Abstract
Tissue transglutaminase (tTG) has been implicated in the pathogenesis of Parkinson disease (PD). However, exactly how tTG modulates the structural and functional properties of alpha-synuclein (alpha-syn) and contributes to the pathogenesis of PD remains unknown. Using site-directed mutagenesis combined with detailed biophysical and mass spectrometry analyses, we sought to identify the exact residues involved in tTG-catalyzed cross-linking of wild-type alpha-syn and alpha-syn mutants associated with PD. To better understand the structural consequences of each cross-linking reaction, we determined the effect of tTG-catalyzed cross-linking on the oligomerization, fibrillization, and membrane binding of alpha-syn in vitro. Our findings show that tTG-catalyzed cross-linking of monomeric alpha-syn involves multiple cross-links (specifically 2-3). We subjected tTG-catalyzed cross-linked monomeric alpha-syn composed of either wild-type or Gln --> Asn mutants to sequential proteolysis by multiple enzymes and peptide mapping by mass spectrometry. Using this approach, we identified the glutamine and lysine residues involved in tTG-catalyzed intramolecular cross-linking of alpha-syn. These studies demonstrate for the first time that Gln(79) and Gln(109) serve as the primary tTG reactive sites. Mutating both residues to asparagine abolishes tTG-catalyzed cross-linking of alpha-syn and tTG-induced inhibition of alpha-syn fibrillization in vitro. To further elucidate the sequence and structural basis underlying these effects, we identified the lysine residues that form isopeptide bonds with Gln(79) and Gln(109). This study provides mechanistic insight into the sequence and structural basis of the inhibitory effects of tTG on alpha-syn fibrillogenesis in vivo, and it sheds light on the potential role of tTG cross-linking on modulating the physiological and pathogenic properties of alpha-syn.
Highlights
Tissue transglutaminase has been implicated in the pathogenesis of Parkinson disease (PD)
This hypothesis was initially supported by in vitro studies demonstrating that Tissue transglutaminase (tTG) catalyzes the polymerization of the ␣-syn-derived non-amyloid component (NAC) peptide via intermolecular covalent cross-linking of residues Gln79 and Lys80 [21] and by other studies suggesting that tTG promotes the fibrillization of amyloidogenic proteins implicated in the pathogenesis of other neurodegenerative diseases such as Alzheimer disease, supranuclear palsy, Huntington disease, and other polyglutamine diseases [22,23,24]
Tissue transglutaminase cross-links a wide range of protein substrates in vitro, including several proteins implicated in the pathogenesis of neurodegenerative diseases, such as A [31], Tau [32, 33], and amyloid precursor protein [34], which are linked to Alzheimer disease; ␣-syn [21, 25, 26], which is linked to Parkinson disease; and polyglutamine domains [35,36,37], which are linked to Huntington disease and related polyglutamine disorders
Summary
Tissue transglutaminase (tTG) has been implicated in the pathogenesis of Parkinson disease (PD). We have identified the primary glutamine and lysine residues involved in tTG-catalyzed, intramolecularly cross-linked monomeric ␣-syn and investigated how crosslinking these residues affects the oligomerization, fibrillization, and membrane binding of ␣-syn in vitro.
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