The FASEB Journal express article 10.1096/fj.03-0098fje. Published online August 15, 2003.
α-Synuclein implicated in Parkinson’s disease is present in extracellular biological fluids, including human plasma Omar M. A. El-Agnaf,*,†† Sultan A. Salem,*, †† Katerina E. Paleologou,* Leanne J. Cooper,* Nigel J. Fullwood,* Mark J. Gibson,† Martin D. Curran,‡ Jennifer A. Court,§ David M. A. Mann,║ Shu-ichi Ikeda,# Mark R. Cookson,** John Hardy,** and David Allsop* *Department of Biological Sciences, Lancaster University, Lancaster, United Kingdom; † Movement Disorders Clinic, Belfast City Hospital, Belfast, United Kingdom; ‡Northern Ireland Regional Histocompatibility and Immunogenetics Laboratory, Belfast City Hospital, Belfast, United Kingdom; §Joint MRC Newcastle University Development for Clinical Brain Ageing, MRC Building, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne, United Kingdom; ║Clinical Neuroscience Research Group, Greater Manchester Neurosciences Centre, Hope Hospital, Salford, United Kingdom; #School of Medicine, Shinshu University, Japan; and **Laboratory of Neurogenetics, National Institute on Aging, Bethesda, Maryland. ††These authors contributed equally to this work. Corresponding author: Omar M. A. El-Agnaf, Department of Biological Sciences, Lancaster University, Lancaster, LA1 4YQ, United Kingdom. E-mail:
[email protected] ABSTRACT Parkinson’s disease (PD) and other related disorders are characterized by the accumulation of fibrillar aggregates of α-synuclein protein (α-syn) inside brain cells. It is likely that the formation of α-syn aggregates plays a seminal role in the pathogenesis of at least some of these diseases, because two different mutations in the gene encoding α-syn have been found in inherited forms of PD. α-Syn is mainly expressed by neuronal cells and is generally considered to exist as a cytoplasmic protein. Here, we report the unexpected identification of α-syn in conditioned culture media from untransfected and α-syn-transfected human neuroblastoma cells, as well as in human cerebrospinal fluid and blood plasma. The method used was immunocapture by using anti-α-syn antibodies coupled to magnetic beads, followed by detection on Western blots. In all cases, α-syn was identified as a single 15 kDa band, which co-migrated with a recombinant form of the protein and reacted with five different antibodies to α-syn. Our findings suggest that cells normally secrete α-syn into their surrounding media, both in vitro and in vivo. The detection of extracellular α-syn and/or its modified forms in body fluids, particularly in human plasma, offers new opportunities for the development of diagnostic tests for PD and related diseases. Key words: amyloid • dementia with Lewy bodies • neurodegeneration
α
-Synuclein (α-syn) is a small protein (~14 kDa) that is expressed at high levels by neuronal cells (1). The first indication of an involvement of α-syn in the pathogenesis of neurodegenerative diseases came from the isolation of a peptide named non-Aβcomponent (NAC) from preparations of amyloid from the brains of patients with Alzheimer’s disease (AD). Amino acid sequencing revealed that NAC comprised 35 amino acids, corresponding to residues 61–95 of α-syn (2). Recently, two mutations, which accelerate α-syn
oligomerization, have been identified in the gene encoding α-syn (3, 4). These two mutations are each associated with rare, inherited forms of Parkinson’s disease (PD) (5, 6). This finding led to the discovery that α-syn is the main component of the intracellular aggregates found in Lewy bodies inside nerve cells in PD and dementia with Lewy bodies (DLB) and in glial cytoplasmic inclusions of multiple system atrophy (MSA) (7–10). Consequently, these diseases are now collectively known as the “synucleinopathies” (7). Substantial evidence suggests that the conversion of α-syn from soluble monomers to aggregated, insoluble forms in the brain is a key event in the pathogenesis of the synucleinopathies [reviewed in (11)]. The function of α-syn remains to be established; however, it has been implicated in the regulation of synaptic plasticity (12) and neuronal differentiation (13, 14), as well as in regulation of dopamine synthesis (15, 16), and it also has chaperone-like activity (17). More recently, evidence has shown that α-syn shares some properties with the family of fatty acid binding proteins and may thus transport fatty acids between the aqueous and membrane phospholipid compartments of the neuronal cytoplasm (18, 19). Recent studies have shown that neuronal cells overexpressing the wild-type α-syn are more resistant to oxidative stress than are untransfected cells (20, 21). It has also been shown that exogenous, non-aggregated α-syn at low concentrations protects neuronal cells against cellular stress conditions, such as serum deprivation, oxidative stress, and excitotoxicity (21), whereas, pre-aggregated α-syn was found to be toxic to neuronal cells (22), possibly via the formation of hydrogen peroxide (23). α-Syn lacks an ER targeting signal sequence and so is generally thought to exist only as a cytoplasmic protein (1, 24). For this reason, there have only been two studies investigating the presence of α-syn extracellularly in human cerebrospinal fluid (CSF) (25, 26). In one of these studies, α-syn could not be detected in the CSF (25), whereas the other study presented unconvincing data for the presence of α-syn in CSF (26). In this report, we have performed careful experiments to investigate the presence of α-syn in biological fluids. Here we present evidence to support the idea that cultured neuronal cells normally secrete α-syn, and, furthermore, we have also identified α-syn in normal human CSF and blood plasma. Our results raise the possibility that the extracellular form of α-syn has a normal physiological function throughout life and may play an important role in the development of PD and related diseases. MATERIAL AND METHODS Brain lysate A frozen post-mortem sample (0.33 g) of frontal cerebral cortex (BA8/9) was obtained from a 73-year-old female without neurological or psychiatric disease. Post-mortem interval was 27 h. This sample was homogenized in 1 ml of CelLytic buffer, consisting of mild detergent, bicine, and 150 mM NaCl (Sigma-Aldrich Company Ltd., Dorset, England, Product Number: C3228), containing a cocktail of protease inhibitors, including AEBSF, aprotinin, E-64, EDTA, and leupeptin (Calbiochem-Novabiochem Corporation, San Diego, CA), and centrifuged at 3000 × g for 30 min. The supernatant was collected and then stored at –80°C. Preparation of α-syn Recombinant α-syn was expressed in Escherichia coli and purified as described previously (1, 3).
Human neuroblastoma cell lines The production of stable cell lines overexpressing wild-type α-syn from parental BE (2)-M17 human dopaminergic neuroblastoma cells has been detailed elsewhere (27). Cells were grown in Dulbecco’s modified Eagle’s medium (Gibco BRL, Rockville, MD) containing [10% (v/v) fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate, and 2 mM glutamine] to 60– 75% confluency in 25 cm2 flasks (Nunc, Rochester, NY). For transfected cells, the media also contained 50 µg/ml of Geneticin (G418; Roche, Basel, Switzerland). Cells were then used to condition 5 ml of OPTI-MEM (Gibco BRL) serum-free medium, containing 100 U/ml penicillin, 100 µg/ml streptomycin sulfate, and 2 mM glutamine, for 6, 24, and 48 h. Conditioned media were collected, and a cocktail of protease inhibitors (Calbiochem-Novabiochem; see above) was added. The medium was clarified by centrifugation at 223 × g for 5 min at 4°C and then stored at –80°C until used for imunoprecipitation. Immunoprecipitation Dynabeads covalently coupled with recombinant protein A/G were derivatized with FL-140 rabbit polyclonal antibody raised against a recombinant full-length human α-syn, which recognizes α-, β-, and γ-syn (Santa Cruz Biotechnology) or 211 mouse monoclonal antibody, which recognizes amino acid residues 121–125 of α-syn of human origin (Santa Cruz Biotechnology), as recommended by the manufacturer (Dynal Biotech Ltd., Wirral, UK). Plasma (400 µl), CSF (500 µl), or culture medium (2 ml) were incubated with the beads overnight at 4°C. The beads were then washed three times with 0.1 M phosphate buffer (pH 8.2). Sodium dodecyl sulfate-PAGE (SDS-Page) and immunoblotting Captured α-syn was eluted from the beads by boiling in NuPAGE sample buffer (Invitrogen Ltd., Paisley, UK) for 10 min. Proteins were separated on NuPAGE Bis-Tris 4–12%, 1 mm gels (Invitrogen). The separated proteins were transferred to nitrocellulose membranes (0.45 µm; Invitrogen) at 125 mA for 45 min. Membranes were boiled for 5 min in phosphate buffered saline, and then blocked with 5% marvel dried skimmed milk, dissolved in PBS-Tween 20 (0.05%; PBST), for 1 h. The membranes were probed with primary antibodies (see Fig. 1), LB509 mouse monoclonal antibody that recognizes α-syn (115–122) (Zymed Laboratories, San Francisco, CA); 211 mouse monoclonal antibody to α-syn (121-125); N-19 goat polyclonal antibody to an N-terminal region of α-syn (5–19) (Santa Cruz Biotechnology); FL-140 rabbit polyclonal antibody, which recognizes α-, β-, and γ-syn; or α-synuclein C-20 goat polyclonal antibody, which recognizes the C-terminal region of α-syn (120–135) (Santa Cruz Biotechnology) as indicated, overnight at 4°C. The membranes were washed several times with PBST, followed by incubation with HRP-conjugated goat anti-rabbit (Dako Ltd., Ely, UK), goat anti-mouse (Dako Ltd.), or chicken anti-goat antibodies (Santa Cruz Biotechnology), as appropriate, for 60 min. The protein bands were visualized by using ECL reagents (Pierce, Rockford, IL) as described by the manufacturer. After exposure of blots to film, the density of bands was determined by scanning with Quantity One-4.1.1 (Bio-Rad, Hercules, CA). Cerebrospinal fluid (CSF) samples Fresh CSF samples were collected from PD patients and normal individuals by Shu-ichi Ikeda (Department of Medicine, School of Medicine, Shinshu University, Matsumoto, Japan). Ethical
approval for collection was obtained for all samples. Post-mortem CSF samples were obtained from the London Neurodegenerative Diseases Brain Bank (Department of Neuropathology, Institute of Psychiatry, King’s College, London, UK), Queen Square Brain Bank for Neurological Disorders (Department of Molecular Pathogenesis, Institute of Neurology, University College London, UK), and The Newcastle Brain Tissue Resource (Joint MRC Newcastle University Development for Clinical Brain Ageing, MRC Building, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne, UK). The samples were stored at – 80°C before analysis. Repeat freeze/thaw cycles were avoided. Plasma samples Blood samples were obtained from clinically diagnosed PD patients attending an outpatient clinic in the Neurology Department of Belfast City Hospital, Belfast, UK. Diagnosis of idiopathic PD was made, in each instance, by a consultant Neurologist (JMG). The diagnosis was made based on a progressive history and more than two of the cardinal signs of PD being present (resting tremor, bradykinesia, rigidity, postural instability). In addition there was absence of any features suggesting an alternative cause for parkinsonism. Control blood samples were obtained from the Haematology Department at Blackpool Victoria Hospital. Ethical approval was obtained for all samples. Approximately 10 ml blood was collected in plastic tubes containing sodium citrate or potassium-EDTA from all subjects, and plasma was separated by centrifuging the blood at 3000 rpm at 4°C for 20 min. Plasma was collected in 0.5 ml plastic tubes and stored frozen at –80°C. The samples were thawed at room temperature directly before analysis. RESULTS To determine whether cells secrete α-syn during normal metabolism, we used human neuroblastoma M17 cells stably transfected with cDNA encoding the wild-type form of α-syn (M17-wt) (27), untransfected M17 cells, or cells transfected with vector alone (M17-vec). Serum-free OPTI-MEM medium conditioned for 6–48 h with each cell type was immunoprecipitated by using magnetic dynabeads coupled to mouse monoclonal anti-α-syn antibody 211. Bound proteins were eluted from the beads, after washing, and were separated on SDS-gels before being transferred to nitrocellulose membranes for Western blotting. The blots were probed with anti-α-syn antibodies Fl-140 (Fig. 2A) or with N-19 or α-synuclein C-20 (data not shown). In all cases, a 15 kDa protein that co-migrated with recombinant α-syn, and also comigrated with α-syn from human brain homogenates, was identified in the conditioned medium (Fig. 2). In accord with its identification as α-syn, the 15 kDa protein was much more abundant in medium conditioned by M17-wt cells than medium conditioned with either M17-vec or untransfected M17 cells (Fig. 2A,B). Prolonged exposure of the blotting membrane revealed detection of the15 kDa band in immunoprecipitates prepared from medium conditioned with M17-vec or M17 cells for as little as 6 h (data not shown), with a progressive increase in the amount of the 15 kDa protein released into the medium over time. The 15 kDa band was not detected when the membrane was probed with control rabbit IgG (data not shown) or when the anti-α-syn antibodies used to probe the blots were pre-incubated with recombinant α-syn (Fig. 2A). A similar 15 kDa band was also detected when Fl-140 antibody was used for immunocapture, and the blots were probed with 211 (Fig. 2B), LB509, N-19, or α-synuclein C20 antibodies (data not shown). In all of these experiments, more than 96% of the cultured cells were found to be viable at the end of the 48 h conditioning period, as tested by trypan blue exclusion. Furthermore, to exclude the possibility that the 15 kDa protein was present as a
component of insoluble membranes released by dying cells, we centrifuged the conditioned culture medium at 100,000 g for 2 h and analyzed the resulting particle-free supernatant. We obtained complete recovery of the 15 kDa protein in this supernatant (data not shown). Taken together, all of these data indicate that the soluble 15 kDa protein normally released by the cells is α-syn and that α-syn in conditioned media is not released by dying cells. The low level of α-syn detected in media conditioned by M17-vec or M17 cells is due to the low expression of α-syn in these cells (27). Using the same experimental approach, we also determined whether α-syn is present in human CSF and plasma. We used FL-140 to capture the protein from the samples, and LB509, 211, N19, and α-synuclein C-20 for detection on the Western blots. Fig. 3 shows the results obtained from 500 µl samples of fresh or post-mortem CSF from non-neurodegenerative disease controls, and from patients with DLB and PD. Recombinant α-syn and human brain lysates were run on the same gels as positive controls, with recombinant β-synuclein (β-syn) and γ-synuclein (γ-syn) as additional negative controls. A 15 kDa protein that co-migrated with recombinant α-syn, and with α-syn present in the brain lysate, was identified in all of the CSF samples tested (Fig. 3). Similar results were obtained when the immunoprecipitation step was performed with 211 and the blots were probed with FL-140, N-19, or α-synuclein C-20 (data not shown). The 15 kDa band was absent in negative controls using mouse or rabbit IgG for capture (data not shown) and also when the membranes were probed with LB509, 211, N-19, or α-synuclein C-20 preadsorbed with recombinant α-syn (Fig. 3). There is only one previous report in the literature of α-syn in CSF, from both normal and PD patients (26). However, this study is not convincing because numerous bands were seen on the Western blots, and the supposed monomeric α-syn was detected only when the same antibody was used for immunoprecipitation and detection. A similar 15 kDa protein to that already described was readily detected in human plasma samples obtained from normal control subjects or from PD patients. Again, this protein could be captured with magnetic beads derivatized with FL-140 or 211, and detected on Western blots by using any of the antibodies LB509, 211, N-19, FL-140, and α-synuclein C-20 (Fig. 4). On Western blots, this protein was indistinguishable from recombinant α-syn and from α-syn present in human brain homogenates. There was no detection of recombinant β-syn or γ-syn (Fig. 4A, B) and the 15 kDa band was absent when rabbit or mouse IgG was used for capture (data not shown) or when the membranes were probed with antibodies to α-syn pre-adsorbed with the recombinant protein (Fig. 4). Similar results were obtained when the plasma was separated from blood collected in either EDTA or citrate buffer. Our results strongly suggest that the 15 kDa band detected in human plasma is also monomeric α-syn. Our initial estimation of αsyn concentration indicates that there is considerable overlap in the amount of α-syn observed in the CSF (~1–2.8 nM) and plasma (~0.5–2 nM) from PD and non-PD groups. Neither β-syn nor γ-syn could be detected on Western blots when we used several specific antibodies for β-syn or γ-syn for immunoprecipitation of the plasma or CSF (data not shown). However, both β-syn and γ-syn were detected when brain lysates were immunoprecipitated and detected with antibodies for β-syn or γ-syn.
DISCUSSION Our results show that α-syn is readily detectable in the CSF and plasma of PD and control patients, and there was considerable overlap in the amount of α-syn observed in the PD and nonPD group. α-Syn lacks a signal sequence for targeting to the ER and so is generally thought to exist only as an intracellular protein (1, 24). In contrast to current thinking, our results provide convincing evidence for the existence of an extracellular form of α-syn, detected here in the culture medium of neuronal cells and in CSF and plasma. Although at this stage we do not know the precise mechanism through which α-syn is released, we do not believe this to be due to necrotic or apoptotic cell death, as immunogold labeling experiments have shown α-syn being released from the surface of healthy cells (unpublished data). With regard to possible mechanisms of secretion, a point of significance is the fact that the C-terminal tail of α-syn contains two di-acidic motifs of the form Asp-X-Glu. This motif is known to be an ER to Golgi directing signal (28) and to interact with the ER coat protein II (COPII) complex (29). Although α-syn does not have an ER directing signal, its amphipathic αhelical, lipid-binding domain has been reported to bind strongly to membranes (18, 30) and even permealize them (31). One could speculate that these membrane-binding properties might allow α-syn to associate with the ER membrane. Then, the di-acidic motifs on α-syn might interact with the COPII complex transporting proteins from the ER to Golgi, and once in the Golgi the default pathway is secretion from the cell. There is even some evidence that the di-acidic motif can also interact with the adaptor protein-3 complex that is involved in Golgi to cell-surface transport (32). Significantly, β-syn and γ-syn, which were not detected extracellularly in the CSF and plasma, do not contain any of these di-acidic motifs (Fig. 5). Evidence that large proteins without ER leader sequences can be secreted comes from work on vimentin, a large (52 kDa) intermediate filament protein that, until recently, had been thought to be confined to the cell cytoskeleton. New work has shown that, despite lacking any ER signal sequence, in some circumstances vimentin can somehow cross into the ER and be secreted from the cell through the ER-Golgi pathway (33). Significantly, like α-syn, vimentin also has a diacidic Asp-X-Glu motif on its C-terminal tail and the authors (33) cite this motif as a partial explanation for the secretion of vimentin through the ER–Golgi pathway. Finally, although we believe that the existence of the two di-acidic motifs on the C-terminal tail of α-syn suggest passage through the ER and Golgi, we cannot exclude the possibility that α-syn is secreted via a nonclassical mechanism, such as endosomal recycling or via exosomes (34). It has been reported that platelets from PD and normal individuals contain α-syn and γ-syn (35, 36). However, neither of these proteins are secreted upon platelet activation (35). Thus, it is unlikely that the α-syn in human plasma originates from platelets. Recently, it has been shown that in a transgenic mouse model of Alzheimer’s disease, after peripheral administration of a monoclonal antibody to the β-amyloid (Aβ) peptide, a rapid increase in plasma Aβ was observed, and the magnitude of this increase was highly correlated with amyloid burden in the hippocampus and cortex (37, 38). These results demonstrate that Aβ can efflux from the brain to the plasma, and so it is possible that a similar mechanism could operate for other neuronal proteins, including α-syn. This type of mechanism could explain the presence of the α-syn in plasma samples. In MSA brains, glial cytoplasmic inclusions in oligodendrocytes contain α-syn
fibrils (9, 10) but α-syn is not expressed in glial cells (39), which suggests that the α-syn deposits found in glial cells of MSA brains may also be due to neuronal secretion. All of these pieces of evidence suggest that neuronal cells normally secrete α-syn into the surrounding media in the brain, and this could circulate to the CSF and then to the blood. One of the most interesting possibilities to arise from this is the potential use of α-syn and/or its derivatives in biological fluids as a biomarker for PD and related disorders. Further detailed studies of the levels of normal, nitrated, phosphorylated, glycosylated, or oligomeric forms of α-syn (40–44) will be required to determine whether this is a viable approach. The development of a reliable biomarker would dramatically accelerate research on the etiology, pathology, disease progression, and therapeutics for synucleinopathies. It has been shown that exogenous, non-aggregated α-syn at nanomolar concentrations can protect neurons from cellular stress conditions, such as serum deprivation, oxidative stress, and excitotoxicity (21), whereas at micromolar concentrations it can induce cell death (21, 22), possibly via hydrogen peroxide formation and oxidative damage (23). Therefore, it will also be important to understand the normal function of the extracellular form of α-syn and its role in the pathogenesis of PD and related disorders. ACKNOWLEDGMENTS We thank Ross Jakes and Michel Goedert (MRC-LMB, Cambridge, UK) for the samples of recombinant α-, β-, and γ-syn. We also thank The London Neurodegenerative Diseases Brain Bank (Department of Neuropathology, Institute of Psychiatry, King’s College, London, UK) and The Queen’s Square Brain Bank for Neurological Disorders (Department of Molecular Pathogenesis, Institute of Neurology, University College London, UK), for the human postmortem CSF samples. This work was supported by a grant from The Parkinson’s Disease Society, UK. The EM was provided by a Wellcome Trust JREI grant 055795/Z/98/ST/RC. S.A.S. was supported by The Higher Education Department, Libya. REFERENCES 1.
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Fig. 1
Figure 1. Schematic diagram of human α-syn, showing regions recognized by four anti-α-syn antibodies used for our study.
Fig. 2
Figure 2. Detection of α-syn in culture medium. Medium conditioned by M17-wt cells (expressing α-syn), M17-vec cells (expressing vector only) or nontransfected M17 cells were collected after 6, 24, and 48 h and then immunoprecipitated with 211 (A) or with FL-140 (B). The captured proteins were separated on Bis-Tris 4–12% SDSPAGE, and Western blotting was performed with FL-140 (A) and 211 (B). In (A), Lanes 1, 12, and 13 contain 3 ng recombinant human α-syn; Lane 2 contains 1 µg of human brain lysate; Lanes 3–5, M17 medium; Lanes 6–8, M17-vec; and Lanes 9–10 and 14–16, M17-wt medium. Lanes 12–16 were probed with FL-140 antibody pre-adsorped with recombinant human α-syn (1 µg α-syn/ml antibody). In (B), Lanes 1–3 and 14 and 15 contain M17-wt medium; Lanes 4– 6, M17-vec; and Lanes 7–9, M17 medium. Lanes 10 and 12 were loaded with 3 ng of recombinant human α-syn; lanes 11 and 13, 1 µg of human brain lysate; Lanes 12–15 were probed with 211 antibody pre-adsorped with recombinant human α-syn (1 µg α-syn/ml antibody).
Fig. 3
Figure 3. Immunoprecipitation of α-syn from human CSF. Human CSF from normal, PD, and DLB cases was incubated with beads cross-linked with primary antibody FL-140, and the resulting immunoprecipitates were fractionated on SDS-PAGE, transferred to nitrocellulose membranes, and probed with several antibodies for α-syn, LB509 (A); 211 (B); N-19 (C); and α-synuclein C-20 (D). In (A and B) lanes 1, 2, 10, and 11 contain PD CSF and lanes 3–5 and 12–14 contain normal CSF; 2 µg of normal human brain lysates (Lanes 6 and 15); 5 ng of recombinant human α-syn (Lanes 7 and 16); 5 ng of recombinant human β-syn (Lane 8); 5 ng of recombinant human γ-syn (Lane 9). Lanes 10–16 were probed with the antibodies pre-adsorped with recombinant human α-syn (1 µg α-syn/ml antibody). In (C and D) Lanes 1– 3 and 8–10 contain normal CSF; lanes 4–6 and 11–13 contain DLB CSF; 5 ng of recombinant human α-syn (Lanes 7 and 15); and 2 µg of normal human brain lysate (Lane 14). Lanes 1–7 were probed with the antibodies pre-adsorped with recombinant human α-syn (1 µg α-syn/ml antibody).
Fig. 4
Figure 4. Immunoprecipitation of α-syn from human plasma. Human plasma from normal and PD patients was incubated with beads cross-linked with primary antibodies FL-140 (A–C) or 211 (D, E), and the resulting immunoprecipitates were fractionated on SDS-PAGE and were immunoblotted with LB509 (A), 211 (B), N-19 (C), FL140 (D), and N-19 (E). In (A and B) 6 ng of recombinant human β-syn (Lane 1); 6 ng of recombinant human γ-syn (Lane 2); 6 ng of recombinant human α-syn (Lane 3); 3 µg of normal human brain lysates (Lane 4); plasma from PD patients (Lanes 5–8 and 13); and age-matched control plasma (Lanes 9–12 and 14) were used. Lanes 13 and 14 were probed with the antibodies pre-adsorped with recombinant human α-syn (1 µg α-syn/ml antibody). In (C) plasma from PD patients (Lanes 1, 2, and 7); age-matched control plasma (Lanes 3, 4, 8, and 9); 6 ng of recombinant human α-syn (Lanes 6 and 10); 3 µg of normal human brain lysates (Lane 5) were used. Lanes 7–10 were probed with N-19 antibody pre-adsorped with recombinant human α-syn (1 µg α-syn/ml antibody). In (D) PD patients plasma (Lanes 1, 5, and 6); age-matched control plasma (Lanes 2, 3, 7, and 8); 6 ng of recombinant human α-syn (Lanes 4 and 9); 3 µg of normal human brain lysates (Lane 10) were used. Lanes 1–4 were probed with FL-140 antibody pre-adsorped with recombinant human α-syn (1 µg α-syn/ml antibody). In (E) plasma from PD patients (Lanes 3, 4, and 7); age-matched control plasma (Lanes 5, 6, 8, and 9); 6 ng of recombinant human α-syn (Lanes 1 and 10); 3 µg of normal human brain lysates (Lanes 2) were used. Lanes 7–10 were probed with N-19 antibody pre-adsorped with recombinant human α-syn (1 µg α-syn/ml antibody).
Fig. 5
Figure 5. Aligned C-terminal amino acid sequences of α-syn, β-syn and γ-syn. Note that only α-syn has two di-acidic motifs of the form Asp-X-Glu (underlined).
