Tissue transglutaminase modulates alpha-synuclein oligomerization

Abstract

We have studied the interaction of the enzyme tissue transglutaminase (tTG), catalyzing cross-link formation between protein-bound glutamine residues and primary amines, with Parkinson's disease-associated alpha-synuclein protein variants at physiologically relevant concentrations. We have, for the first time, determined binding affinities of tTG for wild-type and mutant alpha-synucleins using surface plasmon resonance approaches, revealing high-affinity nanomolar equilibrium dissociation constants. Nanomolar tTG concentrations were sufficient for complete inhibition of fibrillization by effective alpha-synuclein cross-linking, resulting predominantly in intramolecularly cross-linked monomers accompanied by an oligomeric fraction. Since oligomeric species have a pathophysiological relevance we further investigated the properties of the tTG/alpha-synuclein oligomers. Atomic force microscopy revealed morphologically similar structures for oligomers from all alpha-synuclein variants; the extent of oligomer formation was found to correlate with tTG concentration. Unlike normal alpha-synuclein oligomers the resultant structures were extremely stable and resistant to GdnHCl and SDS. In contrast to normal beta-sheet-containing oligomers, the tTG/alpha-synuclein oligomers appear to be unstructured and are unable to disrupt phospholipid vesicles. These data suggest that tTG binds equally effective to wild-type and disease mutant alpha-synuclein variants. We propose that tTG cross-linking imposes structural constraints on alpha-synuclein, preventing the assembly of structured oligomers required for disruption of membranes and for progression into fibrils. In general, cross-linking of amyloid forming proteins by tTG may prevent the progression into pathogenic species.

Figure 1.

tTG concentration-dependent cross-linking of wild-type α-synuclein. Samples of 100 μM α-synuclein incubated with different tTG concentrations at 37°C for 68 h were loaded on a 16.5% SDS-PAGE gel. (A) Lane M: low molecular mass marker; for the other lanes the tTG concentrations are indicated on the top. Arrows A, B, and C indicate specific gel regions described in the text. (B) SDS-PAGE result of progression of 100 μM wild-type α-synuclein cross-linking with 50 nM tTG. Lane Ø: no tTG added; lane M: low molecular mass marker; for the other lanes the incubation times are indicated on the top. Quantitative analysis is shown in Supplemental Figure S1.

Figure 2.

Aggregation of α-synuclein variants in the presence and absence of tTG. (Left panel) ThioT fluorescence assay. Wild-type, A30P, E46K, and A53T α-synuclein were incubated at a concentration of 100 μM without (filled squares), and with 50 nM (filled triangles) and 1.3 μM tTG (open circles). (Right panel) AFM images of α-synuclein aggregation products in the final plateau phase of the Thio T curves. (Left column) In the absence of tTG for wild-type, A30P, E46K, and A53T α-synuclein (A, B, C, and D, respectively). (Right column) After incubation of wild-type, A30P, E46K, and A53T α-synuclein with 1.3 μM tTG (E, F, G, and H, respectively). Image I is a representative example of 100 μM wild-type α-synuclein incubated with 50 nM tTG. Image J: normal wild-type α-synuclein oligomers without tTG. All images are 1.25 × 1.25 μm².

Figure 3.

Characterization of tTG/α-synuclein oligomers. (A) SEC analysis of tTG/α-synucleins reactions with 50 nM (black solid) and 1.3 μM tTG (gray) for 158 h at 37°C; normal monomers (black dashed line). (B) Samples from A without (solid) or with (dashed) 5 M GdnHCl treatment for 30 min at room temperature; gray profiles: 1.3 μM tTG samples from A; black profiles: normal α-synuclein oligomers; (inset) zoom-in on normal oligomer peak eluting at ∼8 mL. All profiles in A and B normalized on area. (C) Fifteen percent SDS-PAGE gel of 1.3 μM tTG/α-synuclein fractions from SEC (A) and comparison with normal α-synuclein monomers and oligomers. Lane M, low molecular mass marker; lane 1, untreated monomers; lane 2, ∼14.6 mL monomeric tTG/α-synuclein fraction; lane 3, ∼12.9 mL intermediate tTG/α-synuclein fraction; lane 4, ∼8 mL oligomeric tTG/α-synuclein fraction; lane 5, normal oligomers. Samples with “+” were heated for 5 min at 95°C before loading. (D) CD spectra of tTG/α-synuclein oligomers and normal α-synuclein oligomers. Black spectra: oligomeric tTG/α-synuclein, SEC fraction eluting at ∼8 mL, before (solid line) and after (dashed line) addition of 0.1% (w/v) SDS. Gray spectrum: normal α-synuclein oligomers.

Figure 4.

Vesicle permeabilization assay. O_n,1: Normal α-synuclein oligomers used at 1 μM; O_t,1, O_t,2, and O_t,4: SEC purified 1.3 μM tTG/α-synuclein oligomers used at 1, 2, and 4 μM, respectively. M_t,4: SEC purified 1.3 μM tTG/α-synuclein monomers used at 4 μM.