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Int J Mol Sci
2022 May 27;2311:. doi: 10.3390/ijms23116058.
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Synuclein Analysis in Adult Xenopus laevis.
Bonaccorsi di Patti MC
,
Angiulli E
,
Casini A
,
Vaccaro R
,
Cioni C
,
Toni M
.
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The α-, β- and γ-synucleins are small soluble proteins expressed in the nervous system of mammals and evolutionary conserved in vertebrates. After being discovered in the cartilaginous fish Torpedo californica, synucleins have been sequenced in all vertebrates, showing differences in the number of genes and splicing isoforms in different taxa. Although α-, β- and γ-synucleins share high homology in the N-terminal sequence, suggesting their evolution from a common ancestor, the three isoforms also differ in molecular characteristics, expression levels and tissue distribution. Moreover, their functions have yet to be fully understood. Great scientific interest on synucleins mainly derives from the involvement of α-synuclein in human neurodegenerative diseases, collectively named synucleinopathies, which involve the accumulation of amyloidogenic α-synuclein inclusions in neurons and glia cells. Studies on synucleinopathies can take advantage of the development of new vertebrate models other than mammals. Moreover, synuclein expression in non-mammalian vertebrates contribute to clarify the physiological role of these proteins in the evolutionary perspective. In this paper, gene expression levels of α-, β- and γ-synucleins have been analysed in the main organs of adult Xenopus laevis by qRT-PCR. Moreover, recombinant α-, β- and γ-synucleins were produced to test the specificity of commercial antibodies against α-synuclein used in Western blot and immunohistochemistry. Finally, the secondary structure of Xenopus synucleins was evaluated by circular dichroism analysis. Results indicate Xenopus as a good model for studying synucleinopathies, and provide a useful background for future studies on synuclein functions and their evolution in vertebrates.
Figure 1. Alignment of syn amino acid sequences. Comparisons among human and Xenopus α- (a), β- (b) and γ- (c) syns, among Xenopus α- and β-syns (d), and among α- and γ-syns (e). (a) Conserved repeats of the apolipoprotein lipid-binding motif [EGS]-KT-K-[EQ]-[GQ]-V-XXXX are underlined. The non-amyloid-component (NAC) region of α-syn, the phosphorylatabletyrosines and serines are highlighted in grey, green and yellow, respectively. The methionines representing binding sites for Mn(II) and other metals are highlighted in red. Negative amino acids in the CT region are indicated in bold. The amino acids that in humans are involved in the pathological mutations linked to Parkinson’s disease are shown in red. The aa stretch GVTAVAQKTVE that is directly involved in the formation of human amyloid fibrils is double underlined. The sequences were aligned with Clustal Omega. Asterisks indicate identity of amino acids; double dots indicate amino acids with the same polarity or size; dots indicate semiconserved substitutions. The epitope recognized by the ab27766 antibody is dotted underlined.
Figure 2. Syn gene expression in the major organs of adult Xenopus. qRT-PCR analysis of α- (a), β- (b) and γ- (c) syn gene expression in the main organs of adult Xenopus. Expression levels were normalized against GAPDH and expressed as fold change relative to brain sample. Br: brain, SC: spinal cord, E: eye, Mu: muscle, He: heart, St: stomach, In: intestine, Li: liver, Sp: spleen Ki: kidney, Lu: lung, Sk: skin.
Figure 3. SDS-PAGE analysis of purified recombinant Xenopus α-syn (Xsynα). (Left panel), purified GST-Xsynα before and after treatment with thrombin. (Right panel), GSH-Sepharose chromatography fractions: GST-Xsynα treated with thrombin, flow-through (FT), Xsynα and GST recovered in the wash and GSH-eluted fractions, respectively; MW: molecular weight markers.
Figure 4. Western blot analysis of α-syn expression by ab27766 antibody. Validation of the antibody on Xenopus (Xα, Xβ, Xγ) and Cyprinus carpio (Cβ and Cγ) recombinant syns (a,b). α-syn expression in the main Xenopus organs (c,h). Red ponceau staining is shown in (a,c,e). α-syn immunolabelling in (b,d,f,g). Negative control (primary antibody omitted) in (h). Br: brain; Ey: eye; He: heart; In: intestine; Ki: kidney; Li: liver; Lu: lung; Mu: skeletal muscle; Ne: nerve; SC: spinal cord; Sk: skin; Sp: spleen; St: stomach; Xα, Xβ and Xγ: Xenopus recombinant α-, β- and γ-syn, respectively; Cβ and Cγ: carp recombinant β- and γ-syn, respectively.
Figure 5. Immunohistochemical analysis of the α-syn distribution. Xenopus brain coronal sections (a–d). Strong α-syn immunoreactivity was found in the interpeduncular nucleus (a,c) and in the visual projections, tractus opticus marginalis (d). Retina (e,f). The strongest α-syn immunoreactivity was found in the thick inner plexiform layer (white arrow) and in the outer plexiform layer (white arrowhead) (e). No immunoreactivity was found in control sections (b,f). The α-syn immunoreactivity was found in motor nerve endings within skeletal muscle (longitudinal (g), and transverse section (h), arrows) and heartmuscle (i,j). α-syn immunolabelled nerve fibres were found also within all layers of the stomach wall (k,l). Some sections have been counterstained with Nuclear Fast Red Solution. IN: interpeduncular nucleus; optma: tractus opticus marginalis. Bar = 100 µm.
Figure 6. Fluorescence spectra of purified recombinant Xenopus syns. Protein concentration was 0.11 mg/mL for α- and β-syn, and 0.24 mg/mL for γ-syn.
Figure 7. CD spectra of purified recombinant Xenopus syns. The proteins were diluted in 10 mM potassium phosphate buffer pH 7, containing 50 mM Na2SO4 (a); SDS was added at 10 mM (b), while CuSO4 was added at 100 µM final concentration (c). The spectra are normalized for protein concentration.
Figure 8. CD spectra of Xenopus α-syn. The protein (10 μM) in 10 mM potassium phosphate buffer pH 7, containing 50 mM Na2SO4, was incubated at 37 °C for the specified times.
Figure 9. Xenopus laevis: a potential model for the study of synucleins.
Figure S1. mRNA and amino acid sequence of Xenopus synucleins available at the NCBI database. The nt sequences of L and S α-syn mRNA are reported in A. The coding regions are represented in bold. The regions against which the forward and reverse RT-PCR primers were designed are highlighted in grey. The deduced aa sequences of L and S α-syn are reported in B. The predicted MW and pI are indicated. The currently available S α-syn mRNA sequence is partial. The sequences were aligned with Clustal Omega at https://www.ebi.ac.uk/Tools/msa/clustalo/. Asterisks indicate identity of amino acids; double dots indicate amino acids with the same polarity or size; dots indicate semiconserved substitutions
Figure S2. mRNA and amino acid sequence of Xenopus synucleins available at the NCBI database. The nt sequences of L and S β-syns mRNA are reported in A. The coding regions are represented in bold. The regions against which the forward and reverse RT-PCR primers were designed are highlighted in grey. The deduced aa sequences of L and S β-syn are reported in B. The predicted MW and pI are indicated. The sequences were aligned with Clustal Omega at https://www.ebi.ac.uk/Tools/msa/clustalo/. Asterisks indicate identity of amino acids; double dots indicate amino acids with the same polarity or size; dots indicate semiconserved substitutions
Figure S3. mRNA and amino acid sequence of Xenopus synucleins available at the NCBI database. The nt sequences of L and S γ-syns mRNA are reported in A. The coding regions are represented in bold. The regions against which the forward and reverse RT-PCR primers were designed are highlighted in grey. The deduced aa sequences of L and S β-syn are reported in B. The predicted MW and pI are indicated. The sequences were aligned with Clustal Omega at https://www.ebi.ac.uk/Tools/msa/clustalo/. Asterisks indicate
identity of amino acids; double dots indicate amino acids with the same polarity or size; dots indicate semiconserved substitutions.
Appel-Cresswell,
Alpha-synuclein p.H50Q, a novel pathogenic mutation for Parkinson's disease.
2013, Pubmed
Appel-Cresswell,
Alpha-synuclein p.H50Q, a novel pathogenic mutation for Parkinson's disease.
2013,
Pubmed
Askanas,
Novel immunolocalization of alpha-synuclein in human muscle of inclusion-body myositis, regenerating and necrotic muscle fibers, and at neuromuscular junctions.
2000,
Pubmed
Binolfi,
Interaction of alpha-synuclein with divalent metal ions reveals key differences: a link between structure, binding specificity and fibrillation enhancement.
2006,
Pubmed
Binolfi,
Site-specific interactions of Cu(II) with alpha and beta-synuclein: bridging the molecular gap between metal binding and aggregation.
2008,
Pubmed
Bisaglia,
A topological model of the interaction between alpha-synuclein and sodium dodecyl sulfate micelles.
2005,
Pubmed
Blum,
Xenopus: An Undervalued Model Organism to Study and Model Human Genetic Disease.
2018,
Pubmed
,
Xenbase
Borodinsky,
Xenopus laevis as a Model Organism for the Study of Spinal Cord Formation, Development, Function and Regeneration.
2017,
Pubmed
,
Xenbase
Braak,
Gastric alpha-synuclein immunoreactive inclusions in Meissner's and Auerbach's plexuses in cases staged for Parkinson's disease-related brain pathology.
2006,
Pubmed
Burré,
Cell Biology and Pathophysiology of α-Synuclein.
2018,
Pubmed
Camponeschi,
Copper(I)-α-synuclein interaction: structural description of two independent and competing metal binding sites.
2013,
Pubmed
Chen,
Recapitulation of zebrafish sncga expression pattern and labeling the habenular complex in transgenic zebrafish using green fluorescent protein reporter gene.
2009,
Pubmed
Chung,
Alpha-synuclein in gastric and colonic mucosa in Parkinson's disease: Limited role as a biomarker.
2016,
Pubmed
Cookson,
The biochemistry of Parkinson's disease.
2005,
Pubmed
Davidson,
Stabilization of alpha-synuclein secondary structure upon binding to synthetic membranes.
1998,
Pubmed
Dikiy,
Folding and misfolding of alpha-synuclein on membranes.
2012,
Pubmed
El-Agnaf,
Aggregates from mutant and wild-type alpha-synuclein proteins and NAC peptide induce apoptotic cell death in human neuroblastoma cells by formation of beta-sheet and amyloid-like filaments.
1998,
Pubmed
Gasser,
Mendelian forms of Parkinson's disease.
2009,
Pubmed
Giasson,
Mutant and wild type human alpha-synucleins assemble into elongated filaments with distinct morphologies in vitro.
1999,
Pubmed
Goedert,
Alpha-synuclein and neurodegenerative diseases.
2001,
Pubmed
Hamilton,
alpha-Synuclein A53T substitution associated with Parkinson disease also marks the divergence of Old World and New World primates.
2004,
Pubmed
Hartman,
Testosterone regulates alpha-synuclein mRNA in the avian song system.
2001,
Pubmed
Hong,
The cDNA cloning and ontogeny of mouse alpha-synuclein.
1998,
Pubmed
Irwin,
Parkinson's disease dementia: convergence of α-synuclein, tau and amyloid-β pathologies.
2013,
Pubmed
Jain,
Comparative Analysis of the Conformation, Aggregation, Interaction, and Fibril Morphologies of Human α-, β-, and γ-Synuclein Proteins.
2018,
Pubmed
Jakes,
Identification of two distinct synucleins from human brain.
1994,
Pubmed
Jao,
Structure of membrane-bound alpha-synuclein from site-directed spin labeling and computational refinement.
2008,
Pubmed
Khounlo,
Membrane Binding of α-Synuclein Stimulates Expansion of SNARE-Dependent Fusion Pore.
2021,
Pubmed
Kiely,
α-Synucleinopathy associated with G51D SNCA mutation: a link between Parkinson's disease and multiple system atrophy?
2013,
Pubmed
Krüger,
Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson's disease.
1998,
Pubmed
Kõressaar,
Primer3_masker: integrating masking of template sequence with primer design software.
2018,
Pubmed
Laemmli,
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
1970,
Pubmed
Larsen,
Threonine 53 in alpha-synuclein is conserved in long-living non-primate animals.
2009,
Pubmed
Lavedan,
The synuclein family.
1998,
Pubmed
Lee-Liu,
The African clawed frog Xenopus laevis: A model organism to study regeneration of the central nervous system.
2017,
Pubmed
,
Xenbase
Lesage,
G51D α-synuclein mutation causes a novel parkinsonian-pyramidal syndrome.
2013,
Pubmed
Li,
Effects of in vitro and in vivo avermectin exposure on alpha synuclein expression and proteasomal activity in pigeons.
2017,
Pubmed
Liu,
[Cloning, subcellular localization and in situ detection of Xenopus laevis beta-synnclein gene].
2011,
Pubmed
,
Xenbase
Ltic,
Alpha-synuclein is expressed in different tissues during human fetal development.
2004,
Pubmed
Lu,
Phosphorylation of α-Synuclein at Y125 and S129 alters its metal binding properties: implications for understanding the role of α-Synuclein in the pathogenesis of Parkinson's Disease and related disorders.
2011,
Pubmed
Luk,
Modeling Lewy pathology propagation in Parkinson's disease.
2014,
Pubmed
Maroteaux,
Synuclein: a neuron-specific protein localized to the nucleus and presynaptic nerve terminal.
1988,
Pubmed
Martínez-Navarrete,
Alpha synuclein gene expression profile in the retina of vertebrates.
2007,
Pubmed
Matsui,
Age- and α-Synuclein-Dependent Degeneration of Dopamine and Noradrenaline Neurons in the Annual Killifish Nothobranchius furzeri.
2019,
Pubmed
Mbefo,
Parkinson disease mutant E46K enhances α-synuclein phosphorylation in mammalian cell lines, in yeast, and in vivo.
2015,
Pubmed
Mehra,
α-Synuclein misfolding and aggregation: Implications in Parkinson's disease pathogenesis.
2019,
Pubmed
Mihajlovic,
Membrane-bound structure and energetics of alpha-synuclein.
2008,
Pubmed
Milanese,
Hypokinesia and reduced dopamine levels in zebrafish lacking β- and γ1-synucleins.
2012,
Pubmed
Narhi,
Both familial Parkinson's disease mutations accelerate alpha-synuclein aggregation.
1999,
Pubmed
Norris,
Alpha-synuclein: normal function and role in neurodegenerative diseases.
2004,
Pubmed
Ostrerova-Golts,
The A53T alpha-synuclein mutation increases iron-dependent aggregation and toxicity.
2000,
Pubmed
Pfefferkorn,
Biophysics of α-synuclein membrane interactions.
2012,
Pubmed
Polymeropoulos,
Mutation in the alpha-synuclein gene identified in families with Parkinson's disease.
1997,
Pubmed
Proukakis,
A novel α-synuclein missense mutation in Parkinson disease.
2013,
Pubmed
Prusiner,
Evidence for α-synuclein prions causing multiple system atrophy in humans with parkinsonism.
2015,
Pubmed
Rospigliosi,
E46K Parkinson's-linked mutation enhances C-terminal-to-N-terminal contacts in alpha-synuclein.
2009,
Pubmed
Sahin,
α-Synucleins from Animal Species Show Low Fibrillation Propensities and Weak Oligomer Membrane Disruption.
2018,
Pubmed
Seleem,
Teratogenicity and neurotoxicity effects induced by methomyl insecticide on the developmental stages of Bufo arabicus.
2019,
Pubmed
Session,
Genome evolution in the allotetraploid frog Xenopus laevis.
2016,
Pubmed
,
Xenbase
Singleton,
alpha-Synuclein locus triplication causes Parkinson's disease.
2003,
Pubmed
Slater,
Xenopus laevis as a model system to study cytoskeletal dynamics during axon pathfinding.
2017,
Pubmed
,
Xenbase
Spillantini,
Parkinson's disease, dementia with Lewy bodies and multiple system atrophy are alpha-synucleinopathies.
1999,
Pubmed
Straka,
Xenopus laevis: an ideal experimental model for studying the developmental dynamics of neural network assembly and sensory-motor computations.
2012,
Pubmed
,
Xenbase
Sulzer,
The physiological role of α-synuclein and its relationship to Parkinson's Disease.
2019,
Pubmed
Sun,
Discovery and characterization of three novel synuclein genes in zebrafish.
2008,
Pubmed
Sung,
Secondary structure and dynamics of micelle bound beta- and gamma-synuclein.
2006,
Pubmed
Tiunova,
Chicken synucleins: cloning and expression in the developing embryo.
2000,
Pubmed
Toni,
Fish Synucleins: An Update.
2015,
Pubmed
Toni,
Metal Dyshomeostasis and Their Pathological Role in Prion and Prion-Like Diseases: The Basis for a Nutritional Approach.
2017,
Pubmed
Toni,
Synuclein expression in the lizard Anolis carolinensis.
2016,
Pubmed
Tulumello,
SDS micelles as a membrane-mimetic environment for transmembrane segments.
2009,
Pubmed
Uversky,
Biophysical properties of the synucleins and their propensities to fibrillate: inhibition of alpha-synuclein assembly by beta- and gamma-synucleins.
2002,
Pubmed
Uéda,
Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease.
1993,
Pubmed
Vaccaro,
Localization of α-synuclein in teleost central nervous system: immunohistochemical and Western blot evidence by 3D5 monoclonal antibody in the common carp, Cyprinus carpio.
2015,
Pubmed
Wakabayashi,
Involvement of the peripheral nervous system in synucleinopathies, tauopathies and other neurodegenerative proteinopathies of the brain.
2010,
Pubmed
Wang,
Characterization of three synuclein genes in Xenopus laevis.
2011,
Pubmed
,
Xenbase
Waxman,
Molecular mechanisms of alpha-synuclein neurodegeneration.
2009,
Pubmed
Weinreb,
NACP, a protein implicated in Alzheimer's disease and learning, is natively unfolded.
1996,
Pubmed
Yang,
Distribution of the Alpha-Synuclein in the Brain and the Primary Organs of the Rhesus Monkey.
2021,
Pubmed
Yuan,
Beta-synuclein protein from Xenopus laevis: overexpression in Escherichia coli of the GST-tagged protein and production of polyclonal antibodies.
2007,
Pubmed
,
Xenbase
Zarranz,
The new mutation, E46K, of alpha-synuclein causes Parkinson and Lewy body dementia.
2004,
Pubmed
