Identification of Alzheimer's Disease Autoantibodies and Their Target

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Identification of Alzheimer’s Disease Autoantibodies and Their Target Biomarkers by Phage Microarrays Pablo San Segundo-Acosta, Ana Montero-Calle, Manuel Fuentes, Alberto Rabano, Mayte Villalba, and Rodrigo Barderas J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.9b00258 • Publication Date (Web): 28 May 2019 Downloaded from http://pubs.acs.org on May 29, 2019

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Journal of Proteome Research

Identification of Alzheimer’s Disease Autoantibodies and Their Target Biomarkers by Phage Microarrays Pablo San Segundo-Acosta†,‡,#, Ana Montero-Calle‡,#, Manuel Fuentes¶,Ç, Alberto Rábano§, Mayte Villalba†, Rodrigo Barderas‡,*

†

Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas,

Universidad Complutense de Madrid, E-28040 Madrid, Spain ‡Chronic

Disease Programme (UFIEC), Instituto de Salud Carlos III, Majadahonda E-28220,

Madrid, Spain ¶Proteomics

Unit, Cancer Research Centre (IBMCC/CSIC/USAL/IBSAL), 37007, Salamanca,

Spain. ÇDepartment

of Medicine and General Cytometry Service-Nucleus, CIBERONC CB16/12/00400,

Cancer Research Centre (IBMCC/CSIC/USAL/IBSAL), 37007, Salamanca, Spain. §Alzheimer

Disease Research Unit, CIEN Foundation, Queen Sofia Foundation Alzheimer

Center, Madrid, Spain

#These *To

authors share authorship.

whom correspondence should be addressed:

Rodrigo Barderas. ORCID ID: 0000-0003-3539-7469 Chronic Disease Programme (UFIEC), Instituto de Salud Carlos III, E-28222 Majadahonda, Madrid, Spain; Tel.: 34-91-8223231; E-mail: [email protected]

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Abstract

The characterization of the humoral response in Alzheimer’s disease (AD) patients might aid in detecting the disease at early stages. We have combined phage display and protein microarrays to identify AD autoantibodies and their target biomarkers. After enrichment of T7 phage display libraries from AD and healthy brain tissue mRNA in AD-specific phages, 1536 monoclonal phages were printed on microarrays to probe them with 8 AD and 8 healthy controls sera. 57 phages showed higher seroreactivity in AD. 13 out of the 44 unique sequences displayed on phages were selected for validation using 68 AD and 52 healthy controls sera. Peptides from Anthrax toxin receptor 1, Nuclear protein 1, Glycogen phosphorylase and Olfactory receptor 8J1 expressed in bacteria as HaloTag fusion proteins showed statistically significant ability to discriminate between AD patients and controls. The identified panel of AD autoantibodies might provide new insights into the blood-based diagnosis of the disease.

KEYWORDS: protein microarrays, phage display, autoantibodies, Alzheimer’s Disease, humoral immune response

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INTRODUCTION

Alzheimer’s disease (AD) is a chronic and unremitting neurodegenerative disorder affecting fundamental brain areas for memory and cognition. It is the most common form of dementia in ageing population, with an estimated prevalence of 10-30% (1, 2). Although the average duration of AD is 10 years since the first clinical symptoms, it has long preclinical and prodromal stages which extend for up to 20 years (3). Two main histopathological hallmarks define AD: extracellular amyloid-β (Aβ) deposits known as amyloid plaques, and intracellular aggregates of hyperphosphorylated Tau known as neurofibrillary tangles (NFTs) (4, 5). However, the exact mechanisms triggering the deposition of both types of aggregates and their role in the disease remain unclear (6-8). Other hallmarks of the disease are oxidative stress and neuroinflammation, which actively contribute to the loss of neuronal structure and function (9, 10). AD diagnosis based on clinical data is currently supported by a set of cerebrospinal fluid (CSF) and imaging biomarkers obtained through expensive and high-invasive techniques only available in specialized clinical settings (11-15). Some of them have been proved to be useful for AD diagnosis in the preclinical and prodromal stages, but there is not a solid correlation between the progress of the disease across the different preclinical stages and the levels of these biomarkers (16, 17). Thus, it is essential to identify new AD-specific biomarkers obtained through low-cost and less invasive techniques, useful for early diagnosis and available for primary care settings. Current investigations in neuroproteomics are mainly focused on the analysis of changes in tissue protein patterns to improve our understanding on the pathological changes involving neurodegenerative diseases and find new candidate biomarkers for early diagnosis (18-22). Specifically, blood-based biomarkers are of particular interest due to their high accessibility, stability and low-cost (23). Among them, autoantibodies and their target proteins have become promising diagnostic tools (24, 25). Mass-spectrometry and protein microarraybased proteomic approaches have been successfully used to identify them in multiple diseases 3



(26-31). Different sets of autoantibodies have been identified in AD, although their role and their origin are not yet well-understood (32). Nevertheless, the recent identification of human dural lymphatic vessels and the better understanding of the glymphatic system provide increasing evidences of the development of an AD-specific humoral response beginning at the preclinical stage (33-36), which may be determinant for early diagnosis. Among different available microarray formats (37-39), phage arrays have been used as an alternative to commercial protein microarrays for autoantibody profiling, especially in cancer (31, 40). This technique takes advantage of the biopanning procedure, in which serum purified IgGs from healthy individuals and patients are used to enrich phage libraries in those phages displaying disease-specific antigens, and then printed on microarrays for further testing. In this work, we used this approach to test two T7 phage display libraries on a microarray format to identify AD-specific targets of autoantibodies. After statistical analysis, peptides displayed on the most reactive phages against IgGs from AD patients were identified, cloned and expressed as Halo-tagged peptides for further validation using 68 AD patients and 52 healthy control serum samples. Among 13 seroreactive target peptides with amino acid sequence identity with olfactory receptors, transcription factors, or ubiquitin ligases identified from phage microarrays and selected for validation, four of them with identity with Anthrax toxin receptor 1 (ANTXR1), Nuclear protein 1 (NUPR1), Glycogen phosphorylase (PYGB) and Olfactory receptor 8J1 (OR8J1) showed significant ability to discriminate AD from controls, with AUCs up to 71.1%.

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METHODS Alzheimer’s Disease and Reference Healthy Control Serum Samples Serum samples were obtained from the CIEN foundation’s Tissue Bank (BT-CIEN) (Table 1 and Table S1).

Table 1. Clinical and pathological information of the serum samples used for phage microarray experiments and validation.

Gender

Microarray screening

Validation

Braak Stageb

Number (n)

Age average ± SD (years)

Age range (years)

Male (n)

Female (n)

III

IV

V

VI

AD

8

84.2±6.7

74-92

1

7

-

2 (25%)

5 (62.5%)

1 (12.5%)

Controls

8

74.9±3.1

72-82

5

3

AD Controls

68 52

84±6.8 72.2±14.1

62-101 36-90

13 18

55 34

5 (7.4%)

4 (5.9%)

50 (73.5)

9 (13.2%)

a,

the distribution according to the age of the patients and the gender of the samples was non-significant. Braak stage as determined post mortem (number of samples and percentage relative to total). AD serum samples were collected when patients suffered from moderate to advance clinical AD (see Table S1). c, the 8 controls used in the microarray screening were also used for validation. b,

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Individuals enrolled in the study were monitored twice a year. In addition to blood collection, during the course of the disease, AD patients were histopathologically confirmed post mortem to verify they were actual AD patients. Blood used in the study was collected prior to histopathological assessment when AD patients were in moderate or advance clinical AD (41). Neuropathological diagnosis and classification of cases and controls was performed on the basis of international consensus criteria (42, 43). The BT-CIEN develops a brain donation program based on Standard Operating Procedures, meeting the ethical and legal requirements established by current legislation regarding the protection of personal data procedures and as regards to the use of samples of human origin for biomedical research. Written informed consent was obtained from all patients. The Institutional Ethical Review Board of the Spanish Research Center for Neurological Diseases Foundation, Complutense University of Madrid and Instituto de Salud Carlos III approved this study on biomarker discovery (CEI PI 49). A total of 76 different serum samples from AD patients (68 for validation and 8 for biopanning and microarray screening) ranging from Braak III to VI, and 52 individual sera from healthy individuals were used (Table 1, and Table S1).

T7 Phage Display Library Construction and Biopanning Two commercial T7 Phage Display libraries displaying the mRNA repertoire of Alzheimer’s disease patients or controls (Novagen) were used in the study according to the manufacturer instructions. For each round of biopanning, 25 μL Protein G-Plus Agarose Beads (sc-2002, Santa Cruz Biotechnology) were washed twice with phosphate buffered-saline (PBS), blocked with blocking buffer (1% BSA-PBS) for 1 hour at 4ºC and incubated 4 hours at 4ºC with 250 μL 1:20 diluted either non-AD (non-AD beads) or AD pooled sera (AD-beads) in blocking buffer. AD sera pool consisted of samples from 1 patient at Braak IV, 4 patients at Braak V and 1 patient at Braak VI. Then, they were washed twice with PBS. To remove normal antigens, 350 μL 1% BSA bacterial lysates containing phages from each T7 library were incubated with nonAD beads for 2 hours at 4ºC. Next, supernatants were incubated with AD-beads overnight at 4ºC. Finally, supernatants were removed, and beads washed ten times with PBS. Bound phages 6


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were eluted using 1% SDS 10 minutes at RT and amplified by E. coli BLT5615 infection for the next round of biopanning. This process was performed four times to enrich the eluted fractions in AD-specific reactive phages.

Monoclonal Phage Amplification, Printing and Use of Phage Microarrays After biopanning, 1,536 phages from the third and four rounds of biopanning were amplified onto 96-well plates, and transferred to five 384-well plates after 1:2 dilution in PBS 0.1% Tween 20 (PBS-T) (44). Size diversity of cDNA-derived inserted sequences and absence of cross-contamination among monoclonal phages (presence of more than one band) were checked by PCR using T7_up2 and T7_down2 primers (31, 45). Then, 1,920 phages and controls were printed onto SuperNitro Substrates Nitrocellulose Coating slides (Arrayit) using a MicroGrid II Spotter (GeneMachines, San Carlos, CA) at 20ºC and 50% humidity. Negative controls consisted of empty spots, PBS-Tween 0.1% or 1 μg/μL bovine serum albumin (BSA, SigmaAldrich). Positive controls consisted of three serial 1:10 dilutions starting from 1 μg/μL human and mouse IgGs. Briefly, microarrays were equilibrated with PBS at room temperature (RT) for 5 minutes and then blocked with PBS-T supplemented with 5% skimmed milk (MPBS-T) for 1 hour at RT with gentle agitation. Then, 1:300 individual human sera in MPBS-T preincubated 2 hours at RT with 20 μg of both, BLT5615 and BL21 E. coli lysates, were added to the arrays and incubated for 90 minutes at RT with 150 rpm rotation. After washing three times with PBST, monoclonal anti-T7 tag antibody (1:200 dilution, Sigma-Aldrich) in MPBS-T was added to the arrays and incubated 1 hour at RT. After washing, human IgG antibodies and T7 phages were detected with Alexa-Fluor 647-labeled goat anti-human IgG (Invitrogen, Carlsbad, CA) diluted 1:2,000 in 3% MPBS-T and Alexa Fluor 555-labeled goat anti-mouse IgG (Invitrogen) diluted 1:40,000 in MPBS-T, respectively. Finally, arrays were washed three times with PBS-T, one last time with PBS and dried by centrifugation at 1,200 rpm for 3 min at RT. Microarray scan was performed on a GenePix 4000B Microarray Scanner (Axon Laboratories, Boston,

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MA). Genepix Pro 7.1 (Axon Laboratories) image analysis software was used for spot intensity quantification.

Phage Purification, DNA Isolation, Sequencing and Protein BLAST Analysis Individual phage clones were firstly purified using saline polyethylene glycol (PEG) precipitation. Briefly, 500 μL of a 5M NaCl solution were added to 4.5 mL cleared bacterial lysates containing phages. After mixing, 1 mL 50% PEG 8000 (Sigma-Aldrich) was added and the solutions mixed by vortex and left overnight at 4ºC on ice. Finally, samples were centrifuged at 4,000 rpm 4ºC and supernatants discarded. Pellets (purified phages) were resuspended in 500 μL PBS. DNA extraction from purified individual phage clones was optimized using a phenol-chloroform-isoamyl alcohol mix after thermal denaturation. Firstly, 100 μg RNase A (Qiagen), 6.25 μL 1 M MgCl2 and 1 Unit of DNase I (Ambion, 1U/uL) were added to 500 μL purified phage clones and incubated at RT for 45 minutes. Then, 20 μL 0.5 M EDTA, 50 μg Proteinase K (Thermo Fisher Scientific, 10 μg/μL) and 10 μL 10% SDS were added, mixed and incubated for 90 minutes at 55ºC. Next, 600 μL Phenol:Chloroform:Isoamyl Alcohol 25:24:1 (v/v/v, Sigma-Aldrich) were added, mixed and centrifuged 12,000 x g at RT. Upper colorless supernatants were recovered, and the previous step repeated once. Finally, DNA from the cleaned upper supernatants was precipitated and purified using ethanol, and resuspended in Milli-Q, DNase-free water. For sequencing, purified phage DNA (1 ng) was used as template for cDNA insert amplification by PCR using forward primer T7_up2 and reverse primer T7_down2. PCR products were analyzed by agarose gel electrophoresis, ethanol purified and directly sequenced with the forward primer T7_up2. Protein BLAST on the NCBI database to find sequence identity to each peptide displayed on the phages in frame with the 10B protein was restricted to Homo sapiens (taxid: 9606). The first hit retrieved from the BlastP 2.6.0 was used to identify the protein sequence displayed on phages.

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Gateway Vector Design, and Cloning, Expression and Purification of Halo-tagged Peptides The developed assay to verify the immunoreactivity of the identified peptides involved the design and construction of the “pDEST-HisHALO” Gateway destination vector for cloning, which allowed for the E. coli expression of proteins at the C-terminal end of an N-terminal 6xHis-tagged HaloTag. The pET15b vector, optimized for bacterial protein expression and presenting a T7 promoter sequence and a bacterial ribosome binding sequence (RBS) upstream of a coding region with an N-terminal 6xHis-tag, was used as template. Then, HaloTag coding sequence from the pFN18a plasmid and the Gateway cassette (containing the attR recombination sites, the ccdB coding sequence and a chloramphenicol-resistance gene) from the pDEST15 vector, were directionally assembled into the linearized pET15b vector through HiFi Assembly (NEBuilder® HiFi DNA Assembly Master Mix, New England BioLabs) after PCR using overlapping oligonucleotides (Figure S1 and Table S2). To clone phage cDNA inserts encoding displayed peptides into pDEST-HisHALO, DNA was PCR amplified using two attB site-containing oligonucleotides complementary to the 10-3b vector region immediately before the cDNA-insertion EcoRI and HindIII sites. After ethanol purification, fragments were inserted using single-step combined BP/LR Gateway reactions (Thermo Fisher Scientific) using pDONR221 and pDEST-HisHALO as donor and destination vectors, respectively (46). pDEST-HisHALO/cDNA constructs were used to transform BL21 (DE3) E. coli cells. 10 mL of LB cultures containing 100 μg/ mL ampicillin and the transformed cells were grown overnight and then, diluted ten times, and grown until OD600nm reached 0.7. Finally, protein production was induced with 0.4 mM isopropyl thio-β-D-thiogalactoside (IPTG) and incubated at 37ºC 250 rpm during 4 hours (exceptionally, expression of the 6xHisHalo-34AM fusion protein was induced at 16 ºC 250 rpm for 24 hours to prevent degradation). Then, cultures were centrifuged at 6,000 x g for 20 minutes at 4ºC and the supernatant discarded. The pellet was reconstituted in lysis buffer supplemented with 10 mM Phenylmethanesulfonyl fluoride (PMSF) and 1x Protease Inhibitor Cocktail (Roche), and sonicated (3 cycles of 30 seconds with 2 9



minutes rest in between) on a JP SELECTA Sonicator (Ultrasons). HaloTag fusion peptides overexpressed in soluble fractions were purified by gravity-flow chromatography from the soluble fraction of cell lysates using Super Ni-NTA agarose resin (Generon SpA) following the manufacturer instructions with slight modifications (lysis Buffer: 20 mM Phosphate, 300 mM NaCl, pH 8.0; Wash buffer: 20 mM Phosphate, 300 mM NaCl, 2.5 mM Imidazol, pH 8.0; and elution buffer: 20 mM Sodium Phosphate, NaCl 300 mM, 500 mM Imidazol, pH 8.0; soluble fractions were incubated with resin overnight at 4ºC). Protein expression and purification were analyzed by Coomassie Blue staining and Western blot (WB) after 10% SDS-PAGE under reducing conditions, incubating with 1:3,000 mouse monoclonal anti-6xHis-tag antibody (MA1-21315, Thermo Fisher Scientific) followed by 1:2,000 horseradish peroxidase-labeled goat polyclonal antibody against mouse IgG (Sigma Aldrich). PBS-T supplemented with 3% skimmed milk was used as blocking buffer. Chemiluminescent signal was developed using ECL reagent (Thermo Fisher Scientific). Purified protein concentration was calculated by measuring the absorbance at 280 nm using a DU-7 spectrometer (Beckman, Barcelona, Spain) after theoretical extinction coefficient calculation with the ProtParam tool from ExPASy.

Luminescence Beads Immunoassay The seroreactivity of the purified fusion peptides was tested using a self-tuned assay based on the capture of HaloTag fusion proteins for subsequent immunological assays. Firstly, Magne® HaloTag® beads (Promega) were equilibrated in lysis buffer and subsequently incubated overnight at 4ºC with the purified HaloTag peptides with continuous rocking. For each measurement a relation of 0.625 μg of Halo protein per 0.31 μL of MagneBeads® solution was made. For covalent binding, a mixture of the required amount of protein and MagneBeads was made taking into account the number of replicates and measurements to be performed. After covalent binding, MagneBeads® were washed three times with 200 μL lysis buffer for 5 minutes at RT and 250 rpm shaking. Then, unbound proteins were removed adding 100 μL elution 2 buffer (Glycine 0.1M pH 2.7) and incubating for 5 minutes at RT and 250 rpm shaking. 10


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MagneBeads® where blocked with PBS-T supplemented with 3% BSA (BPBS-T) for 1 hour at RT and 250 rpm shaking, and transferred onto separate wells of Bio-Plex 96-well plates (BioPlex ProTM Flat bottom plates, Bio-Rad) to perform the subsequent incubations with serum samples or indicated antibodies. Serum samples were diluted 1:100 in PBS-T and pre-incubated with 150 μg/mL BL21(DE3) extract and 10 μg/mL control protein overnight at 4ºC. Then, Halo fusion peptides on the plate-wells were incubated with serum samples 1 hour at RT and 150 rpm shaking. After washing three times with PBS-T, bound human IgG was detected with a subsequent incubation during 1 hour at RT and 150 rpm shaking of 1:10,000 diluted Goat AntiHuman IgG Fc secondary antibody conjugated with biotin (Thermo Fisher Scientific) followed by an incubation with 1:1,000 HRP-Streptavidin (RayBiotech), washing three times in between. Finally, the signal was developed with ECL chemiluminescent reagent (Thermo Fisher Scientific), and recorded onto the Spark multimode microplate (TECAN).

Bioinformatics, Statistical Analysis and Protein Modeling Analysis, normalization, and quantification of all microarray images were performed using GenepixPro 7.1 software (Axon Laboratories). The median values of the spots and background were determined, and interarray median normalization was performed (30, 31, 45, 47). To identify seroreactive AD-specific phages by comparing AD patients and healthy individual groups, a t-test using pomelo II (http://pomelo2.iib.uam.es/) was performed, where p-values were obtained by permutation testing using 200,000 permutations. Pomelo II generated a heatmap, showing the phages with a false discovery rate (FDR) value below 0.15 and an unadjusted p-value below 0.05 to minimize for the presence of false positive results (15% of significant tests might be false positives instead of 5% of all positive phages if using only the pvalue). MultiExperiment Viewer Analysis (MeV) was used for visualization. Plots, mean, standard error of the mean (SEM), signal to noise (S/N) ratios of phage microarrays, coefficient of variation between technical (intra-assay) and biological (inter-assay) replicates of the luminescence beads immunoassay and Student’s t-test calculations were performed with Microsoft Excel 2013 and GraphPad Prims 5 programs. Non parametric Mann-Whitney U test 11



values were calculated using the R program (version 3.5.0) and ROC curves constructed with the R program (Version 3.5.0) using the ModelGood and the Epi packages. NUPR1 structure was modeled using the Iterative Threading ASSEmbly Refinement (ITASSER Suite) (48). OR8J1 and Halo-tagged peptide construct were modeled using the ExPASy Swiss Model Tool (PDB: 5tvn.1 and PDB: 5vnp.1.A as templates, respectively) and topological prediction confirmed by TMHMM (49). For PYGB, the solved structure (PDB: 5IKO) was used. All structures were visualized with PyMOL Molecular Graphics System (Version 2.0, Schrödinger, LLC). Residue-based side chain solvent accessible surface area and percentage of solvent accessibility were calculated in Parameter Optimized Surfaces (POPS) Version 1.0.6 (50).

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RESULTS In the present study, we have performed the profiling of T7 phage display libraries from AD and healthy brain tissue by combining phage display and protein microarrays to identify autoantibodies and their target amino acid sequences with diagnostic ability in Alzheimer’s Disease. A schematic representation of the workflow of the study is depicted in Figure 1.

Figure 1. Workflow for the identification of specific targets of autoantibodies in AD patients’ sera using phage microarrays. Two libraries of T7 phages displaying the cDNA from healthy and AD brain tissues were subjected to four rounds of biopanning. In each round, libraries were incubated with healthy individuals’ IgGs, bound phages removed and then, unbound phages incubated with IgGs from AD patients sera. Bound phages were eluted and amplified using BLT5615 E. coli cultures to enrich the libraries in AD-specific peptides. Then, individual phages were isolated and amplified on 96-well plates and finally transferred to 384-well plates for microarray printing. After image analysis and microarray quantification, peptides displayed on the most immunoreactive phages against individual and pooled serum samples from AD patients were identified using t-test permutations by Pomelo II. Thirteen out of 44 unique peptides were cloned and expressed in E. coli as 6xHisHalo-tagged proteins using a selfdesigned pDEST vector. Finally, after Ni-NTA affinity purification, their immunoreactivity against AD patients’ sera and healthy subjects was analyzed using a fine-tuned luminescence beads immunoassay to determine their AD diagnostic ability.

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Profiling of Alzheimer Disease Sera with Phage Microarrays T7 phage display brain libraries were individually enriched in specific phages by biopanning using purified IgG from the sera of Alzheimer disease patients and controls (Figure 1). Four rounds of biopanning were performed to enrich in AD-specific phages. In each round of biopanning, a negative selection using sera from control individuals to remove non-specific phages was carried out. Then, phage libraries were enriched in AD-specific phages by their selection with sera from AD patients and infection of E. coli cells. A total of 1,536 individual phages from the third and fourth rounds of biopanning were manually picked, and printed in duplicate together with positive and negative controls onto nitrocellulose microarrays. Then, to set up the optimal sera dilution to be used in the microarrays, we separately probed the microarrays with a pool of sera from AD patients and controls at a 1:150 and 1:300 dilution (Figure 2). While the dynamic range of the IgG intensity response was 3 orders of magnitude at both dilutions of sera, the S/N ratio was 13% better using the 1:300 dilution in comparison to the 1:150 dilution. Better results were observed for a 1:300 dilution regarding signal-to-noise ratio. Then, the 1:300 dilution was used to probe separately 16 arrays with 8 individual sera from AD patients and 8 from controls. To increase the number of potentially identified AD-specific IgG-seroreactive phages from the protein microarrays; we separately analyzed microarrays incubated with pool of sera and individual sera. Following image quantification and normalization, the comparison of AD and control groups was performed as previously described (Figure 2) (30, 31, 45, 47). First, AD/control signal intensity ratios were calculated for the arrays incubated with the pool of sera. A ≥1.4 ratio AD/control and signal intensity for the IgG channel higher than 250 in the AD group was used as cut-off for selection of phages. Twenty-nine individual phages were selected (Figure 2b). Second, individual arrays probed with 8 AD and 8 control sera were analyzed using a t-test analysis with 200,000 permutations using Pomelo II and visualized with MeV. Nineteen individual phage clones showed statistically significant higher reactivity in AD group than in the control group with a FDR

Identification of Alzheimer's Disease Autoantibodies and Their Target

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