Ca2+ Binding to α-Synuclein Regulates Ligand Binding and Oligomerization
2001; Elsevier BV; Volume: 276; Issue: 25 Linguagem: Inglês
10.1074/jbc.m101181200
ISSN1083-351X
AutoresMorten S. Nielsen, Henrik Vorum, Evo Lindersson, Poul Henning Jensen,
Tópico(s)Neuroscience and Neuropharmacology Research
Resumoα-Synuclein is a protein normally involved in presynaptic vesicle homeostasis. It participates in the development of Parkinson's disease, in which the nerve cell lesions, Lewy bodies, accumulate α-synuclein filaments. The synaptic neurotransmitter release is primarily dependent on Ca2+-regulated processes. A microdialysis technique was applied showing that α-synuclein binds Ca2+ with an IC50 of about 2–300 μm and in a reaction uninhibited by a 50-fold excess of Mg2+. The Ca2+-binding site consists of a novel C-terminally localized acidic 32-amino acid domain also present in the homologue β-synuclein, as shown by Ca2+binding to truncated recombinant and synthetic α-synuclein peptides. Ca2+ binding affects the functional properties of α-synuclein. First, the ligand binding of 125I-labeled bovine microtubule-associated protein 1A is stimulated by Ca2+ ions in the 1–500 μm range and is dependent on an intact Ca2+ binding site in α-synuclein. Second, the Ca2+ binding stimulates the proportion of125I-α-synuclein-containing oligomers. This suggests that Ca2+ ions may both participate in normal α-synuclein functions in the nerve terminal and exercise pathological effects involved in the formation of Lewy bodies. α-Synuclein is a protein normally involved in presynaptic vesicle homeostasis. It participates in the development of Parkinson's disease, in which the nerve cell lesions, Lewy bodies, accumulate α-synuclein filaments. The synaptic neurotransmitter release is primarily dependent on Ca2+-regulated processes. A microdialysis technique was applied showing that α-synuclein binds Ca2+ with an IC50 of about 2–300 μm and in a reaction uninhibited by a 50-fold excess of Mg2+. The Ca2+-binding site consists of a novel C-terminally localized acidic 32-amino acid domain also present in the homologue β-synuclein, as shown by Ca2+binding to truncated recombinant and synthetic α-synuclein peptides. Ca2+ binding affects the functional properties of α-synuclein. First, the ligand binding of 125I-labeled bovine microtubule-associated protein 1A is stimulated by Ca2+ ions in the 1–500 μm range and is dependent on an intact Ca2+ binding site in α-synuclein. Second, the Ca2+ binding stimulates the proportion of125I-α-synuclein-containing oligomers. This suggests that Ca2+ ions may both participate in normal α-synuclein functions in the nerve terminal and exercise pathological effects involved in the formation of Lewy bodies. Parkinson's disease bis(sulfosuccinimidyl)suberate α-synuclein microtubule-associated protein Parkinson's disease (PD)1 and other common neurodegenerative disorders, e.g. dementia with Lewy bodies and the Lewy body variant of Alzheimer's disease, are characterized by the development of the proteinaceous inclusions called Lewy bodies in the degenerating nerve cells (1McKeith I.G. Perry E.K. Perry R.H. Neurology. 1999; 53: 902-905Crossref PubMed Google Scholar). Lewy bodies comprise α-synuclein (AS)-containing filaments, and purified AS readily forms amyloid-like filaments in vitro (2Spillantini M.G. Schmidt M.L. Lee W.M. Trojanowski J.Q. Jakes R. Goedert M. Nature. 1997; 388: 839-840Crossref PubMed Scopus (5871) Google Scholar, 3Baba M. Nakajo S. Tu P.H. Tomita T. Nakaya K. Lee V.M. Trojanowski J.Q. Iwatsubo T. Am. J. Pathol. 1998; 152: 879-884PubMed Google Scholar, 4Crowther R.A. Jakes R. Spillantini M.G. Goedert M. FEBS Lett. 1998; 436: 309-312Crossref PubMed Scopus (343) Google Scholar, 5Conway K.A. Lee S.J. Rochet J.C. Ding T.T. Williamson R.E. Lansbury Jr., P.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 571-576Crossref PubMed Scopus (1305) Google Scholar). Moreover, missense mutations in the AS gene cause heritable autosomal dominant PD (6Polymeropoulos M.H. Lavedan C. Leroy E. Ide S.E. Dehejia A. Dutra A. Pike B. Root H. Rubenstein J. Boyer R. Stenroos E.S. Chandrasekharappa S. Athanassiadou A. Papapetropoulos T. Johnson W.G. 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Kauffmann S. Hofele K. Spooren W.P. Ruegg M.A. Lin S. Caroni P. Sommer B. Tolnay M. Bilbe G. J. Neurosci. 2000; 20: 6021-6029Crossref PubMed Google Scholar, 11Feany M.B. Bender W.W. Nature. 2000; 404: 394-398Crossref PubMed Scopus (1659) Google Scholar). It has been proposed that the pathogenic mechanisms triggered by AS rely on structural changes occurring during the transition from the monomeric to the β-folded filamentous state (12Goldberg M.S. Lansbury Jr., P.T. Nat. Cell Biol. 2000; 2: E115-E119Crossref PubMed Scopus (440) Google Scholar). AS is a member of the synuclein family, which, in man, is dominated by α-, β-, and γ-synuclein (13Duda J.E. Lee V.M. Trojanowski J.Q. J. Neurosci. Res. 2000; 61: 121-127Crossref PubMed Scopus (247) Google Scholar). The synucleins are acidic proteins of about 140 amino acids that display a "natively unfolded" structure (14Weinreb P.H. Zhen W. Poon A.W. Conway K.A. Lansbury P.T. Biochemistry. 1996; 35: 13709-13715Crossref PubMed Scopus (1283) Google Scholar). The N-terminal part of the proteins is highly conserved and contains several KTKEGV consensus repeats, whereas the C-terminal portion is less well conserved and possesses no known structural elements (15Clayton D.F. George J.M. J. Neurosci. Res. 1999; 58: 120-129Crossref PubMed Scopus (373) Google Scholar). The differences in its primary structure are reflected in segregated functional domains, e.g. brain vesicles bind to the N-terminal part, whereas the microtubule-associated proteins tau and microtubule-associated protein 1B bind to the C-terminal part (16Jensen P.H. Nielsen M.S. Jakes R. Dotti C. Goedert M. J. Biol. Chem. 1998; 273: 26292-26294Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar, 17Jensen P.H. Hager H. Nielsen M.S. Højrup P. Gliemann J. Jakes R. J. Biol. Chem. 1999; 274: 25481-25489Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 18Jensen P.H. Islam K. Kenney J.M. Nielsen M.S. Power J. Gai W.P. J. Biol. Chem. 2000; 275: 21500-21507Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The AS gene is dispensable for normal development and breeding as demonstrated in AS knockout mice (19Abeliovich A. Schmitz Y. Farinas I. Choi-Lundberg D. Ho W.H. Castillo P.E. Shinsky N. Verdugo J.M. Armanini M. Ryan A. Hynes M. Phillips H. Sulzer D. Rosenthal A. Neuron. 2000; 25: 239-252Abstract Full Text Full Text PDF PubMed Scopus (1366) Google Scholar). However, these mice do exhibit subtle changes in the contents of certain neurotransmitters and in synaptic transmission (19Abeliovich A. Schmitz Y. Farinas I. Choi-Lundberg D. Ho W.H. Castillo P.E. Shinsky N. Verdugo J.M. Armanini M. Ryan A. Hynes M. Phillips H. Sulzer D. Rosenthal A. Neuron. 2000; 25: 239-252Abstract Full Text Full Text PDF PubMed Scopus (1366) Google Scholar), and antisense suppression in primary nerve cell cultures causes a reduced distal pool of synaptic vesicles (20Murphy D.D. Rueter S.M. Trojanowski J.Q. Lee V.M. J. Neurosci. 2000; 20: 3214-3220Crossref PubMed Google Scholar). This indicates that AS plays a role in the cellular signaling events, an observation that is in agreement with biochemical studies demonstrating that AS can affect phospholipase D2 and protein kinases and modulate phosphorylation of nerve cell proteins (17Jensen P.H. Hager H. Nielsen M.S. Højrup P. Gliemann J. Jakes R. J. Biol. Chem. 1999; 274: 25481-25489Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 21Jenco J.M. Rawlingson A. Daniels B. Morris A.J. Biochemistry. 1998; 37: 4901-4909Crossref PubMed Scopus (377) Google Scholar, 22Ostrerova N. Petrucelli L. Farrer M. Mehta N. Choi P. Hardy J. Wolozin B. J. Neurosci. 1999; 19: 5782-5791Crossref PubMed Google Scholar). Ca2+ ions regulate a plethora of cellular processes. This functionality has been refined in neurons, where the propagation of action potentials over long distances and the fine-tuned neurotransmitter release from nerve terminals represent such Ca2+-regulated processes (23Ghosh A. Greenberg M.E. Science. 1995; 268: 239-247Crossref PubMed Scopus (1231) Google Scholar, 24Goda Y. Südhof T.C. Curr. Opin. Cell Biol. 1997; 9: 513-518Crossref PubMed Scopus (60) Google Scholar, 25Berridge M.J. Neuron. 1998; 21: 13-26Abstract Full Text Full Text PDF PubMed Scopus (1725) Google Scholar). The actions of Ca2+ ions are mediated by several mechanisms. The Ca2+-calmodulin complex and its diverse downstream signaling pathways represent common cellular mechanisms (26Chin D. Means A.R. Trends Cell Biol. 2000; 10: 322-328Abstract Full Text Full Text PDF PubMed Scopus (1096) Google Scholar). More neuron-specific Ca2+-regulated proteins are represented by synaptic vesicle-associated proteins and abundant Ca2+-binding neuronal proteins like parvalbumin and calbindin (27Baimbridge K.G. Celio M.R. Rogers J.H. Trends Neurosci. 1992; 15: 303-308Abstract Full Text PDF PubMed Scopus (1185) Google Scholar). The latter group may function as a slow buffer that modulates synaptic plasticity (28Caillard O. Moreno H. Schwaller B. Llano I. Celio M.R. Marty A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13372-13377Crossref PubMed Scopus (295) Google Scholar). The importance of cellular Ca2+ homeostasis is highlighted by the central role of Ca2+ ions in apoptotic processes and neuronal excitotoxicity (29Choi D.W. J. Neurobiol. 1992; 23: 1261-1276Crossref PubMed Scopus (2037) Google Scholar, 30Berridge M.J. Bootman M.D. Lipp P. Nature. 1998; 395: 645-648Crossref PubMed Scopus (1752) Google Scholar). The purpose of the present study was to investigate the binding of Ca2+ to AS to ascertain whether Ca2+ can regulate normal and pathological AS functions. 45Ca and 125I were obtained from Amersham Pharmacia Biotech. All reagents were of analytic grade, unless stated otherwise. The synthetic peptide AS-(109–140), corresponding to amino acid residues 109–140 in human AS, was from Shaefer-N (Copenhagen, Denmark). The novel deletion mutants AS-(1–110) and AS-(1–125) were produced by PCR-based mutagenesis as described previously for AS-(1–95) and AS-(55–140) (18Jensen P.H. Islam K. Kenney J.M. Nielsen M.S. Power J. Gai W.P. J. Biol. Chem. 2000; 275: 21500-21507Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The constructs were verified by DNA sequencing. The mutant proteins were expressed inEscherichia coli and purified essentially as described for wild type AS (31Jensen P.H. Hojrup P. Hager H. Nielsen M.S. Jacobsen L. Olesen O.F. Gliemann J. Jakes R. Biochem. J. 1997; 323: 539-546Crossref PubMed Scopus (134) Google Scholar). The peptides were more than 95% pure as assessed by Coomassie Blue staining (Fig. 2, middle panel, A, inset), and their identities were verified by mass spectrometry (data not shown). Purified bovine microtubule-associated protein (MAP)-1A was kindly provided by Dr. Khalid Islam (32Pedrotti B. Islam K. Biochemistry. 1994; 33: 12463-12470Crossref PubMed Scopus (30) Google Scholar). It consisted essentially of the pure ∼360-kDa heavy chain (Fig. 3 , top panel, inset, lane 1). The MAP-1A was iodinated to a specific activity of about 250 mCi/mg using chloramin T as the oxidizing agent, as described previously for MAP-1B (18Jensen P.H. Islam K. Kenney J.M. Nielsen M.S. Power J. Gai W.P. J. Biol. Chem. 2000; 275: 21500-21507Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The electrophoretic migration of the iodinated MAP-1A consisted of a single slow-migrating band corresponding to the nonlabeled protein (Fig. 3 , top panel, inset, lane 2). All protein concentrations were determined using the Bio-Rad protein assay using bovine serum albumin as standard.Figure 3Ca2+ binding to AS regulates the interaction with MAP-1A. Top panel,recombinant AS, immobilized in microtiter plates, was incubated with 50 pm125I-MAP-1A and increasing concentrations of unlabeled MAP-1A (●) or AS (▴). Theordinate represents the percentage of bound/free (B/F) ligand, and the abcissa represents the concentration of free ligand. The points represent the mean ± 1 S.D. of four replicates in one of four similar experiments. Inset, lane 1, purified bovine MAP-1A (4 μg) was mixed with 10,000 cpm 125I-MAP-1A, resolved by 8–16% reducing SDS-polyacrylamide gel, and stained with Coomassie Blue.Lane 2, autoradiogram of the same gel. The molecular size markers (in kDa) are shown on the left. Middle panel, the Ca2+ dependence of 125I-MAP-1A binding to AS. The binding (as described in the top panel) was determined in the presence of increasing concentrations of Ca2+ ions. The ordinate represents the ratio between bound MAP-1A and the control binding of MAP-1A in the absence of Ca2+ ions, and the abscissa represents the Ca2+ concentration. The points represent the mean ± 1 S.D. of four replicates in one of three similar experiments. Bottom panel, A, effect of Ca2+ and Mg2+ on the binding of 125I-MAP-1A to AS. The binding was determined in the absence (Control) and presence of the indicated concentrations of divalent cations. Bottom panel, B, the binding of 125I-MAP-1A to immobilized full-length AS-(1–140) and the C-terminal-truncated AS peptides 1–125, 1–110, and 1–95 was determined in the presence of 1.5 mm Ca2+. The columns in Aand B represent the mean ± 1 S.D. of four replicates in one of three similar experiments. The equal immobilization of the different AS peptides was verified by their similar binding of the125I-labeled ASY-3 antibody that recognizes the N terminus of AS.View Large Image Figure ViewerDownload Hi-res image Download (PPT) First buffers and protein stock solutions were passed through a Chelex 100 column (Bio-Rad) to remove Ca2+ ions to negligible levels (33Vorum H. Madsen P. Rasmussen H.H. Etzerodt M. Svendsen I. Celis J.E. Honoré B. Electrophoresis. 1996; 17: 1787-1796Crossref PubMed Scopus (24) Google Scholar). Next, solutions containing 1 mm45Ca2+ and different concentrations of unlabeled Ca2+ were prepared with and without a constant concentration of AS-(1–140), AS-(1–125), AS-(1–110), AS-(1–95), β-synuclein, and γ-synuclein. All experiments were performed at 4 °C in a solution containing 150 mm KCl and 20 mm HEPES, pH 7.4. The concentration of the synucleins varied from 20 to 300 μm. Binding was measured by equilibrium dialysis. For equilibrium dialysis, 30-μl plexiglass chambers were used (34Kragh-Hansen U. Vorum H. Clin. Chem. 1993; 39: 202-208Crossref PubMed Scopus (62) Google Scholar). Each chamber was divided into two equal compartments by a cellulose membrane cut from dialysis tubing (Spectrum, Houston, Texas; cutoff, 3,500 Da). The left-side compartments contained 25 μl of calcium-containing samples, with or without the synucleins, and the right-side compartments contained 25 μl of buffer. Control experiments showed that equilibrium was established within 2 h (data not shown). Accordingly, the chambers were emptied after 9 h, before the samples were assayed for radioactivity and protein. The Ca2+ concentration was determined by liquid scintillation counting with a LKB Wallac 1209 Rackbeta counter (Turku, Finland). No quenching of the radioactivity of45Ca2+ by the synucleins was observed. The recovery of 45Ca2+ was 97%, demonstrating that no significant adsorption of calcium to the cellulose membrane or dialysis chamber had occurred. The radioactivity of Ca2+-containing solutions that had not been dialyzed was taken to represent the known concentration of total Ca2+. In the binding experiments, the concentrations of bound and free Ca2+ were calculated by using the radioactivity samples taken from the synuclein-containing chambers (representing bound plus free Ca2+) and the corresponding synuclein-free chambers (representing free Ca2+). The concentrations of the synucleins were measured by spectroscopy at 280 nm using the extinction coefficient calculated for each of them. The protein content in the isolated samples was determined by SDS-polyacrylamide gel electrophoresis and silver staining to assure the absence of degradation and leakage through the membrane. The 125I-MAP-1A binding to AS peptides immobilized in Polysorb microtiter plates (Nunc, Copenhagen, Denmark) was performed essentially as described previously for tau (17Jensen P.H. Hager H. Nielsen M.S. Højrup P. Gliemann J. Jakes R. J. Biol. Chem. 1999; 274: 25481-25489Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). The binding buffer consisted of 150 mm KCl, 20 mm HEPES, pH 7.4, 0.01% extensively dialyzed bovine serum albumin, 0.1 mm EDTA, and 0.1 mm EGTA supplemented with various concentrations of CaCl2 and MgCl2. The even immobilization of the C-terminally truncated AS peptides was verified by their similar specific binding of a 125I-labeled affinity-purified antibody (ASY-3) raised against a synthetic peptide corresponding to the N-terminal 31 residues of AS (data not shown). AS and C-terminal-truncated peptides (1 μm) supplemented with 500 pm of the corresponding 125I-labeled AS were incubated for 2 h at 20 °C in 150 mm KCl, 20 mm 4-morpholinepropanesulfonic acid, pH 7.4, 0.1 mm EDTA, 0.1 mm EGTA, and 0.5 mmdithioerythreitol in the absence and presence of Ca2+. The distribution of monomers, oligomers, and higher aggregates was subsequently stabilized by the addition of a short-length hydrophilic chemical cross-linker, bis(sulfosuccinimidyl)suberate (BS3) (1 mm), for 15 min, and then the cross-linker was quenched by the addition of an equal volume of Tris-containing SDS, dithioerythreitol loading buffer. The samples were subsequently resolved by reducing gradient SDS-polyacrylamide gel electrophoresis followed by visualization by autoradiography. 45Ca2+ equilibrium dialysis was performed to determine whether AS is a Ca2+-binding protein. Fig. 1 demonstrates that human recombinant AS binds Ca2+ with a half-saturation of about 300 μm. The saturation of the binding approaches 0.5 mol Ca2+/mol AS at 1 mm Ca2+, which indicates the presence of a single binding site. Mg2+ (8 mm) fails to inhibit the 45Ca2+tracer binding (1 μm) significantly as compared with the ∼85% inhibition obtained by 1.5 mm unlabeled Ca2+. Hence, the binding site displays a Ca2+selectivity among the dominating intracellular divalent cations (Fig.2, middle panel, A). Truncated recombinant AS peptides with deletions of the N-terminal 29- and 54-amino acid residues and the C-terminal 45-amino acid residues were used for initial localization of the Ca2+-binding site. Fig. 2, middle panel, B demonstrates that only the C-terminal truncation inhibited the binding, whereas the N-terminal truncation has no effect. No inhibition of the Ca2+ binding is observed when testing the mutations causing PD (A30P and A53T) (Fig.2, middle panel, A). Acidic amino acid residues often participate in the binding of Ca2+ ions as noted in the EF-hand, C2-domain and the low-affinity Ca2+-binding sites in S100 proteins (35Branden C. Tooze J. Introduction to Protein Structure. Garland Publishing, New York1991: 22-23Google Scholar, 36Rizo J. Sudhof T.C. J. Biol. Chem. 1998; 273: 15879-15882Abstract Full Text Full Text PDF PubMed Scopus (700) Google Scholar, 37Tjoelker L.W. Seyfried C.E. Eddy R.L. Byers M.G. Shows T.B. Calderon J. Schreiber R.B. Gray P.W. Biochemistry. 1994; 33: 3229-3236Crossref PubMed Scopus (92) Google Scholar), and such residues account for 33% of the C-terminal 45 residues. Fig. 2,top panel, demonstrates a striking identity in the spacing of 10 of the 12 acidic residues in the C-terminal 32 residues of α- and β-synuclein. γ-Synuclein, however, shows no such similarity. The similarity in the spacing of acidic residues is reflected at the functional level, where α- and β-synuclein, but not γ-synuclein, bind Ca2+ (Fig. 2, middle panel, A). The acidic residues in the C terminus of α- and β-synuclein are organized as a tandem repeat of 16 amino acids (Fig. 2, top panel), and the integrity of this structure may be required for the binding of Ca2+ ions. This hypothesis was explored by examining the expression and purification of recombinant truncated AS peptides lacking (i) the C-terminal repeat AS-(1–125) and (ii) both repeats AS-(1–110) and AS-(1–95) (Fig. 2, middle panel, B, inset). Fig. 2, middle panel, B shows that removal of the single C-terminal repeat in AS-(1–125) inhibits the Ca2+ binding to the level of the peptides lacking both repeats or the entire 45 C-terminal residues. Moreover, a synthetic peptide corresponding to the tandem repeat structure, AS-(109–140), binds 45Ca2+ to the same extent as wild type AS (Fig. 2, middle panel, B), and its binding isotherms reveal indistinguishable affinities for Ca2+ (Fig. 2, bottom panel). Accordingly, the C-terminal repeat structure in α- and β-synuclein is necessary and sufficient to bind Ca2+ and represents a bona fide Ca2+-binding domain. The propensity of Ca2+ ions to modulate ligand binding to AS was analyzed in terms of the effect of such binding of (i) the amyloidogenic Aβ(1–40) peptide and (ii) the microtubule-associated proteins tau and MAP-1B. Ca2+ ions have no significant effect on these interactions (data not shown). MAP-1A is a novel AS ligand, as demonstrated by the binding of125I-labeled bovine MAP-1A to immobilized AS (Fig.3, top panel). The association of 50 pm125I-MAP-1A reaches a plateau within 9 h at 4 °C (data not shown), and all incubations are therefore performed for 16 h. The interaction is specific, as demonstrated by the inhibition of 125I-MAP-1A binding by both unlabeled MAP-1A and AS, and it exhibits a high affinity (IC50 ∼ 30 nm; Fig. 3, top panel). The binding of MAP-1A to AS is enhanced by Ca2+ ions (Fig.3, middle panel), and a maximal stimulatory effect of about 90% is obtained at concentrations greater than 0.5 mm(Fig. 3, middle panel), with a half-maximal stimulation at about 0.3 mm Ca2+ (Fig. 3, middle panel). Both Mg2+ and Ca2+ (1.5 mm) stimulate MAP-1A binding to AS, but the effect of Ca2+ alone is about 60% greater than that for Mg2+ ions alone; when combined, their effect is synergistic (Fig. 3, bottom panel, A). Disruption of the Ca2+-binding domain in AS obtained by removal of the C-terminal 15, 30, and 45 amino acid residues completely abrogates the Ca2+-stimulatory effect on MAP-1A binding (Fig. 3,bottom panel, B) and demonstrates that the Ca2+effect was indeed based on the AS moiety. Removal of the Ca2+-binding site in AS increases the binding of MAP-1A to the truncated AS peptide (data not shown), but it abrogates the stimulatory Ca2+ effect (Fig. 3, bottom panel, B). This indicates a negative regulatory effect of the C-terminal segment of AS on the MAP-1B interaction that is alleviated by binding of Ca2+ ions. The stimulatory effect of Mg2+ ions on MAP-1A binding may thus be mediated via the MAP-1A moiety. Many Ca2+ effects are mediated through the binding of Ca2+ to calmodulin, but AS does not bind to calmodulin-Sepharose in either the absence or presence of Ca2 (data not shown). Abnormal filamentous AS is a characteristic of diseased brain tissue, and AS aggregation represents a nucleation-dependent process, where the nucleation by oligomeric AS species may represent a rate-limiting step. We used the short-length hydrophilic chemical cross-linker BS3 to covalently stabilize AS oligomers in the absence and presence of Ca2. This analysis is likely to underestimate the oligomeric content because the cross-linking efficiency is <100%. However, the method has the advantage of visualizing molecules associated through low-affinity interactions. Gel filtration methods and other time-consuming procedures for separating oligomerized and monomeric species may not be able to reveal such interactions due to dissociation during the procedures. No significant AS-(1–140) oligomers are present without cross-linking (Fig. 4). The same applies to AS-(1–125) and AS-(1–110) (data not shown). Supplementing the AS solution with 1 mm BS3 for 15 min before reducing SDS-polyacrylamide gel electrophoresis causes the formation of125I-labeled bands compatible with AS dimers, trimers, and higher oligomers with a higher oligomeric content among the C-terminal-truncated peptides (Fig. 4). Saturation of the Ca2+ binding site (1.5 mm) increases the oligomeric content of AS-(1–140) 2-fold for dimers and 2.5-fold for trimers, and higher aggregates, whereas no Ca2effect is observed for the truncated peptides. The oligomers are not an artifact of the iodination of AS because the distinct oligomeric pattern is absent without the presence of 1 μm unlabeled AS. Accordingly, Ca2+ binding to the C-terminal tandem repeat domain favors the formation of AS oligomers. The present study identifies a novel Ca2+-binding motif in the C terminus of AS. The binding of Ca2+ alters the interactions between AS molecules in the process of oligomerization and between AS and certain nerve cell proteins as exemplified by MAP-1A. AS binds Ca2+ ions selectively as compared with the predominant cytosolic divalent cation Mg2+, which suggests that AS functions can be regulated by Ca2+ ions in a cellular context. Several synuclein genes are expressed in man; the most predominant of these are AS, β-synuclein, and γ-synuclein (15Clayton D.F. George J.M. J. Neurosci. Res. 1999; 58: 120-129Crossref PubMed Scopus (373) Google Scholar). The localization of α- and β-synuclein in normal nervous tissue is restricted to the nerve terminals (38Jakes R. Spillantini M.G. Goedert M. FEBS Lett. 1994; 345: 27-32Crossref PubMed Scopus (879) Google Scholar, 39Iwai A. Masliah E. Yoshimoto M. Ge N. Flanagan L. de Silva H.A. Kittel A. Saitoh T. Neuron. 1995; 14: 467-475Abstract Full Text PDF PubMed Scopus (1095) Google Scholar), in contrast to γ-synuclein, which is localized in the somatodendritic compartment (40Buchman V.L. Adu J. Pinõn L.G.P. Ninkina N.N. Davies A.M. Nat. Neurosci. 1998; 1: 101-103Crossref PubMed Scopus (102) Google Scholar). The nerve terminal localization parallels the Ca2+ binding properties of the proteins because α- and β-synuclein, but not γ-synuclein, bind Ca2+. We therefore wish to suggest a functional significance of Ca2+ binding to AS in the nerve terminals where high local Ca2+ concentrations are reached (41Llinas R. Sugimori M. Silver R.B. Science. 1992; 256: 677-679Crossref PubMed Scopus (699) Google Scholar) and AS regulates complex nerve terminal processes related to neurotransmitter homeostasis and maintenance of the distal pool of synaptic vesicles (19Abeliovich A. Schmitz Y. Farinas I. Choi-Lundberg D. Ho W.H. Castillo P.E. Shinsky N. Verdugo J.M. Armanini M. Ryan A. Hynes M. Phillips H. Sulzer D. Rosenthal A. Neuron. 2000; 25: 239-252Abstract Full Text Full Text PDF PubMed Scopus (1366) Google Scholar, 20Murphy D.D. Rueter S.M. Trojanowski J.Q. Lee V.M. J. Neurosci. 2000; 20: 3214-3220Crossref PubMed Google Scholar). The Ca2+-binding motif is localized to the C-terminal 32 residues of AS and comprises an acidic tandem repeat rich in proline residues. This structure is sufficient and necessary to confer Ca2+ binding activity and requires the presence of both repeats as demonstrated both by the full binding activity of the synthetic peptide AS-(109–140) and the absence of binding to AS-(1–125). The Ca2+-binding domain does not resemble any of the hitherto recognized Ca2+-binding structures such as the EF-hand, the C2-domain, or the less defined low-affinity binding sites in the SH-100 class of proteins, with the exception of the clustering of negatively charged residues (35Branden C. Tooze J. Introduction to Protein Structure. Garland Publishing, New York1991: 22-23Google Scholar, 36Rizo J. Sudhof T.C. J. Biol. Chem. 1998; 273: 15879-15882Abstract Full Text Full Text PDF PubMed Scopus (700) Google Scholar, 37Tjoelker L.W. Seyfried C.E. Eddy R.L. Byers M.G. Shows T.B. Calderon J. Schreiber R.B. Gray P.W. Biochemistry. 1994; 33: 3229-3236Crossref PubMed Scopus (92) Google Scholar). AS is natively unfolded, and circular dichroism spectroscopy does not reveal any structural changes in the absence or presence of Ca2+ (14Weinreb P.H. Zhen W. Poon A.W. Conway K.A. Lansbury P.T. Biochemistry. 1996; 35: 13709-13715Crossref PubMed Scopus (1283) Google Scholar). However, it is not always necessary for Ca2+ ions to cause gross structural changes for functional effects to arise, as shown for the C2A domain in synaptotagmin I, where Ca2+ works as an electrostatic switch that facilitates binding to syntaxin I and acidic phospholipids (42Shao X. Davletov B.A. Sutton R.B. Südhof T.C. Rizo J. Science. 1996; 273: 248-251Crossref PubMed Scopus (292) Google Scholar, 43Davletov B.A. Südhof T.C. J. Biol. Chem. 1993; 268: 26386-26390Abstract Full Text PDF PubMed Google Scholar). The IC50 for the Ca2+ binding to AS is about 300 μm, and Ca2+ concentrations close to this magnitude are only encountered in normal nerve cells close to Ca2+ channels at the plasma membrane during propagation of action potentials and at neurotransmitter release (41Llinas R. Sugimori M. Silver R.B. Science. 1992; 256: 677-679Crossref PubMed Scopus (699) Google Scholar). However, cofactors may increase the Ca2+ affinity and thus increase the potential significance of the Ca2+ binding in analogy with the approximate 1000-fold increase in the apparent Ca2+ affinity of the synaptotagmin C2A domain upon phospholipid binding (43Davletov B.A. Südhof T.C. J. Biol. Chem. 1993; 268: 26386-26390Abstract Full Text PDF PubMed Google Scholar). Candidate cofactors are the kinases casein kinase-1, casein kinase-2, src, and fyn that have been implicated in the phosphorylation of Ser129 and Tyr125 (44Okochi M. Walter J. Koyama A. Nakajo S. Baba M. Iwatsubo T. Meijer L. Kahle P.J. Hass C. J. Biol. Chem. 2000; 275: 390-397Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar, 45Nakamura T. Yamashita H. Takahashi T. Nakamura S. Biochem. Biophys. Res. Commun. 2001; 280: 1085-1092Crossref PubMed Scopus (107) Google Scholar, 46Ellis C.E. Schwartzberg P.L. Grider T.L. Fink D.W. Nussbaum R.L. J. Biol. Chem. 2001; 276: 3879-3884Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Tyr125 is conserved from fish and birds to man. Such phosphorylation will increase the negative charge of the Ca2+-binding domain and thereby potentially increase the Ca2+ affinity. α-Synuclein and β-synuclein are soluble proteins with vesicle-binding properties that are localized to nerve terminals. Their local concentration is very high because they constitute about 0.1% of the total protein in rat brain extracts (47Shibayama-Imazu T. Okahash I. Omata K. Nakajo S. Ochiai H. Nakai Y. Hama T. Nakamura Y. Nakaya K. Brain Res. 1993; 622: 17-25Crossref PubMed Scopus (108) Google Scholar), and this may make them suited to be presynaptic Ca2+ buffers. The Ca2+ binding to the C-terminal domain in AS stimulates binding of the novel ligand MAP-1A, and this domain probably plays a negative regulatory role because its removal increases MAP-1A binding (data not shown) but abrogates the stimulatory Ca2+ effect. MAP-1A belongs to the same group of microtubule-associated proteins as the AS ligands tau and microtubule-associated protein-1B (17Jensen P.H. Hager H. Nielsen M.S. Højrup P. Gliemann J. Jakes R. J. Biol. Chem. 1999; 274: 25481-25489Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 18Jensen P.H. Islam K. Kenney J.M. Nielsen M.S. Power J. Gai W.P. J. Biol. Chem. 2000; 275: 21500-21507Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), and several characteristics favor a physiological interaction between MAP-1A and AS. First, their developmental expression profiles are parallel with a low to absent expression in the fetal period, followed by increased expression during postnatal development (48Hsu L.J. Mallory M. Xia Y. Veinbergs I. Hashimoto M. Yoshimoto M. Thal L.J. Saitoh T. Masliah E. J. Neurochem. 1998; 71: 338-344Crossref PubMed Scopus (154) Google Scholar, 49Petersen K. Olesen O.F. Mikkelsen J.D. Neuroscience. 1999; 91: 651-659Crossref PubMed Scopus (120) Google Scholar, 50Bayer T.A. Jakala P. Hartmann T. Egensperger R. Buslei R. Falkai P. Beyreuther K. Neuroreport. 1999; 10: 2799-2803Crossref PubMed Scopus (53) Google Scholar, 51Fink J.K. Jones S.M. Esposito C. Wilkowski J. Genomics. 1996; 35: 577-585Crossref PubMed Scopus (27) Google Scholar). Second, both proteins are predominantly carried as part of the slow component b of axonal transport, indicating subcellular contacts to the same transporting structures (52Jensen P.H. Li J.-Y. Dahlström A. Dotti C. Eur. J. Neurosci. 1999; 11: 3369-3376Crossref PubMed Scopus (88) Google Scholar, 53Nixon R.A. Fischer I. Lewis S.E. J. Cell Biol. 1990; 110: 437-448Crossref PubMed Scopus (30) Google Scholar). Third, a significant part of the transported proteins is incorporated into stationary axonal structures (52Jensen P.H. Li J.-Y. Dahlström A. Dotti C. Eur. J. Neurosci. 1999; 11: 3369-3376Crossref PubMed Scopus (88) Google Scholar, 53Nixon R.A. Fischer I. Lewis S.E. J. Cell Biol. 1990; 110: 437-448Crossref PubMed Scopus (30) Google Scholar). The functional significance of such a putative interaction remains unsolved, but AS is known to change the functional properties of its ligands (17Jensen P.H. Hager H. Nielsen M.S. Højrup P. Gliemann J. Jakes R. J. Biol. Chem. 1999; 274: 25481-25489Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 21Jenco J.M. Rawlingson A. Daniels B. Morris A.J. Biochemistry. 1998; 37: 4901-4909Crossref PubMed Scopus (377) Google Scholar). AS-containing filaments accumulate in Lewy bodies during the year-long process of neurodegeneration in PD. In vitro, filament formation is a nucleation-dependent process, as demonstrated, where preformed oligomers/filaments can seed the growth of filaments (54Wood S.J. Wypych J. Steavenson S. Louis J.C. Citron M. Biere A.L. J. Biol. Chem. 1999; 274: 19509-19512Abstract Full Text Full Text PDF PubMed Scopus (586) Google Scholar). Accordingly, if oligomer formation represents a rate-limiting step, then even a small increase in their rate of formation, regulated by pathogenic factors, may enhance filament growth significantly (12Goldberg M.S. Lansbury Jr., P.T. Nat. Cell Biol. 2000; 2: E115-E119Crossref PubMed Scopus (440) Google Scholar). Known factors with this property are: (i) AS mutations linked to familial Parkinson's disease (5Conway K.A. Lee S.J. Rochet J.C. Ding T.T. Williamson R.E. Lansbury Jr., P.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 571-576Crossref PubMed Scopus (1305) Google Scholar), and (ii) proteolytic activities directed against the AS C-terminal because C-terminally truncated AS preparations more readily form fibrils (4Crowther R.A. Jakes R. Spillantini M.G. Goedert M. FEBS Lett. 1998; 436: 309-312Crossref PubMed Scopus (343) Google Scholar), contain a higher proportion of oligomers, as revealed by chemical cross-linkers (Fig. 4), and such peptides are recovered from pathological brain tissue and isolated Lewy bodies (3Baba M. Nakajo S. Tu P.H. Tomita T. Nakaya K. Lee V.M. Trojanowski J.Q. Iwatsubo T. Am. J. Pathol. 1998; 152: 879-884PubMed Google Scholar, 18Jensen P.H. Islam K. Kenney J.M. Nielsen M.S. Power J. Gai W.P. J. Biol. Chem. 2000; 275: 21500-21507Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Increased Ca2+ concentrations represent a novel fibrillogenic factor, as demonstrated by the increased oligomeric content upon binding of Ca2+ to the tandem repeat domain in AS. This makes AS resemble synaptotagmin VII whose oligomerization is stimulated by Ca2+(55Fukuda M. Mikoshiba K. J. Biol. Chem. 2000; 275: 28180-28185Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). High levels of AS filaments have been reported in preparations of recombinant protein (56Hashimoto M. Hsu L.J. Sisk A. Xia Y. Takeda A. Sundsmo M. Masliah E. Brain Res. 1998; 799: 301-306Crossref PubMed Scopus (247) Google Scholar). However, this study was performed with prolonged incubation, elevated temperature, and acidic pH as compared with our 2-h incubation at pH 7.4. The low oligomeric content in Ca2+-stimulated wild type AS is, by contrast, in accordance with gel filtration experiments demonstrating oligomers with low solubility (<10%) even after 66 days of incubation at pH 7.4 (5Conway K.A. Lee S.J. Rochet J.C. Ding T.T. Williamson R.E. Lansbury Jr., P.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 571-576Crossref PubMed Scopus (1305) Google Scholar). The inhibitory role of the C-terminal part of AS on fibril formation may rely on an electrostatic repulsion from these negatively charged segments. The molecular mechanism exploited by proteolysis and Ca2+ binding would then be similar because both remove negative charges from the C terminus. Conclusively, our study extends our knowledge of AS functions in relation to both normal and pathological nerve cell paradigms by linking AS functions to the important cellular messenger Ca2+. This may facilitate future studies on the still poorly understood mechanisms underlying the gain in toxic function by AS in neurodegenerative disorders. We thank Dr. Khalil Islam for generously providing purified bovine MAP-1A. We also thank Lis Hygom for excellent technical assistance.
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