′-Tetramethylbenzidine) TMB solution that was added for each well and incubated for 15-30 min. Equal volume of stopping solution (2 M H2SO4) was added to the plate and absorbance (optical density) of plate was read at 450 nm.
(68) ELISA with Beads
(69) Beads linked Peptide (6×10.sup.4 beads/sample) were mixed with different concentrations of patient extracted immunoglobulins for 16 h at 4° C. in a rotator. The beads were washed twice with PBS by centrifugation at 14,000 rpm for 2 min at room temperature and were resuspended in 100 μl of PBS. For the detection, we used a secondary antibody anti human (recognized constant region of the patient antibody conjugated with HRP and (3,3′,5,5′-Tetramethylbenzidine) TMB solution that was added for each well and incubated for 15-30 min. Equal volume of stopping solution (2 M H2SO4) was added to the plate and absorbance (optical density) of plate was read at 450 nm.
(70) Detection of Peptide/Immunoglobulins Derived from Patients Interaction on Beads by Flow Cytometry
(71) Beads linked Peptide (6×10.sup.4 beads/sample) were mixed with different concentrations of patient extracted immunoglobulins for 16 h at 4° C. in a rotator. The beads were washed twice with PBS by centrifugation at 14,000 rpm for 2 min at room temperature and were resuspended in 100 μl of PBS. Then, the samples was incubated with anti-human secondary antibodies conjugated with FITCH and beads were analyzed on a BD FACS Calibur (Becton-Dick-inson, Franklin Lakes, N.J.), and the data analyzed on FlowJo (Treestar, Ashland, Oreg.) software.
(72) Determination of Reactive Oxygen Species
(73) ROS levels were determined using the membrane-permeant fluorogenic probe 5,6-carboxy-2′,7′-dichlorofluoresceindiacetate, DCHFDA (Molecular Probes, Leiden, the Netherlands). The assay was based on the fluorescence detection of dichlorofluorescein (DCF), formed by ROS-mediated oxidation of the non-fluorescent precursor, dichlorofluorescin.
(74) MO3-13 cells were grown to semi-confluence in 24 multiwell plates (45000 cell/well) and incubated for 18 h in medium containing 0.2% FBS before the experiments. The cells were washed twice with FBS free medium and incubated with 50 μM of DDSK for 30 min at 37° C. and then with 200 μg/ml of IgG from Ctr or MS patients (Damiano et al., 2013) for 30 min at 37° C. The cells were incubated with 10 μM DCHF-DA for 10 min and washed three times with PBS containing 10 mM glucose, 1.2 mM MgCl2 and 1.2 mM CaCl.sub.2. DCF fluorescence was measured using the plate reader Fluoroskan Ascent FL fluorometer (Thermo Electron Oy, Vantaa, Finland) and data analyzed by Ascent software.
(75) To evaluate the effects of 5Ht on ROS levels, a dose of 50 μM of the substance was added to the cells after DCHF-DA incubation and DCF fluorescence was measured at different time intervals.
(76) Semi-Quantitative PCR Analysis
(77) Total RNA was extracted using Trizol reagent according to the protocol provided by the manufacturer (Sigma-Aldrich). Total RNA (1 μg) was reverse transcribed using Transcriptor First Strand cDNA Syn-thesis Kit (Roche Applied Science, Monza, Italy) by oligo-dT primers for 30 min at 55° C. in a 20 μl reaction volume. Semi-quantitative PCR was performed using Hot Master TaqDNA Polymerase (SPRIME) in 20 μl final volume containing 0.2 mM dNTP, 0.2 μM of the specific primers and 100 ng of sample cDNA. The PCR conditions used were 94° C. 2 min, (94° C. 30 s, 60° C. 30 s, 70° C. 30 s) and 70° C. 5 min. The reactions were carried out at 35 number of cycles. Primers used in these experiments are the following:
(78) TABLE-US-00002 Human NOX1: (SEQ ID NO: 159) (F), TTA ACA GCA CGC TGA TCC TG (SEQ ID NO: 160) (R), CAC TCC AGTGAG ACC AGC AA. Human cytochrome b-245. beta polypeptide (CYBB, alias NOX2): (SEQ ID NO: 161) (F), GGA GTT TCA AGA TGC GTG GAA ACT A (SEQ ID NO: 162) (R), GCC AGA CTC AGAGTT GGA GAT GCT. Human NOX 3: (SEQ ID NO: 163) (F), CCA GGG CAG TAC ATC TTG GT (SEQ ID NO: 164) (R), CCG TGTTTC CAG GGA GAG TA. Human NOX4: (SEQ ID NO: 165) (F), GCT TAC CTC CGA GGA TCA CA (SEQ ID NO: 166) (R), CGG GAGGGT GGG TAT CTA A. Human NOX 5: (SEQ ID NO: 167) (F), ATC AAG CGG CCC CCT TTT TTT CAC (SEQ ID NO: 168) (R), CTCATT GTC ACA CTC CTC GAC AGC. Human 5HT2A: (SEQ ID NO: 169) (F) TCATCATGGCAGTGTCCCTA (SEQ ID NO: 170) (R), TGAGGGAGGAAGCTGAAAGA. B-actin: (SEQ ID NO: 171) (F) TCACCCTGAAGTACCCCATC (SEQ ID NO: 172) (R), GGCTGGAAGAGTGCCTCA.
Plasmid
(79) h-5HT2aR/EGFP construct: h-5HT2aR gene (NCBI Accession Number: NP_000612) has been inserted in the pEGFP-N3 vector from Clontech. The cDNA is cloned between BamHI and BglII sites in the MCS.
(80) Transfections
(81) The cells were transfected with h-5HT2aR/EGFP construct. One day before transfection, 450.000 cells (HEK293) were plated in 35 mm dishes in growth medium so that cells will be 70-90% confluent at the time of transfection. For each transfection sample, complexes were prepared as follows: 1 γ/λ DNA (5-HT2a receptor conjugated to EGFP) was added to 80 μl of growth medium without serum and antibiotics. 4 μl of Lipofectamine™ 2000 was added to 80 μl of growth medium without serum and antibiotics. The diluted DNA was combined with diluted Lipofectamine™ 2000, mixed gently and incubated for 45 minutes at room temperature. Then the complexes were added to cells. Cells were incubated at 37° C. in a CO2 incubator for 18-48 hours prior to testing.
Western Blotting Analysis
(82) Antibodies.
(83) DUOX 1 and 2 proteins were detected with a rabbit polyclonal antibody raised against the peptide sequence ETELTPQRLQC (SEQ ID NO: 174) located inside the first intracellular loop of human DUOX1 (Damiano et al., PlosOne). P-ERK1/2 (sc-7383) and HaRas (sc-520) antibodies were purchased by Santa Cruz; NOX3 (ab81864) and h-5HT2aR antibodies (ab85496) were purchased by Abcam.
(84) Total cells lysates were obtained in RIPA buffer (50 mM Tris-HCl, pH 7.5, NaCl 150 mM, 1% NP40, 0.5% deoxycholate, 0.1% SDS) containing 2.5 mM Na-pyrophosphate, 1 mM β-glycerophosphate, 1 mM NaVO4, 1 mM NaF, 0.5 mM PMSF, and a cocktail of protease inhibitors (Roche, USA). The cells were kept for 15 min at 4° C. and disrupted by repeated aspiration through a 21-gauge needle. Cell lysates were centrifuged for 10 min at 13000 rpm and the pellets were discarded. Fifty micrograms of total proteins were subjected to SDS-PAGE under reducing conditions. After electrophoresis, the proteins were transferred onto a nitrocellulose filter membrane (Bio-Rad Laboratories, UK) with a Trans-Blot Cell (Bio-Rad Laboratories, UK) and transfer buffer containing 25 mM Tris, 192 mM glycine, 20% methanol. Membranes were placed in 5% non-fat milk in phosphate-buffered saline, 0.5% Tween 20 (TBST) at 4° C. for 2 h to block the nonspecific binding sites. Filters were incubated with specific antibodies before being washed three times in TBST and then incubated with a peroxidase-conjugated secondary antibody (Santa Cruz). After washing with TBST, peroxidase activity was detected with the ECL system (GE-Healthcare, UK).
(85) The filters were also probed with an anti α-tubulin antibody (Sigma, USA). Protein bands were revealed by ECL and, when specified, quantified by densitometry using ImageJ software. Densitometric values were normalized to α-tubulin.
(86) Since, depending on the cell type or tissue, bands of different sizes can appear by Western blotting for 5HT2aR (fragments or protein complexes), to determine which bands are specific, before proceeding with the staining protocol, the antibody was incubated with an excess of peptide that correspond to the epitope recognized by the antibody. By comparing the staining from the blocking antibody vs the antibody alone it has been possible to evidence the specific 5HT2aR staining.
(87) Peptide Synthesis and Screening Assays
(88) The linear and CLIPS peptides are synthesized based on the amino acid sequence of the target protein using standard Fmoc-chemistry and deprotected using trifluoric acid with scavengers. The constrained peptides are synthesized on chemical scaffolds in order to reconstruct conformational epitopes, using Chemically Linked Peptides on Scaffolds (CLIPS) technology (Timmerman et al. (2007). For example, the single looped peptides are synthesized containing a dicysteine, which was cyclized by treating with alpha, alpha′-dibromoxylene and the size of the loop is varied by introducing cysteine residues at variable spacing. If other cysteines besides the newly introduced cysteines are present, they are replaced by alanine. The side-chains of the multiple cysteines in the peptides are coupled to CLIPS templates by reacting onto credit-card format polypropylene PEPSCAN cards (455 peptide formats/card) with a 0.5 mM solution of CLIPS template such as 1,3-bis (bromomethyl) benzene in ammonium bicarbonate (20 mM, pH 7.9)/acetonitrile (1:1(v/v)). The cards are gently shaken in the solution for 30 to 60 minutes while completely covered in solution. Finally, the cards are washed extensively with excess of H.sub.2O and sonicated in distrupt-buffer containing 1 percent SDS/0.1 percent beta-mercaptoethanol in PBS (pH 7.2) at 70° C. for 30 minutes, followed by sonication in H.sub.2O for another 45 minutes.
(89) The binding of antibody to each peptide is tested in a PEPSCAN-based ELISA. The 455-well credit card format polypropylene cards containing the covalently linked peptides are incubated with primary antibody solution for example consisting of 1/1000 diluted serum in blocking solution, for example 4% horse serum, 5% ovalbumin (w/v) in PBS/1% Tween. After washing, the peptides are incubated with a 1/1000 dilution of antibody peroxidase conjugate for one hour at 25° C. After washing, the peroxidase substrate 2,2′-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 2 microlitres of 3 percent H.sub.2O.sub.2 are added. After one hour, the color development are measured. The color development are quantified with a charge coupled device (CCD)-camera and an image processing system (Slootstra et al., 1996).
(90) The raw data are optical values obtained by a CCD-camera calibrated to export absorption values compatible with a standard 96-well plate ELISA-reader. First the CCD-camera makes a picture of the card before peroxidase coloring and then again a picture after the peroxidase coloring. These two pictures are substracted from each other yielding a binding value for each peptide. This data is entered in the Peplab™ database for secure storage and retrieval.
(91) All raw data are provided in an excel file and the full technical report include plots of the binding acitivity to all peptides and a 3D visualization of all epitope candidates identified.
(92) Methods are described in Timmerman et al. (2007). Functional reconstruction and synthetic mimicry of a conformational epitope using CLIPS™ technology). Structural aspects of antibody-antigen interaction revealed through small random peptide libraries (Slootstra et al., 1996).
(93) Co-Culture of Cortical Neurons and OPCs
(94) Primary cultures of cortical OPCs and neurons are prepared as described by Cheli et al. (2015). For OPCs preparation, cerebral hemispheres from 1 day old mice are mechanically dissociated and plated on poly-D-lysine-coated flasks in Dulbecco's modified Eagle's medium and Ham's F12 (1:1 v/v), containing 100 μg/ml gentamicin and supplemented with 4 mg/ml dextrose anhydrous, 3.75 mg/ml HEPES buffer, 2.4 mg/ml sodium bicarbonate and 10% fetal bovine serum (FBS). After 24 h the medium is changed and the cells are grown in DMEM/F12 supplemented with insulin (5 μg/ml), human transferrin (50 μg/ml), sodium selenite (30 nM), D-Biotin (10 mM), 0.1% BSA (Sigma), 1% FBS and 1% horse serum. After 9 days, OPCs are purified from the mixed glial culture by the differential shaking and adhesion procedure and allowed to grow on poly-Dlysine-coated coverslips in culture media plus PDGF (10 ng/ml) and bFGF (10 ng/ml). OPCs are kept in mitogens (PDGF and bFGF) for 2 days and then induced to exit from the cell cycle and differentiate by switching the cells to a mitogen-free medium (mN2).
(95) Cortical neurons are prepared from the brains of 1- to 2-day-old mouse. Brain cortices are isolated and dissociated by digestion with a solution of 0.05% trypsin (Sigma) containing DNase I (0.06%) in Neurobasal medium for 10 min at 37° C. The digestion reaction is stopped with Neurobasal medium containing 10% fetal bovine serum and triturated by repeated passages (20 times) through a 10 ml pipette. The cell suspension is filtered through a sterile cell strainer (70 μm) into a 50 ml centrifuge tube. The cells are pelleted by centrifugation at 200 g for 5 min, and resuspended in Neurobasal medium plus 2% (v/v) B27 supplemented with 0.25 mM GlutaMax I, 0.25 mM glutamine (Invitrogen), and 100 μg/ml gentamicin. High-density cultures (5×10.sup.5 cells, ˜2500 cells/mm2) are plated onto 20 mm.sup.2 tissue culture wells coated with poly-d-lysine. The neurons are kept at 37° C. in 95% air 5% CO2 for 7 days in vitro and used for co-cultures. After 7 days in vitro, cortical neuron cultures consist of neurons essentially free from non-neural cells.
(96) Co-cultures are prepared by the addition of OPCs to the cultures of cortical neurons at a density of 3×10.sup.5 cells/ml. These cultures are maintained in DMEM/F12, 1% FBS for 7 and 14 days (Cheli et al., 2015).
(97) The neuron-OPC co-colture model allows the evaluation of the myelination stage of mature OL, by confocal microscopy categorizing them in three different stages: (1) cells that only extend processes but do not contact with neurofilaments; (2) cells that establish contact with neurofilaments but do not myelinate; and (3) cells that wrap axons and have at least two internodes connected to the cell body (Barateiro and Fernandes, 2014).
(98) Scratch Assay
(99) MO3-13 cell migration is assessed by in vitro scratch assay, based on the creation of an artificial gap on a confluent cell monolayer. MO3-13 cells are grown in complete medium and when cells reach 70-80% confluence, a wound is made across the cell layer using a cell scraper. Then cultures are washed in complete medium and are allowed to migrate for 24-48 h and the number of cells that moved across the injury line is counted at microscope.
(100) Fluorimetric Measurement of Intracellular Ca.sup.2+
(101) Intracellular Ca.sup.2+levels are measured fluorimetrically using the membrane-permeable Ca.sup.2+-sensitive dye Fluo-3-AM. Briefly, cells are washed twice with TTS buffer (137 mN NaCl, 2.7 mM KCl, 1.0 mM MgCl.sub.2, 1.8 mM CaCl.sub.2, 0.2 mM NaH.sub.2PO.sub.4, 12 Mm NaHCO.sub.3, 5.5 mM glucose, pH 7.4) and incubated with 10 μm Fluo-3-AM and 0.02% pluronic acid for 1 h at room temperature in the dark. Cells are then washed twice with TTS, before adding the substances to be tested and the changes in fluorescence is measured at different time intervals using Fluoroskan Ascent fluorescent plate reader (ThermoElectron Oy, Vantaa, Finland) and data analyzed by Ascent software.
(102) Induction of EAE (Experimental Autoimmune Encephalomyelitis) as Murine Model for Human Multiple Sclerosis
(103) Mice will be immunized subcutaneously with 100 ml of emulsified incomplete Freund's adjuvant supplemented with 500 mg of Mycobacterium tuberculosis H37Ra (Difco) and 100 mg of MOG35-55, and will receive an intraperitoneal injection of 200 ng of pertussis toxin (List Biological Laboratories) at the time of immunization and 48 hours later. The mice will be observed daily for clinical signs and scored as described (Shi et al., 2011). Mice will be euthanized, and brains and spinal cords will be removed and fixed by immersion with a 10% neutral-buffered formalin solution and decalcified. Fixed tissues will be embedded in paraffin, sectioned, and stained with H&E and serial histological sections will be stained also immunohistochemically to determine the distribution and types of inflammatory cells in the brain and spinal cord and the demyelination. Spinal cord pathology will be assigned by scores with an experienced pathologist as described previously (Shi et al., 2011)
(104) Annexin V/Propidium Iodide Apoptosis Assay
(105) 2-4×10.sup.6 cells are resuspend in 100 μL 1×Annexin V binding buffer. 5 μL Annexin V Alexa Fluor 488 was added to the samples and incubated in the dark for 15 minutes at room temperature. 100 μL of 1×Annexin V binding buffer and 4 μL of PI at final PI concentration of 2 μg/mL are added to each reaction tube.
(106) The samples are incubated in the dark for 15 minutes at room temperature. The cells are washed with 500 μL 1×Annexin V binding buffer. Samples centrifuged at 335×g for 10 minutes and supernatant decanted. Cells are resuspended in 500 μL 1×Annexin V binding buffer and 500 μL 2% formaldehyde to create a 1% formaldehyde (fixative) solution. Tubes are mixed by gentle flicking and fixed on ice for 10 minutes.
(107) The cells are washed with PBS and diluted with RNase A at the final concentration of 50 μg/mL and incubated for 15 min at 37° C. The tubes are centrifuged at 425×g for eight minutes and samples are analyzed by cytofluorimetry.
(108) Statistical Analysis
(109) Statistical differences were evaluated using a Student's t-test for unpaired samples.
(110) Results
(111) IgG from MS Patients Interact with 5Ht2A Receptor
(112) 5-HT2aR protein expression in MO3-13 cells was evaluated by indirect immunofluorescence and flow cytometry (
(113) To confirm the direct interaction between IgG MS and 5-HT2AR, we performed flow cytometric surface binding experiments on HEK293 cells transfected in transient with the h-5HT2aR/EGFP construct. Neither the IgG from control subjects (IgN), nor those from MS patients (IgG MS) significantly bound to the surface of mock transfected cells (
(114) NOX3 Interacts with 5HT2aR
(115) As shown in
(116) The inventors focused their attention on NOX3 since this isoform shows a higher percentage of identity (58.8) with NOX2 than NOX5 (32.6%). NOX2 is expressed in oligodendrocytes in vivo (Cavaliere et al., 2013). In particular, NOX3 extracellular domains show a certain degree of identity with NOX2 extracellular domains. This is not the case for NOX5.
(117) The inventors first evaluated whether NOX3 directly interacts with 5HT2aR by immunoprecipitation of cell extract with anti h-NOX3 antibody followed by Western blotting with anti h-5HT2aR antibody. As shown in
(118) IgG from MS Patients Interact with NOX3
(119) The inventors further evaluated whether IgG from MS patients interact with NOX3 in MO3-13 cells. To this aim MO3-13 cells were immunoprecipitated with IgG from Control and MS subjects and then immunoblotted with anti h-NOX3 antibody. As shown in
(120) Overall, our data suggest that IgGs from MS patients bind to 5HT2aR and to NOX3. Since NOX3 binds to 5HT2aR, it is possible that a membrane protein complex constituted by 5HT2aR, NOX3 and the IgGs from MS is present in MO3-13 cells and in vivo.
(121) Precision Epitope Mapping of NOX2 and 5-HT2A Receptor Extracellular Domains with the Serum from Multiple Sclerosis Affected Patients
(122) Approximately 1250 different peptides including linear and CLIPS peptides have been designed and synthesize based on NOX2 heavy chain amino acid sequence (SEQ ID 1) and other 1250 peptides were designed and synthesize based on the human 5-HT2A receptor amino acid sequence (SEQ ID 2). In particular, in both cases, only the extracellular domains of the proteins have been used for the design of the two peptide libraries. For NOX2 were selected the regions 30-48 (SEQ ID 3), 124-169 (SEQ ID 4) and 222-261 (SEQ ID 5) while for 5-HT2Ar were selected the regions 1-76 (SEQ ID 6), 133-148 (SEQ ID 7), 215-233 (SEQ ID 8), 347-362 (SEQ ID 9).
(123) TABLE-US-00003 Sequence ID SEQ ID 1: >sp|P04839|CY24B_HUMAN Cytochrome b- 245 heavy chain OS = Homo sapiens GN = CYBB PE = 1 SV = 2 (NOX2) MGNWAVNEGLSIFVILVWLGLNVFLFVWYYRVYDIPPKFFYTRKLLGSAL ALARAPAACLNFNCMLILLPVCRNLLSFLRGSSACCSTRVRRQLDRNLTF HKMVAWMIALHSAIHTIAHLFNVEWCVNARVNNSDPYSVALSELGDRQNE SYLNFARKRIKNPEGGLYLAVTLLAGITGVVITLCLILIITSSTKTIRRS YFEVFWYTHHLFVIFFIGLAIHGAERIVRGQTAESLAVHNITVCEQKISE WGKIKECPIPQFAGNPPMTWKWIVGPMFLYLCERLVRFWRSQQKVVITKV VTHPFKTIELQMKKKGFKMEVGQYIFVKCPKVSKLEWHPFTLTSAPEEDF FSIHIRIVGDWTEGLFNACGCDKQEFQDAWKLPKIAVDGPFGTASEDVFS YEVVMLVGAGIGVTPFASILKSVWYKYCNNATNLKLKKIYFYWLCRDTHA FEWFADLLQLLESQMQERNNAGFLSYNIYLTGWDESQANHFAVHHDEEKD VITGLKQKTLYGRPNWDNEFKTIASQHPNTRIGVFLCGPEALAETLSKQS ISNSESGPRGVHFIFNKENF SEQ ID 2: >sp|P28223|5HT2A_HUMAN 5-hydroxy- tryptamine receptor 2A OS = Homo sapiens GN = HTR2A PE = 1 SV = 2 MDILCEENTSLSSTTNSLMQLNDDTRLYSNDFNSGEANTSDAFNWTVDSE NRTNLSCEGCLSPSCLSLLHLQEKNWSALLTAVVIILTIAGNILVIMAVS LEKKLQNATNYFLMSLAIADMLLGFLVMPVSMLTILYGYRWPLPSKLCAV WIYLDVLFSTASIMHLCAISLDRYVAIQNPIHHSRFNSRTKAFLKIIAVW TISVGISMPIPVFGLQDDSKVFKEGSCLLADDNFVLIGSFVSFFIPLTIM VITYFLTIKSLQKEATLCVSDLGTRAKLASFSFLPQSSLSSEKLFQRSIH REPGSYTGRRTMQSISNEQKACKVLGIVFFLFVVMWCPFFITNIMAVICK ESCNEDVIGALLNVFVWIGYLSSAVNPLVYTLFNKTYRSAFSRYIQCQYK ENKKPLQLILVNTIPALAYKSSQLQMGQKKNSKQDAKTTDNDCSMVALGK QHSEEASKDNSDGVNEKVSCV SEQ ID 3: CY (NOX2) Loop 1: YRVYDIPPKFFYTRKLLGS SEQ ID 4: CY (NOX2) Loop 2: EWCVNARVNNSDPYSVALSELGDRQNESYLNFARKRIKNPEGGLYL SEQ ID 5: CY (NOX2) Loop 3: HGAERIVRGQTAESLAVHNITVCEQKISEWGKIKECPIPQ SEQ ID 6: N-terminal of 5HT2aR: 1-76 (76 aa) MDILCEENTSLSSTTNSLMQLNDDTRLYSNDFNSGEANTSDAFNWTVDSE NRTNLSCEGCLSPSCLSLLHLQEKNW SEQ ID 7: Loop 1 of 5HT2aR: 133-148 (16 aa) LTILYGYRWPLPSKLC SEQ ID 8: Loop 2 of 5HT2aR: 215-233 (19 aa) LQDDSKVFKEGSCLLADDN SEQ ID 9: Loop 3 of 5HT2aR: 347-362 (16 aa) VICKESCNEDVIGALL
(124) All peptides have been synthesized in a peptide array format and the binding of 20 different sera to all peptide libraries were measured in an ELISA based set up following the procedure described in detail in the method section “Peptide synthesis and screening assays”. In particular 2 sets of experiments were performed on the 2 libraries by using 2 different dilutions of the human patients sera. In the first set of experiment the 20 sera were used at 1:2500 dilutions, while in the second experiment the dilution of the sera was 1:1000. The binding of the different sera to all peptides was quantified and analyzed in detail (Tables 1 and 2,
(125)
(126) TABLE-US-00004 TABLE 1 List of peptide sequences from 5HT2A receptor library that are significantly recognized by the MS sera (n = 9) compared to CTRLs (n = 10)(p < 0.05). Peptide Sequence SEQ ID 1 CNSLMQLNDDTRLYCMDILSEENTSLSSC SEQ ID NO: 10 HTR.MAT 2 CLTILYGYRWPLPSCMDILSEENTSLSSC SEQ ID NO: 11 HTR.MAT 3 LTILYGYRWPAASKL SEQ ID NO: 12 HTR.LIN15AA 4 CLYGYRWPLPSKLSCMDILSEENTSLSSC SEQ ID NO: 13 HTR.MAT 5 YRWPLPSKL SEQ ID NO: 14 HTR.LIN9 6 CNSLMQLNDDTRLYCLSSEGSLSPSSLSC SEQ ID NO: 15 HTR.MAT 7 CLQDDSKVFKEGSSCMDILSEENTSLSSC SEQ ID NO: 16 HTR.MAT 8 LTILYGYRWPLPSKL SEQ ID NO: 17 HTR.LIN15AA 9 CMQLNDDTRLYSNDCMDILSEENTSLSSC SEQ ID NO: 18 HTR.MAT 10 CLSPSSLSLLHLQECMDILSEENTSLSSC SEQ ID NO: 19 HTR.MAT 11 CSTTNSLMQLNDDTCMDILSEENTSLSSC SEQ ID NO: 20 HTR.MAT 12 CLTILYGYRWPLPSCKESSNEDVIGALLC SEQ ID NO: 21 HTR.MAT 13 CTVDSENRTNLSSECMDILSEENTSLSSC SEQ ID NO: 22 HTR.MAT 14 GYRWPLPSK SEQ ID NO: 23 HTR.LIN9 15 CMQLNDDTRLYSNDCSGEANTSDAFNWTC SEQ ID NO: 24 HTR.MAT 16 CDSKVFKEGSSLLACKESSNEDVIGALLC SEQ ID NO: 25 HTR.MAT 17 CENTSLSSTTNSLMCMDILSEENTSLSSC SEQ ID NO: 26 HTR.MAT 18 CSLSSTTNSLMQLNCMDILSEENTSLSSC SEQ ID NO: 27 HTR.MAT 19 CNSLMQLNDTRLYCSGEANTSDAFNWTC SEQ ID NO: 28 HTR.MAT 20 CLYGYRWPLPSKLSCKESSNEDVIGALLC SEQ ID NO: 29 HTR.MAT 21 CMDILSEENTSLSSCMQLNDDTRLYSNDC SEQ ID NO: 30 HTR.MAT 22 CSDAFNWTVDSENRCMDILSEENTSLSSC SEQ ID NO: 31 HTR.MAT 23 TVDSENRTNLAAEGC SEQ ID NO: 32 HTR.LIN15AA 24 CNDDTRLYSNDFNSCMDILSEENTSLSSC SEQ ID NO: 33 HTR.MAT 25 CTVDSENRTNLSSECRTNLSSEGSLSPSC SEQ ID NO: 34 HTR.MAT 26 CLSSEGSLSPSSLSCMDILSEENTSLSSC SEQ ID NO: 35 HTR.MAT 27 CLSPSSLSLLHLQECTVDSENRTNLSSEC SEQ ID NO: 36 HTR.MAT 28 CDSKVFKEGSSLLACMDILSEENTSLSSC SEQ ID NO: 37 HTR.MAT 29 TILYGYRWPLPSKLC SEQ ID NO: 38 HTR.LIN15AA 30 CFNWTVDSENRTNLCMDILSEENTSLSSC SEQ ID NO: 39 HTR.MAT 31 STTNSLMQLNAATRL SEQ ID NO: 40 HTR.LIN15AA 32 CLYGYRWPLPSKLSCYSNDFNSGEANTSC SEQ ID NO: 41 HTR.MAT 33 QDDSKVFKEGAALLA SEQ ID NO: 42 HTR.LIN15AA 34 CNSLMQLNDDTRLYCYSNDFNSGEANTSC SEQ ID NO: 43 HTR.MAT 35 CLTILYGYRWPLPSKLC SEQ ID NO: 44 HTR.P2_15AA 36 CLYGYRWPLPSKLSCSDAFNWTVDSENRC SEQ ID NO: 45 HTR.MAT 37 CDSKVFKEGSSLLACSGEANTSDAFNWTC SEQ ID NO: 46 HTR.MAT 38 CVFKEGSSLLADDNCKESSNEDVIGALLC SEQ ID NO: 47 HTR.MAT 39 CLSEENTSLSSTTNCMDILSEENTSLSSC SEQ ID NO: 48 HTR.MAT 40 CLTILYGYRWPLPSCLYGYRWPLPSKLSC SEQ ID NO: 49 HTR.MAT 41 CLTILYGYRWPAASKLC SEQ ID NO: 50 HTR.P2_15AA 42 CTRLYSNDFNSGEACKESSNEDVIGALLC SEQ ID NO: 51 HTR.MAT 43 CLYGYRWPLPSKLSCSGEANTSDAFNWTC SEQ ID NO: 52 HTR.MAT 44 CMDILSEENTSLSSCSKESSNEDVIGALC SEQ ID NO: 53 HTR.MAT 45 CSSLSLLHLQEKNWCMDILSEENTSLSSC SEQ ID NO: 54 HTR.MAT 46 CDSKVFKEGSSLLACSENRTNLSSEGSLC SEQ ID NO: 55 HTR.MAT 47 CVFKEGSSLLADDNCSDAFNWTVDSENRC SEQ ID NO: 56 HTR.MAT 48 CLQDDSKVFKEGSSCKESSNEDVIGALLC SEQ ID NO: 57 HTR.MAT MEAN SEM Peptide Sequence MEAN MS SEM MS CTRL CTRL P-value 1 CNSLMQLNDDTRLYCMDILSEENTSLSSC 445.78 64.60 252.10 39.78 0.01823 (SEQ ID NO: 10) 2 CLTILYGYRWPLPSCMDILSEENTSLSSC 421.56 68.75 228.85 35.30 0.01984 (SEQ ID NO: 11) 3 LTILYGYRWPAASKL (SEQ ID NO: 12) 653.28 125.82 322.35 46.78 0.01994 4 CLYGYRWPLPSKLSCMDILSEENTSLSSC 449.78 75.33 246.80 41.88 0.02701 (SEQ ID NO: 13) 5 YRWPLPSKL (SEQ ID NO: 14) 566.00 113.14 285.70 42.28 0.02714 6 CNSLMQLNDDTRLYCLSSEGSLSPSSLSC 418.89 63.39 249.55 36.07 0.02908 (SEQ ID NO: 15) 7 CLQDDSKVFKEGSSCMDILSEENTSLSSC 434.67 78.17 233.80 39.09 0.02977 (SEQ ID NO: 16) 8 LTILYGYRWPLPSKL (SEQ ID NO: 17) 647.17 123.46 340.05 54.05 0.03031 9 CMQLNDDTRLYSNDCMDILSEENTSLSSC 379.67 62.21 212.40 38.38 0.03169 (SEQ ID NO: 18) 10 CLSPSSLSLLHLQECMDILSEENTSLSSC 305.00 47.33 183.45 25.89 0.03322 (SEQ ID NO: 19) 11 CSTTNSLMQLNDDTCMDILSEENTSLSSC 321.28 53.13 186.85 29.61 0.03644 (SEQ ID NO: 20) 12 CLTILYGYRWPLPSCKESSNEDVIGALLC 629.14 113.96 350.93 55.40 0.03662 (SEQ ID NO: 21) 13 CTVDSENRTNLSSECMDILSEENTSLSSC 300.11 59.60 160.80 24.09 0.03779 (SEQ ID NO: 22) 14 GYRWPLPSK (SEQ ID NO: 23) 623.00 129.12 322.80 51.78 0.03847 15 CMQLNDDTRLYSNDCSGEANTSDAFNWTC 497.78 104.53 257.25 39.91 0.03889 (SEQ ID NO: 24) 16 CDSKVFKEGSSLLACKESSNEDVIGALLC 604.03 102.90 347.50 57.46 0.03901 (SEQ ID NO: 25) 17 CENTSLSSTTNSLMCMDILSEENTSLSSC 316.22 59.29 175.00 27.76 0.03938 (SEQ ID NO: 26) 18 CSLSSTTNSLMQLNCMDILSEENTSLSSC 323.72 56.11 188.90 27.71 0.03996 (SEQ ID NO: 27) 19 CNSLMQLNDTRzLYCSGEANTSDAFNWTC 564.11 112.62 299.55 51.82 0.04118 (SEQ ID NO: 28) 20 CLYGYRWPLPSKLSCKESSNEDVIGALLC 689.03 123.08 390.93 64.64 0.04121 (SEQ ID NO: 29) 21 CM DILSEENTSLSSCMQLNDDTRLYSNDC 392.28 70.22 223.85 36.73 0.04279 (SEQ ID NO: 30) 22 CSDAFNWTVDSENRCMDILSEENTSLSSC 394.00 81.52 207.20 34.73 0.04293 (SEQ ID NO: 31) 23 TVDSENRTNLAAEGC (SEQ ID NO: 32) 587.33 95.96 348.10 58.23 0.04345 24 CNDDTRLYSNDFNSCMDILSEENTSLSSC 350.67 67.59 196.45 28.93 0.04389 (SEQ ID NO: 33) 25 CTVDSENRTNLSSECRTNLSSEGSLSPSC 507.22 85.61 296.95 50.10 0.04415 (SEQ ID NO: 34) 26 CLSSEGSLSPSSLSCMDILSEENTSLSSC 320.72 57.25 188.85 26.70 0.04533 (SEQ ID NO: 35) 27 CLSPSSLSLLHLQECTVDSENRTNLSSEC 427.89 65.71 263.00 41.97 0.04538 (SEQ ID NO: 36) 28 CDSKVFKEGSSLLACMDILSEENTSLSSC 429.00 84.12 236.60 38.29 0.04573 (SEQ ID NO: 37) 29 TILYGYRWPLPSKLC (SEQ ID NO: 38) 651.50 115.23 378.20 61.96 0.04627 30 CFNWTVDSENRTNLCMDILSEENTSLSSC 312.00 61.58 174.90 25.14 0.04697 (SEQ ID NO: 39) 31 STTNSLMQLNAATRL (SEQ ID NO: 40) 611.83 91.02 385.15 58.43 0.04718 32 CLYGYRWPLPSKLSCYSNDFNSGEANTSC 555.67 120.54 288.50 48.26 0.04721 (SEQ ID NO: 41) 33 QDDSKVFKEGAALLA (SEQ ID NO: 42) 439.11 71.77 263.70 43.61 0.04734 34 CNSLMQLNDDTRLYCYSNDFNSGEANTSC 459.72 91.78 268.10 43.39 0.04736 (SEQ ID NO: 43) 35 CLTILYGYRWPLPSKLC (SEQ ID NO: 44) 792.94 137.92 475.00 68.01 0.04762 36 CLYGYRWPLPSKLSCSDAFNWTVDSENRC 511.11 90.49 298.55 48.45 0.04794 (SEQ ID NO: 45) 37 CDSKVFKEGSSLLACSGEANTSDAFNWTC 623.06 127.57 333.75 59.05 0.04808 (SEQ ID NO: 46) 38 CVFKEGSSLLADDNCKESSNEDVIGALLC 594.61 105.51 348.13 55.91 0.04850 (SEQ ID NO: 47) 39 CLSEENTSLSSTTNCMDILSEENTSLSSC 303.50 51.61 184.40 26.46 0.04929 (SEQ ID NO: 48) 40 CLTILYGYRWPLPSCLYGYRWPLPSKLSC 879.72 160.75 508.45 83.43 0.04973 (SEQ ID NO: 49) 41 CLTILYGYRWPAASKLC 782.33 149.17 449.80 66.54 0.05001 (SEQ ID NO: 50) 42 CTRLYSNDFNSGEACKESSNEDVIGALLC 561.03 100.74 333.60 47.96 0.05023 (SEQ ID NO: 51) 43 CLYGYRWPLPSKLSCSGEANTSDAFNWTC 649.67 137.64 344.80 60.02 0.05041 (SEQ ID NO: 52) 44 CM DI LSEE NTSLSSCSKESSNE DV IGALC 432.33 68.96 266.55 41.79 0.05050 (SEQ ID NO: 53) 45 CSSLSLLHLQEKNWCMDILSEENTSLSSC 318.17 49.59 198.50 30.50 0.05066 (SEQ ID NO: 54) 46 CDSKVFKEGSSLLACSENRTNLSSEGSLC 512.94 90.33 296.70 54.19 0.05072 (SEQ ID NO: 55) 47 CVFKEGSSLLADDNCSDAFNWTVDSENRC 399.89 65.84 244.50 37.80 0.05093 (SEQ ID NO: 56) 48 CLQDDSKVFKEGSSCKESSNEDVIGALLC 632.28 111.59 373.15 61.16 0.051561 (SEQ ID NO: 57) HTR.MAT: double looped conformational peptide, HTR.LIN15AA: linear peptide of 15 amino acids, HTR.LIN9: linear peptide of 9 amino acids, HTR.P2_15AA: single looped conformational peptide of 15 amino acids.
(127) In double looped peptides, three cysteine residues were added, two as first and last amino acid and one in the middle of the sequence. Then peptides of the invention may the whole sequence or the fragments located between two cysteine residues or the sequence with only one cysteine at either end of the sequence. In single looped peptides, two cysteine residues were added, as first and last amino acid. Then peptides of the invention may the whole sequence or the fragment located between two cysteine residues or the sequence with only one cysteine at either end of the sequence.
(128) TABLE-US-00005 TABLE 2 List of peptide sequences from NOX2 library that are significantly recognized by the MS sera (n = 9) compared to CTRLs (n = 10)(p < 0.05). Peptide Sequence SEQ ID NO: 1 NFARKRIKN SEQ ID NO: 58 CB245.LIN9 2 LNFARKRIK SEQ ID NO: 59 CB245.LIN9 3 CNFARKRIKNC SEQ ID NO: 60 CB245.P2_9 4 CSYLNFARKRIKNPEGCQNESYLNFARKRIKNC SEQ ID NO: 61 CB245.MAT 5 NESYLNFARKAAKNP SEQ ID NO: 62 CB245.LIN15AA 6 QNESYLNFARKRIKN SEQ ID NO: 63 CB245.LIN15AA 7 CVSEQKISEWGKIKESCQNESYLNFARKRIKNC SEQ ID NO: 64 CB245.MAT 8 CNFARKRIKNPEGGLYCQNESYLNFARKRIKNC SEQ ID NO: 65 CB245.MAT 9 CVRGQTAESLAVHNITCQNESYLNFARKRIKNC SEQ ID NO: 66 CB245.MAT 10 FARKRIKNP SEQ ID NO: 67 CB245.LIN9 11 GDRQNESYLNAGRKR SEQ ID NO: 68 CB245.LIN15AA 12 CQKISEWGKIKESPIPCQNESYLNFARKRIKNC SEQ ID NO: 69 CB245.MAT 13 CGDRQNESYLNFARKRCQNESYLNFARKRIKNC SEQ ID NO: 70 CB245.MAT 14 SYLNFARKRIAAPEG SEQ ID NO: 71 CB245.LIN15AA 15 CEWSVNARVNNSDPYSCPYSVALSELGDRQNEC SEQ ID NO: 72 CB245.MAT 16 CHGAERIVRGQTAESLCQNESYLNFARKRIKNC SEQ ID NO: 73 CB245.MAT 17 CESLAVHNITVSEQKICQNESYLNFARKRIKNC SEQ ID NO: 74 CB245.MAT 18 CERIVRGQTAESLAVHCQNESYLNFARKRIKNC SEQ ID NO: 75 CB245.MAT 19 RQNESYLNFARKRIK SEQ ID NO: 76 CB245.LIN15AA 20 NFARKRIKNPAAGLY SEQ ID NO: 77 CB245.LIN15AA 21 DPYSVALSELAARQN SEQ ID NO: 78 CB245.LIN15AA 22 CESLAVHNITVSEQKICSYLNFARKRIKNPEGC SEQ ID NO: 79 CB245.MAT 23 CSELGDRQNESYLNFACQNESYLNFARKRIKNC SEQ ID NO: 80 CB245.MAT 24 CNITVSEQKISEWGKICQNESYLNFARKRIKNC SEQ ID NO: 81 CB245.MAT 25 CHGAERIVRGQTAESLCSYLNFARKRIKNPEGC SEQ ID NO: 82 CB245.MAT 26 KISEWGKIK SEQ ID NO: 83 CB245.LIN9 27 CVSEQKISEWGKIKESCEQKISEWGKIKESPIC SEQ ID NO: 84 CB245.MAT 28 CGDRQNESYLNFARKRCGDRQNESYLNFARKRC SEQ ID NO: 85 CB245.MAT 29 CNESYLNFARKAAKNPC SEQ ID NO: 86 CB245.P2_15AA 30 CLNFARKRIKC SEQ ID NO: 87 CB245.P2_9 31 QKISEWGKIKAAPIP SEQ ID NO: 88 CB245.LIN15AA 32 CHGAERIVRGQTAESLCNFARKRIKNPEGGLYC SEQ ID NO: 89 CB245.MAT 33 CQNESYLNFARKRIKNC SEQ ID NO: 90 CB245.P2_15AA 34 LNFARKRIKNAAGGL SEQ ID NO: 91 CB245.LIN15AA 35 CLNFARKRIKNAAGGLC SEQ ID NO: 92 CB245.P2_15AA 36 CQKISEWGKIKESPIPCTVSEQKISEWGKIKEC SEQ ID NO: 93 CB245.MAT 37 TVCEQKISEWGKIKE SEQ ID NO: 94 CB245.LIN15AA 38 CRQNESYLNFARKRIKC SEQ ID NO: 95 CB245.P2_15AA 39 NITVCEQKISAAGKI SEQ ID NO: 96 CB245.LIN15AA 40 CNFARKRIKNPEGGLYCSYLNFARKRIKNPEGC SEQ ID NO: 97 CB245.MAT 41 CQKISEWGKIKESPIPCVSEQKISEWGKIKESC SEQ ID NO: 98 CB245.MAT 42 CNITVSEQKISEWGKICSYLNFARKRIKNPEGC SEQ ID NO: 99 CB245.MAT 43 CQTAESLAVHNITVSECQNESYLNFARKRIKNC SEQ ID NO: 100 CB245.MAT 44 CHGAERIVRGQTAESLCQKISEWGKIKESPIPC SEQ ID NO: 101 CB245.MAT 45 GKIKECPIP SEQ ID NO: 102 CB245.LIN9 46 CEWSVNARVNNSDPYSCQNESYLNFARKRIKNC SEQ ID NO: 103 CB245.MAT 47 LGDRQNESYLNFARK SEQ ID NO: 104 CB245.LIN15AA 48 CNITVSEQKISAAGKIC SEQ ID NO: 105 CB245.P2_15AA 49 CNFARKRIKNPEGGLYCSLAVHNITVSEQKISC SEQ ID NO: 106 CB245.MAT 50 ESYLNFARK SEQ ID NO: 107 CB245.LIN9 51 CKISEWGKIKC SEQ ID NO: 108 CB245.P2_9 52 NESYLNFARKRIKNP SEQ ID NO: 109 CB245.LIN15AA 53 CVALSELGDRQNESYLCQNESYLNFARKRIKNC SEQ ID NO: 110 CB245.MAT 54 CQNESYLNFARKRIKNCVSEQKISEWGKIKESC SEQ ID NO: 111 CB245.MAT 55 CVSEQKISEWGKIKESCGDRQNESYLNFARKRC SEQ ID NO: 112 CB245.MAT 56 QNESYLNFARAAIKN SEQ ID NO: 113 CB245.LIN15AA 57 CSEQKISEWGKIKESPC SEQ ID NO: 114 CB245.P2_15AA 58 CVSEQKISEWGKIKESCVSEQKISEWGKIKESC SEQ ID NO: 115 CB245.MAT 59 WGKIKECPI SEQ ID NO: 116 CB245.LIN9 60 CNFARKRIKNPEGGLYCGDRQNESYLNFARKRC SEQ ID NO: 117 CB245.MAT 61 VNARVNNSDPAAVAL SEQ ID NO: 118 CB245.LIN15AA 62 YLNFARKRIKAAEGG SEQ ID NO: 119 CB245.LIN15AA 63 GDRQNESYLNFARKR SEQ ID NO: 120 CB245.LIN15AA 64 WCVNARVNNSDPYSV SEQ ID NO: 121 CB245.LIN15AA 65 CSYLNFARKRIKNPEGCITVSEQKISEWGKIKC SEQ ID NO: 122 CB245.MAT 66 CAVHNITVSEQKISEWCVSEQKISEWGKIKESC SEQ ID NO: 123 CB245.MAT 67 CPYSVALSELGDRQNECITVSEQKISEWGKIKC SEQ ID NO: 124 CB245.MAT 68 CSYLNFARKRIKNPEGCGDRQNESYLNFARKRC SEQ ID NO: 125 CB245.MAT 69 CAVHNITVSEQKISEWCQNESYLNFARKRIKNC SEQ ID NO: 126 CB245.MAT 70 LGDRQNESYLAAARK SEQ ID NO: 127 CB245.LIN15AA 71 CVNARVNNSDPYSVALCVSEQKISEWGKIKESC SEQ ID NO: 128 CB245.MAT 72 CFNVEWSVNARVNNSDCGDRQNESYLNFARKRC SEQ ID NO: 129 CB245.MAT 73 CNSDPYSVALSELGDRCVSEQKISEWGKIKESC SEQ ID NO: 130 CB245.MAT 74 CGDRQNESYLNFARKRCSYLNFARKRIKNPEGC SEQ ID NO: 131 CB245.MAT 75 DPYSVALSELGDRQN SEQ ID NO: 132 CB245.LIN15AA 76 CAVHNITVSEQKISEWCSYLNFARKRIKNPEGC SEQ ID NO: 133 CB245.MAT 77 CVRGQTAESLAVHNITCITVSEQKISEWGKIKC SEQ ID NO: 134 CB245.MAT 78 CQNESYLNFARKRIKNCSYLNFARKRIKNPEGC SEQ ID NO: 135 CB245.MAT 79 CESLAVHNITVSEQKICGDRQNESYLNFARKRC SEQ ID NO: 136 CB245.MAT 80 CRVNNSDPYSVALSELCQNESYLNFARKRIKNC SEQ ID NO: 137 CB245.MAT 81 CSYLNFARKRIKNPEGCNFARKRIKNPEGGLYC SEQ ID NO: 138 CB245.MAT 82 CQTAESLAVHNITVSECVSEQKISEWGKIKESC SEQ ID NO: 139 CB245.MAT 83 YLNFARKRI SEQ ID NO: 140 CB245.LIN9 84 CFNVEWSVNARVNNSDCVSEQKISEWGKIKESC SEQ ID NO: 141 CB245.MAT 85 CVSEQKISEWGKIKESCTVSEQKISEWGKIKEC SEQ ID NO: 142 CB245.MAT 86 CNSDPYSVALSELGDRCITVSEQKISEWGKIKC SEQ ID NO: 143 CB245.MAT 87 CNITVSEQKISEWGKICGDRQNESYLNFARKRC SEQ ID NO: 144 CB245.MAT 88 CLGDRQNESYLAAARKC SEQ ID NO: 145 CB245.P2_15AA 89 QKISEWGKI SEQ ID NO: 146 CB245.LIN9 90 CITVSEQKISEAAKIKC SEQ ID NO: 147 CB245.P2_15AA 91 CVRGQTAESLAVHNITCVHNITVSEQKISEWGC SEQ ID NO: 148 CB245.MAT 92 CQKISEWGKIKESPIPCVHNITVSEQKISEWGC SEQ ID NO: 149 CB245.MAT 93 CQNESYLNFARKRIKNCNFARKRIKNPEGGLYC SEQ ID NO: 150 CB245.MAT 94 CRVNNSDPYSVALSELCVSEQKISEWGKIKESC SEQ ID NO: 151 CB245.MAT 95 CGDRQNESYLNFARKRCVSEQKISEWGKIKESC SEQ ID NO: 152 CB245.MAT 96 CQKISEWGKIKAAPIPC SEQ ID NO: 153 CB245.P2_15AA 97 CVNARVNNSDPYSVALCTVSEQKISEWGKIKEC SEQ ID NO: 154 CB245.MAT 98 CEWSVNARVNNSDPYSCVSEQKISEWGKIKESC SEQ ID NO: 155 CB245.MAT 99 CVRGQTAESLAVHNITCSLAVHNITVSEQKISC SEQ ID NO: 156 CB245.MAT MEAN Sequence MEAN MS SEM MS CTRL SEM CTRL P-value NFARKRIKN (SEQ ID NO: 58) 613.833 117.974 288.95 50.127 0.017533 LNFARKRIK (SEQ ID NO: 59) 557.056 92.576 294.6 50.896 0.020529 CNFARKRIKNC (SEQ ID NO: 60) 748.389 145.481 374.6 48.888 0.020984 CSYLNFARKRIKNPEGCQNESYLNFARKRIKNC 778.278 141.548 398.9 62.496 0.021068 (SEQ ID NO: 61) NESYLNFARKAAKNP (SEQ ID NO: 62) 505.278 81.335 279.1 42.463 0.021146 QNESYLNFARKRIKN (SEQ ID NO: 63) 610.778 110.062 309.65 53.910 0.021235 CVSEQKISEWGKIKESCQNESYLNFARKRIKNC 804.556 152.312 402.9 62.907 0.021494 (SEQ ID NO: 64) CNFARKRIKNPEGGLYCQNESYLNFARKRIKNC 801.056 148.456 410.85 61.525 0.021931 (SEQ ID NO: 65) CVRGQTAESLAVHNITCQNESYLNFARKRIKNC 824.500 143.332 438.35 67.804 0.022090 (SEQ ID NO: 66) FARKRIKNP (SEQ ID NO: 67) 666.111 125.706 337.4 54.419 0.023461 GDRQNESYLNAGRKR (SEQ ID NO: 68) 547.278 96.746 294.2 42.636 0.023828 CQKISEWGKIKESPIPCQNESYLNFARKRIKNC 855.111 151.464 458.3 72.573 0.025816 (SEQ ID NO: 69) CGDRQNESYLNFARKRCQNESYLNFARKRIKNC 831.278 163.826 412.7 72.303 0.026839 (SEQ ID NO: 70) SYLNFARKRIAAPEG (SEQ ID NO: 71) 472.000 83.677 259.75 37.175 0.027985 CEWSVNARVNNSDPYSCPYSVALSELGDRQNEC 428.167 79.768 225.5 35.843 0.028038 (SEQ ID NO: 72) CHGAERIVRGQTAESLCQNESYLNFARKRIKNC 748.111 136.357 407.2 56.805 0.028309 (SEQ ID NO: 73) CESLAVHNITVSEQKICQNESYLNFARKRIKNC 689.556 135.802 354.9 53.509 0.028995 (SEQ ID NO: 74) CERIVRGQTAESLAVHCQNESYLNFARKRIKNC 833.444 149.785 455.55 68.507 0.029550 (SEQ ID NO: 75) RQNESYLNFARKRIK (SEQ ID NO: 76) 458.833 88.112 241.2 38.449 0.031258 NFARKRIKNPAAGLY (SEQ ID NO: 77) 604.167 112.468 323.85 51.645 0.031431 DPYSVALSELAARQN (SEQ ID NO: 78) 565.389 92.915 325.05 50.274 0.031704 CESLAVHNITVSEQKICSYLNFARKRIKNPEGC 639.611 114.168 357.1 51.488 0.031924 (SEQ ID NO: 79) CSELGDRQNESYLNFACQNESYLNFARKRIKNC 773.167 154.146 398.9 65.715 0.033178 (SEQ ID NO: 80) CNITVSEQKISEWGKICQNESYLNFARKRIKNC 770.889 150.182 408.8 62.723 0.033686 (SEQ ID NO: 81) CHGAERIVRGQTAESLCSYLNFARKRIKNPEGC 755.722 135.689 417.5 66.730 0.033710 (SEQ ID NO: 82) KISEWGKIK (SEQ ID NO: 83) 404.500 76.258 223.9 29.152 0.034179 CVSEQKISEWGKIKESCEQKISEWGKIKESPIC 811.056 151.397 443.5 66.739 0.034184 (SEQ ID NO: 84) CGDRQNESYLNFARKRCGDRQNESYLNFARKRC 775.500 157.009 404.05 61.921 0.035130 (SEQ ID NO: 85) CNESYLNFARKAAKNPC (SEQ ID NO: 86) 702.222 119.948 411.15 54.568 0.035259 CLNFARKRIKC (SEQ ID NO: 87) 713.333 148.240 374.7 47.602 0.036234 QKISEWGKIKAAPIP (SEQ ID NO: 88) 701.889 142.859 364.3 59.774 0.036968 CHGAERIVRGQTAESLCNFARKRIKNPEGGLYC 733.889 136.475 404.6 63.679 0.037064 (SEQ ID NO: 89) CQNESYLNFARKRIKNC (SEQ ID NO: 90) 667.389 130.810 361.75 51.543 0.037142 LNFARKRIKNAAGGL (SEQ ID NO: 91) 354.778 70.317 187.85 30.409 0.037220 CLNFARKRIKNAAGGLC (SEQ ID NO: 92) 691.444 137.690 367 57.492 0.037383 CQKISEWGKIKESPIPCTVSEQKISEWGKIKEC 846.833 155.062 476.45 69.583 0.037396 (SEQ ID NO: 93) TVCEQKISEWGKIKE (SEQ ID NO: 94) 514.944 89.577 289.4 50.414 0.037701 CRQNESYLNFARKRIKC (SEQ ID NO: 95) 653.944 131.478 350.1 49.641 0.037862 NITVCEQKISAAGKI (SEQ ID NO: 96) 564.500 103.965 314.2 49.516 0.038184 CNFARKRIKNPEGGLYCSYLNFARKRIKNPEGC 752.333 139.571 416.6 66.438 0.038310 (SEQ ID NO: 97) CQKISEWGKIKESPIPCVSEQKISEWGKIKESC 815.972 154.878 450.975 66.905 0.038417 (SEQ ID NO: 98) CNITVSEQKISEWGKICSYLNFARKRIKNPEGC 738.444 144.682 397.7 62.491 0.038524 (SEQ ID NO: 99) CQTAESLAVHNITVSECQNESYLNFARKRIKNC 697.667 143.031 367.45 55.429 0.038717 (SEQ ID NO: 100) CHGAERIVRGQTAESLCQKISEWGKIKESPIPC 734.917 132.945 407.625 70.500 0.038779 (SEQ ID NO: 101) GKIKECPIP (SEQ ID NO: 102) 594.611 111.300 324.35 56.061 0.038930 CEWSVNARVNNSDPYSCQNESYLNFARKRIKNC 714.944 148.086 372.8 58.717 0.039223 (SEQ ID NO: 103) LGDRQNESYLNFARK (SEQ ID NO: 104) 457.556 87.314 252.4 38.376 0.039506 CNITVSEQKISAAGKIC (SEQ ID NO: 105) 828.167 161.273 460.55 60.796 0.040192 CNFARKRIKNPEGGLYCSLAVHNITVSEQKISC 599.000 117.360 330.8 44.992 0.040197 (SEQ ID NO: 106) ESYLNFARK (SEQ ID NO: 107) 535.667 101.551 296.3 46.513 0.040403 CKISEWGKIKC (SEQ ID NO: 108) 672.833 131.134 367.95 57.029 0.041005 NESYLNFARKRIKNP (SEQ ID NO: 109) 602.889 112.832 333.75 55.479 0.041060 CVALSELGDRQNESYLCQNESYLNFARKRIKNC 761.778 149.165 406.75 72.796 0.041142 (SEQ ID NO: 110) CQNESYLNFARKRIKNCVSEQKISEWGKIKESC 747.139 139.947 419.075 63.876 0.041252 (SEQ ID NO: 111) CVSEQKISEWGKIKESCGDRQNESYLNFARKRC 846.778 166.956 462.45 69.927 0.041538 (SEQ ID NO: 112) QNESYLNFARAAIKN (SEQ ID NO: 113) 421.722 74.794 244 36.768 0.041742 CSEQKISEWGKIKESPC (SEQ ID NO: 114) 501.167 101.057 271.75 39.588 0.041995 CVSEQKISEWGKIKESCVSEQKISEWGKIKESC 756.639 139.081 432.35 63.156 0.042031 (SEQ ID NO: 115) WGKIKECPI (SEQ ID NO: 116) 501.389 85.494 303.25 37.670 0.042036 CNFARKRIKNPEGGLYCGDRQNESYLNFARKRC 800.500 164.282 425.15 67.492 0.042277 (SEQ ID NO: 117) VNARVNNSDPAAVAL (SEQ ID NO: 118) 426.444 89.633 219.15 39.717 0.042623 YLNFARKRIKAAEGG (SEQ ID NO: 119) 299.167 48.645 181.3 26.445 0.042780 GDRQNESYLNFARKR (SEQ ID NO: 120) 325.333 71.433 163.95 27.971 0.042865 WCVNARVNNSDPYSV (SEQ ID NO: 121) 283.889 51.504 163.8 24.175 0.043310 CSYLNFARKRIKNPEGCITVSEQKISEWGKIKC 796.222 141.260 457.4 74.781 0.043355 (SEQ ID NO: 122) CAVHNITVSEQKISEWCVSEQKISEWGKIKESC 697.000 132.608 392.5 58.017 0.043483 (SEQ ID NO: 123) CPYSVALSELGDRQNECITVSEQKISEWGKIKC 824.056 151.125 469.75 74.499 0.044343 (SEQ ID NO: 124) CSYLNFARKRIKNPEGCGDRQNESYLNFARKRC 786.389 154.795 430.35 70.557 0.044707 (SEQ ID NO: 125) CAVHNITVSEQKISEWCQNESYLNFARKRIKNC 685.667 140.191 370.15 57.325 0.044934 (SEQ ID NO: 126) LGDRQNESYLAAARK (SEQ ID NO: 127) 478.278 93.425 262.75 43.574 0.045056 CVNARVNNSDPYSVALCVSEQKISEWGKIKESC 772.583 148.494 436.575 63.579 0.045484 (SEQ ID NO: 128) CFNVEWSVNARVNNSDCGDRQNESYLNFARKRC 792.889 167.256 423.15 63.958 0.046274 (SEQ ID NO: 129) CNSDPYSVALSELGDRCVSEQKISEWGKIKESC 764.528 146.689 434.925 61.821 0.046277 (SEQ ID NO: 130) CGDRQNESYLNFARKRCSYLNFARKRIKNPEGC 730.222 144.653 401.95 64.840 0.046636 (SEQ ID NO: 131) DPYSVALSELGDRQN (SEQ ID NO: 132) 300.556 52.221 177.25 27.831 0.046679 CAVHNITVSEQKISEWCSYLNFARKRIKNPEGC 648.556 118.334 375.55 57.805 0.047030 (SEQ ID NO: 133) CVRGQTAESLAVHNITCITVSEQKISEWGKIKC 758.278 135.230 440.5 71.450 0.047178 (SEQ ID NO: 134) CQNESYLNFARKRIKNCSYLNFARKRIKNPEGC 711.556 140.645 396.7 60.423 0.047697 (SEQ ID NO: 135) CESLAVHNITVSEQKICGDRQNESYLNFARKRC 730.278 143.452 409.15 61.650 0.047716 (SEQ ID NO: 136) CRVNNSDPYSVALSELCQNESYLNFARKRIKNC 743.167 156.061 398.05 62.948 0.047897 (SEQ ID NO: 137) CSYLNFARKRIKNPEGCNFARKRIKNPEGGLYC 659.944 126.954 371.8 59.312 0.048308 (SEQ ID NO: 138) CQTAESLAVHNITVSECVSEQKISEWGKIKESC 590.111 111.059 339.65 50.662 0.048582 (SEQ ID NO: 139) YLNFARKRI (SEQ ID NO: 140) 601.667 108.516 345.85 59.745 0.048726 CFNVEWSVNARVNNSDCVSEQKISEWGKIKESC 784.944 149.515 447.325 69.485 0.049109 (SEQ ID NO: 141) CVSEQKISEWGKIKESCTVSEQKISEWGKIKEC 760.333 137.654 446.5 66.920 0.049174 (SEQ ID NO: 142) CNSDPYSVALSELGDRCITVSEQKISEWGKIKC 781.611 136.738 456.45 78.433 0.049315 (SEQ ID NO: 143) CNITVSEQKISEWGKICGDRQNESYLNFARKRC 816.722 167.231 446.7 70.860 0.049530 (SEQ ID NO: 144) CLGDRQNESYLAAARKC (SEQ ID NO: 145) 698.944 131.913 402.85 60.271 0.049600 QKISEWGKI (SEQ ID NO: 146) 508.333 92.706 298.2 44.546 0.049820 CITVSEQKISEAAKIKC (SEQ ID NO: 147) 703.000 147.115 383.25 56.600 0.049837 CVRGQTAESLAVHNITCVHNITVSEQKISEWGC 445.278 79.537 263.5 39.692 0.049916 (SEQ ID NO: 148) CQKISEWGKIKESPIPCVHNITVSEQKISEWGC 442.611 82.066 263.25 32.814 0.049972 (SEQ ID NO: 149) CQNESYLNFARKRIKNCNFARKRIKNPEGGLYC 697.056 139.880 392.3 55.275 0.050219 (SEQ ID NO: 150) CRVNNSDPYSVALSELCVSEQKISEWGKIKESC 731.111 139.692 422.275 60.170 0.050319 (SEQ ID NO: 151) CGDRQNESYLNFARKRCVSEQKISEWGKIKESC 805.278 155.053 461.6 67.947 0.050467 (SEQ ID NO: 152) CQKISEWGKIKAAPIPC (SEQ ID NO: 153) 710.778 140.027 402.15 59.861 0.050660 CVNARVNNSDPYSVALCTVSEQKISEWGKIKEC 799.667 154.908 455.6 69.048 0.050729 (SEQ ID NO: 154) CEWSVNARVNNSDPYSCVSEQKISEWGKIKESC 760.083 148.508 433.775 62.562 0.050743 (SEQ ID NO: 155) CVRGQTAESLAVHNITCSLAVHNITVSEQKISC 546.444 110.735 304.75 47.813 0.053099 (SEQ ID NO: 156) CB245.MAT: double looped conformational peptide, CB245.LIN15AA: linear peptide of 15 amino acids, CB245.LIN9: linear peptide of 9 amino acids, CB245.P2_15AA: single looped conformational peptide of 15 amino acids.
(129) In double looped peptides, three cysteine residues were added, two as first and last amino acid and one in the middle of the sequence. Then peptides of the invention may the whole sequence or the fragments located between two cysteine residues. In single looped peptides, two cysteine residues were added, as first and last amino acid. Then peptides of the invention may the whole sequence or the fragment located between two cysteine residues or the sequence with only one cysteine at either end of the sequence.
(130)
(131)
(132) Elisa
(133) Dose-Response Curves of the Interaction Between MS IgG and DDSK Peptide
(134) The inventors have demonstrated the immunoglobulins of Multiple Sclerosis patients bind serotonin receptor. To evaluate the peptide concentration to use for ELISA assay we performed a dose-response curve. The immunoglobulins of Multiple Sclerosis patients and Control (200 μg) were incubated with different concentrations (50-100-250 μM) of peptide DDSKVFKEGS (named DDSK in the figures) and the absorbance (optical density) of plate was read at 450 nm.
(135) MS IgG Recognize Specially Receptor's Peptide
(136) The MS IgG binds peptides derived from serotonin receptor. To confirm this data, the inventors performed indirect Elisa using two different peptides derived from serotonin receptor compared to scrambled.
(137) The immunoglobulins of Multiple Sclerosis patients and Control (200 μg) were incubated with 100 μM of specific or control peptide and the absorbance (optical density) of plate was read at 450 nm.
(138) DDSK Peptide Shows High Sensitivity and Specificity to IgG MS
(139) The inventors have demonstrate the MS IgG binds specific peptide derived from receptor.
(140) To evaluate the sensitivity of the binding, the inventors performed a ROC curve starting from 28 samples each one repeated at least 4 times. In
(141) IgG from MS Patients Interfere with 5HT2aR Signaling
(142) To evaluate whether the interaction of IgG from MS patients with the membrane 5HT2aR/NOX3 complex could affect cell signaling downstream the receptor, the inventors performed experiments with HEK293 cells transfected in transient with 5HT2aR. The cells were stimulated with IgG from Control or MS patients prior to the stimulation with the endogenous receptor agonist 5-Ht, and then P-ERK1/2 levels were measured. As shown in the
(143) The inventors also provided evidences about the interference of IgG from MS patients with 5HT2aR signaling in human oligodendrocyte cell line MO3-13. Cells were stimulated with IgG from control or MS patients in the presence and absence of risperidone, a serotonin receptor antagonist. In the presence of the substance, IgG from MS patients failed to induce P-ERK1/2 levels thus confirming that autoantibodies present in sera of MS patients increase P-ERK1/2 levels acting on serotonin receptor (
(144) Overall, the present results suggest that 5HT2aR-bound autoantibodies present in MS patients may exert an ethiopathogenic role in MS. Therefore, compounds interfering with the immunoglobulins-receptor binding can be used for the treatment of MS.
(145) IgG from MS Patients Increase Reactive Oxygen Species (ROS), DUOX1/2, P-ERK1/2, HaRas and NOX3 Protein Levels and DDSK Peptide Reverts the Effect
(146) 5Ht receptors rely on ROS for downstream signaling (Kruk et al., 2013). Therefore, we measured ROS levels, as DCF fluorescence, in MO3-13 cells stimulated with IgG from Control or MS patients in the absence or presence of the DDSK peptide. As shown in
(147) In addition to NOX3 and NOX5, MO3-13 cells express also DUOX1 and 2 isoforms (Damiano et al., 2012, PloS); their protein levels are very sensitive to ROS. Other downstream ROS targets are P-ERK, and H-Ras (Refs). For these reasons we also measured DUOX1/2 (
(148) On the contrary, incubation of the cells with IgG from control subjects decreased DUOX1/2 and P-ERK1/2, while did not affect HaRas or NOX3 protein levels and DDSK HaRas did not significantly modify the levels of all the protein analyzed.
(149) Altogether, these experiments suggest that peptides with sequences homologue to the extracellular domains of h-5HT2aR are able to counteract the effect of IgMS on 5HT2aR/NOX/ROS signaling pathway in human oligodendrocytes, thus representing a promising specific therapeutic tool for the treatment of Multiple Sclerosis.
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