Inhibiting aggregation of alpha-synuclein with human single chain antibody fragments.
The alpha-synuclein protein has been strongly correlated with Parkinson's disease (PD) and is a major component of the hallmark Lewy body aggregates associated with PD. Two different mutations in the alpha-synuclein gene as well as increased gene dosage of wild-type alpha-synuclein all associate with early onset cases of PD; and transgenic animal models overexpressing alpha-synuclein develop PD symptoms. Alpha-synuclein, a natively unfolded protein, can adopt a number of different folded conformations including a beta-sheet form that facilitates formation of numerous aggregated morphologies, including long fibrils, spherical and linear protofibrils, and smaller aggregates or oligomers. The roles of the various morphologies of alpha-synuclein in the progression of PD are not known, and different species have been shown to be toxic. Here we show that single chain antibody fragments (scFv's) isolated from naïve phage display antibody libraries can be used to control the aggregation of alpha-synuclein. We isolated an scFv with nanomolar affinity for monomeric alpha-synuclein (K(D) = 2.5 x 10(-8) M). When co-incubated with monomeric alpha-synuclein, the scFv decreased not only the rate of aggregation of alpha-synuclein, but also inhibited the formation of oligomeric and protofibrillar structures. The scFv binds the carboxyl terminal region of alpha-synuclein, suggesting that perturbation of this region can influence folding and aggregation of alpha-synuclein in vitro along with the previously identified hydrophobic core region of alpha-synuclein (residues 61-95, particularly residues 71-82). Since the scFv has been isolated from an antibody library based on human gene sequences, such scFv's can have potential therapeutic value in controlling aggregation of alpha-synuclein in vivo when expressed intracellularly as intrabodies in dopaminergic neurons.
Emadi
Sharareh
S
Department of Chemical and Materials Engineering, Arizona State University, Tempe, Arizona 85287, USA.
Liu
Ruitian
R
Yuan
Bin
B
Schulz
Philip
P
McAllister
Chad
C
Lyubchenko
Yuri
Y
Messer
Anne
A
Sierks
Michael R
MR
D000595:Amino Acid Sequence; D052160:Benzothiazoles; D001666:Binding Sites, Antibody / Q000235:genetics; D018604:Epitope Mapping; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure; D017912:Inovirus / Q000235:genetics; D007700:Kinetics; D018625:Microscopy, Atomic Force; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors* / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure; D019151:Peptide Library; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000648:ultrastructure; D012995:Solubility; D051843:Synucleins; D013844:Thiazoles / Q000378:metabolism; D051844:alpha-Synuclein
D052160:Benzothiazoles; D005456:Fluorescent Dyes; D007135:Immunoglobulin Variable Region; D009419:Nerve Tissue Proteins; D019151:Peptide Library; D011994:Recombinant Proteins; C497604:SNCA protein, human; D051843:Synucleins; D013844:Thiazoles; D051844:alpha-Synuclein; C009462:thioflavin T