A polyalanine antibody for the diagnosis of oculopharyngeal muscular dystrophy and polyalanine-related diseases

Production of polyalanine antibody

A 19 GCA repeat sequence was cloned into the multiple cloning site of pGEX-4T1 (Sigma #GE28-9545-49, BamH1 and Xho1 ligation). The recombinant pGEX-4T1 vector was transformed in E. coli BL-21 to produce GST-polyalanine fusion proteins. The same 19 GCA repeat sequence was cloned in the multiple cloning site of pMAL-c2X vector (NEB #E8200, BamH1 and Sal1 ligation); this second recombinant vector was also transformed into E. coli BL21to produce MBP-polyalanine fusion proteins. The presence and orientation of the 19 GCA inserts of the two vectors was confirmed using restriction enzymes, agarose gel electrophoresis and Sanger sequencing. Expression vectors from the bacterial clones confirmed to correctly express the two fusion proteins were transformed into E. coli DH5α to be expanded for the affinity purification of GST-polyalanine and MBP-polyalanine through glutathione-Sepharose and amylose resin columns, respectively.

1 mg of purified GST-polyala was injected subcutaneously in two rabbits (following the collection of a pre-immune blood samples to have a pre-immune antiserum). Four immune response boosts were subsequently made (4 weeks apart) using the same dose. Bleeds were collected every month for 6 months (8.5 ml/kg). Animals were to be euthanized if the titer was insufficient 6 months after the initial immunization. Through the entire process care was taken to make sure the animals suffering was minimal. Animals were observed 15 minutes post-injections, and also on a daily for responses at the injection sites and signs of overall distress. When blood samples were collected the animals were tranquilized by injecting xylazine (5mg/kg) and ketamine (20–35 mg/kg) intramuscularly. At the end of the antisera production, the animals were euthanized by exsanguination under general anaesthesia and the antisera were prepared for storage at -80°C. Animals were obtained from Charles River Laboratories and maintained (one per cage) at the Comparative Medicine and Animal Resources Center (McIntyre, McGill University).

The specific and further purification of the antisera against polyalanine was established using Western blots loaded with MBP-polyalanine fusion proteins transferred on PVDF membranes (Sigma #P2563). The use of this PVDF transfer and immobilization approach insured two aspects: First, the antisera were detecting the polyalanine regions of the fusion peptide used for immunization and not the GST region. Second, the region of the PVDF membranes corresponding to the polyalanine signal could be cut out to elute antibodies that specifically recognized the polyalanine. Aliquots of PVDF eluted polyalanine antibodies were used in the immunodetections of material expressing polyalanine (and control).

Transgenic Drosophila lines

Stocks used in this study were previously described¹⁶. Full-length ATXN3 cDNAs bearing _wtCAG_{14, exp}CAG_{92, exp}CAA₉₆, STOP-CAG₉₄ or STOP-CAA₉₄ repeats were subcloned in pUAST (some vectors have a STOP codon upstream of the repeat). Epitope tags were added to each reading frame: Myc for main-frame, HA for −1 frame and His for +1 frame. Vectors sequenced before injection into w¹¹¹⁸ Drosophila eggs; a step followed by selection of positive transformants, mapping and balancing (Genetic Services, Inc.). Flies bearing transgenic constructs in a homozygous state were maintained at 25°C. Adult males were crossed to virgin gmr-GAL4 flies to obtain lines expressing transgenes in developing eyes (_wtCAG₁₄/gmr-GAL4, _expCAG₉₂/gmr-GAL4, _expCAA₉₆/gmr-GAL4, STOP-CAG₉₄/gmr-GAL4 and STOP-CAA₉₄/gmr-GAL4 genotypes). To obtain isogenic control flies, w¹¹¹⁸male flies were crossed with virgin gmr-GAL4.

Cell culture and transfections

Epstein-Barr Virus (EBV) immortalized lymphoblastoid cell lines (LCL) were prepared from patient and control individuals in the laboratory of Dr Rouleau. All subjects signed an informed consent, which is part of the experimental protocol approved by the Research Ethical Board (REB) of McGill University Health Research Centre (#NEU-14-051, June 11 2015). LCL and HeLa cells were cultured at 37°C in a humid atmosphere enriched with 5% CO₂. HeLa cells (ATCC #ATCC® CCL-2^TM ) cat were grown in Dulbecco’s Modified Eagle Medium (Gibco), supplemented with 10% fetal bovine serum (Gibco) and 1% Penicillin/Streptomycine/Glutamine (Gibco), while the lymphoblastoid cells were grown in Iscove’s Modified Dulbecco’s Medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), 1% Penicillin/Streptomycine/Glutamine (Gibco) and Fungizone antimycotic (Gibco).

For transient transfections, HeLa cells were transfected at 70% confluency for 48 hours with GFP-tagged hPABPN1 plasmid DNA containing various length alanine expansions (0, 10, 13, 17, 30, and 40) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. These constructs were graciously provided by Dr. Bernard Brais (McGill University), and previously described²³.

Western blots

48 hours post transfection, HeLa cells were collected in ice-cold phosphate buffered saline (PBS), and lysed in radioimmunoprecipitation (RIPA) buffer [50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS)] supplemented with protease inhibitors (Roche), and sonicated for five 1 sec pulses. For OPMD patient and control individual lymphoblastoid cell lines, protein extractions were performed using the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Scientific) according to the manufacturer’s instructions.

Membranes were blocked for 24 hours at 4°C in a PBS-T (0.1% Tween-20 in PBS) solution containing 5% milk (instant skim milk powder) and 5% bovine serum albumin (BSA; Fisher), and incubated overnight at 4°C in PBS-T (5% milk and 5% BSA) with one of the following primary antibodies: rabbit monoclonal anti-PABPN1 antibody (1:1,000; Abcam #ab75855); mouse monoclonal anti-GFP antibody (1:5,000; Clontech #632381); or the rabbit polyclonal antibody 4340 (1:500-3,000) developed by the laboratory of Dr McPherson. Membranes were then washed three times for 10 min in PBS-T, incubated for 2 hours at room temperature with the appropriate horseradish peroxidase (HRP) conjugated secondary antibody [donkey anti-mouse IgG antibody (1:5,000; Jackson ImmunoResearch), or donkey anti-rabbit IgG antibody (1:2,500; Jackson ImmunoResearch # 111-035-144), followed by three 10 min washes in PBS-T. Immunodetection was performed using the enhanced chemiluminescence (ECL) system (Perkin Elmer), and membranes were exposed to HyBlot CL autoradiography film (Denville Scientific Inc.).

Human immunohistochemistry

Formalin-fixed paraffin-embedded OPMD patient and control individual cerebellum samples were sectioned (5 μm) and placed on glass slides. The sections were deparaffinized, rehydrated, and incubated in an antigen retrieval solution (DAKO) at 85°C for 1 hour. Sections were cooled to room temperature, and washed three times in PBS. Immunohistochemical detection was carried out by permeabilizing sections in 0.2% Triton-X100 in PBS for 30 min, followed by blocking in PBS containing 10% normal goat serum (NGS; Gibco) for 1 hour, and incubating with primary antibodies overnight at room temperature [mouse monoclonal anti-ubiquitin antibody (1:1,000, Millipore), rabbit polyclonal antibody 4340 (1:500)]. Biotinylated secondary antibodies were used at a 1:500 dilution, and amplified using the ABC Elite kit (Vector). Reaction product was revealed using the DAB Substrate kit (Vector), mounted with VectaMount (Vector), and visualised on a Leica CTR6000 fluorescence microscope. All subjects signed an informed consent which is part of the experimental protocol approved by the Research Ethical Board (REB) of McGill University Health Research Centre (#NEU-14-051, June 11 2015).

LCL from HD and SCA3 patients, and control individuals were washed in PBS and deposited onto glass slides using a StatSpin Cytofuge 2 (Beckman Coulter) at 7,000 rpm for 4 min. Slides were dried for 30 min at room temperature, and fixed in 4% PFA for 20 min. Permeabilization, blocking, and incubation in primary antibody [rabbit polyclonal antibody 4340 (1:500)] was performed as described above. Cells were then incubated for 2 hours at room temperature in HRP-conjugated secondary antibody [donkey anti-rabbit IgG antibody (1:500; Jackson ImmunoResearch)]. Reaction product was revealed using the Vector VIP Substrate kit, mounted with VectaMount (Vector), and visualized on a Leica CTR6000 fluorescence microscope.

Drosophila immunohistochemistry

Adult flies were decapitated (3 days post eclosion), with heads immediately placed in Tissue-Tek (Sakura) and on dry ice to freeze. Ten micron sections were obtained by cryosectioning on a Leica CM3050S cryostat, dried for 30 min at room temperature, and then fixed in 4% paraformaldehyde (PFA) for 15 min. Permeabilization, blocking, and incubation in primary antibodies [mouse monoclonal anti-SCA3 antibody (1:1,000, Chemicon), rabbit polyclonal antibody 4340 (1:500)] was performed as described above. Sections were then incubated with the appropriate fluorescent secondary antibodies for 1 hour (anti-mouse or anti-rabbit fluorescent tagged secondary antibodies, 1:500, Alexafluor) and mounted with Mowiol. Visualization of immunofluorescence staining was carried out on a Leica CTR6000 fluorescence microscope.

An earlier version of this article can be found as a thesis on the Université de Montréal Institutional Repository (https://papyrus.bib.umontreal.ca/xmlui/handle/1866/13553).

Generation of a polyclonal antibody sensitive to polyalanine at the pathological threshold in OPMD

We generated an antibody (4340) against a 19-mer peptide composed of 18 alanines followed by a glycine. In order to evaluate the usefulness of this antibody, it was critical to determine the number of alanine repeats it could detect. Using OPMD as the disease model and Western blot immunodetection as a first assay, our analyses revealed that the antibody was able to produce a strong signal from whole protein lysates prepared from HeLa cells that transiently expressed a vector encoding a GFP-tagged hPABPN1 cDNA bearing alanine repeat lengths of 13, 17, 30, and 40 (Figure 1a). In contrast, only a weak signal was observed from lysates prepared from cells expressing the same cDNA if it encoded a 10-alanine repeat, and no signal could be observed from lysates prepared from cells that were either expressing a cDNA with no polyalanine tract (0-alanine) or that were untransfected (Figure 1a). To test whether the signals detected were the putative GFP-hPABPN1- alanine proteins, we probed the same samples with an antibody against GFP, and observed corresponding bands at ~75 kDa (Figure 1b). This suggests that the ~75 kDa bands detected by both antibodies correspond to the same protein, whereas the ~55 kDa bands detected by our antibody alone appear to be an unspecific contaminating signal.

Figure 1. Testing of Ab4340 sensitivity in hPABPN1 transfected cells.

Western blot immunodetections of polyalanine (a) and GFP (b) from HeLa cells transiently expressing GFP-hPABPN1 vectors where the cDNA contained various lengths of alanine repeats. Lane 1: untransfected cells; Lane 2: expression of GFP-hPABPN1-0Ala; Lane 3: expression of GFP-hPABPN1-10Ala; Lane 4: expression of GFP-hPABPN1-13Ala; Lane 5: expression of GFP-hPABPN1-17Ala; Lane 6: expression of GFP-hPABPN1-30Ala; and Lane 7: expression of GFP-hPABPN1-40Ala. (*) refers to an unspecific contaminant signal. Ab4340 strongly detected GFP-hPABPN1 protein containing 13 or more alanine repeats [(a), Lanes 4–6)], but showed a weaker ability to detect an alanine repeat length of 10 [(a), Lane 3] despite adequate GFP expression [(b), Lanes 2–7]. (c–h) Double-labelling immunofluorescence detection of alanine (red) and GFP (green) in HeLa cells fixed 48 hours post transfection with the same constructs used for the Western blot analysis. Strong detection of alanine-containing aggregates was achieved with repeat lengths of 10-alanine and greater (d–h), whereas no detection was made in cells not expressing alanine (c). Scale bar, 25 µm.

HeLa cells that were transfected with the same expression vectors which were used for the Western blot analyses were also used to test the sensitivity of Ab4340 to polyalanine tracts through an in vitro immunofluorescence assay. The fusion of an N-terminal GFP-tag to each construct made it possible to visualize protein expression using fluorescence microscopy. Intranuclear expression with a strong GFP signal was observed across all constructs (Figure 1c to 1h). Using the 4340 antibody, we were able to specifically target the alanine-containing proteins and detect their expression in cells transfected with the expression vectors that encoded repeat lengths of 10, 13,17, 30, and 40 alanines (Figure 1d to 1h). No alanine signal was detected following the expression of the 0-alanine construct (Figure 1c). The alanine-containing protein appears in aggregates, colocalizing with the GFP-expressing INIs (Figure 1d to 1h). These findings suggest that Ab4340 is more sensitive in detecting alanine expansions using an immunofluorescence assay (immunocytochemistry) than Western blot immunodetection.

Differentiation can be made between OPMD and control patient samples

The results from our Western blot immunodetections based on HeLa cells transiently expressing hPABPN1 cDNA with different polyalanine tracts demonstrate that the sensitivity of Ab4340 coincides with the pathological threshold known to cause OPMD. To determine whether or not it could be used to discriminate between samples obtained from OPMD patients and control individuals, we performed another series of Western blot immunodetections for which the protein lysates were prepared from lymphoblastoid cell lines (LCLs). Furthermore, we used our 4340 antibody in immunohistochemistry assays of cerebellar sections from OPMD patients and controls.

Western blots probed with Ab4340 reveal a strong signal at ~60 kDa in nuclear lysates prepared from OPMD patient material (Figure 2a, lanes 3–6), whereas no bands were detected in nuclear lysates prepared from unaffected individuals (Figure 2a, lanes 1 and 2). These same lysates were probed with an antibody directed against PABPN1, and a corresponding band at ~60 kDa was observed (Figure 2b). This indicates that the ~60 kDa bands detected by the two antibodies are the same predicted PABPN1-alanine protein. In contrast to the results obtained from HeLa cells, no unspecific contaminant signal was observed from patient lymphoblastoid cell lines.

Figure 2. Testing the ability of Ab4340 to differentiate between OPMD patient and control individual samples.

Western blot immunodetections of alanine (a) and PABPN1 (b) from nuclear extracts prepared from LCLs. Lanes 1 and 2: extracts from control individuals; and Lanes 3–6: extracts from OPMD patients. The 4340 antibody cleanly detected alanine-containing proteins exclusively from OPMD patient extracts [(a), Lanes 3–6], despite strong detection of PABPN1 in all patient extracts (b). Immunohistochemical detection of ubiquitin (c,e) and polyalanine (d,f) containing proteins in cerebellar neurons of an OPMD patient (c,d) and control individual (e,f). Both antibodies immunostained intranuclear structures in the OPMD patient’s sample (c,d), whereas only ubiquitin immunostaining was achieved in the control patient’s sample (e). Scale bar, 2.5 µm.

Immunohistochemistry detections made using Ab4340 and an antibody directed against ubiquitin revealed strongly stained intranuclear structures in cerebellar neurons of the OPMD patient (Figure 2c and 2d). The ubiquitin-detecting antibody also revealed intranuclear signals in sections prepared using tissue sections of a control individual (Figure 2e); however, when Ab4340 was used on similar sections no intranuclear signal was observed (Figure 2f).

Alanine-containing proteins are detected in a transgenic Drosophila model of SCA3, and lymphoblastoid cells of SCA3 and HD patients

To test whether our antibody could detect polyalanine-containing proteins in polyglutamine diseases that have a propensity to present frameshifting, we investigated SCA3 and HD. Using expCAG₉₂ and isogenic control flies from our previously reported transgenic ATXN3 Drosophila model¹⁶, we made immunohistochemical detections with both our 4340 antibody and one directed against ataxin-3. Alanine-containing proteins (red) were observed exclusively within the eyes of expCAG₉₂ flies (Figure 3a). In these same flies, ataxin-3 containing aggregates (green) were present throughout the eye, confirming transgene expression (Figure 3a). No ataxin-3 containing proteins were detected in the isogenic control flies (Figure 3b).

Figure 3. Detection of polyalanine in a transgenic Drosophila model of SCA3, and lymphoblastoid cells of an SCA3 and HD patient.

(a,b) Double-labelling immunofluorescence detection of alanine (red) and GFP (green) in an expCAG₉₂ transgenic fly (a) and an isogenic control (b), showing polyalanine- and ubiquitin-labeled aggregates exclusive to the transgenic line (a). Scale bar, 25 µm. (c–e) Immunocytochemical detection of polyalanine containing proteins in lymphoblastoid cells of an SCA3 patient (c), HD patient (d), and control individual (e). The 4340 antibody immunostained intranuclear inclusions in both the SCA3 (c, arrows) and HD (d, arrows) patient cells, whereas no intranuclear staining was present in the control patient lymphoblast cell line (e). Scale bar, 2.5 µm.

Immunocytochemical detections were also made with LCLs derived from SCA3 patients, HD patients, and control individuals. Ab4340 detected alanine-containing protein aggregates in LCLs from both SCA3 (Figure 3c, arrows) and HD (Figure 3d, arrows) patients, whereas no aggregates were observed in the control individual’s LCLs (Figure 3e). When comparing the number of cells presenting aggregates among the SCA3 and HD patients, their occurrence were observed more frequently in HD patient LCLs.