Paradoxical Inhibition of Protein Aggregation and Precipitation by Transglutaminase-catalyzed Intermolecular Cross-linking

Abstract

Cross-linking of proteins catalyzed by tissue transglutaminase has been suggested to play key roles in a variety of cellular events, including cell apoptosis and human pathogenesis (e.g. polyglutamine and Alzheimer diseases). It has often been suggested that tissue transglutaminase enhances aggregation and precipitation of damaged or pathogenic proteins. To ascertain whether this is accurate, we investigated the effects of tissue transglutaminase-catalyzed modulation on the aggregation of structurally damaged and unfolded proteins. Our results indicated that the aggregation and precipitation of some unfolded proteins were inhibited by transglutaminasecatalyzed reaction, although the effect was strongly dependent upon the target protein species. To elucidate the molecular events underlying the inhibitory effect, extensive analysis was performed with regard to reduced ␤-lactoglobulin using a number of techniques, including chromatography and spectroscopy. The results indicated that cross-linking yields high molecular weight soluble polymers but inhibits the growth of insoluble aggregates. The cross-linked ␤-lactoglobulin retained stable secondary structures with a hydrophobic core. We concluded that the transglutaminase-catalyzed intermolecular crosslinking did not necessarily enhance protein aggregation but could sometimes have a suppressive effect. The results of the present study suggested that tissue transglutaminase modifies aggregation and deposition of damaged or pathogenic proteins in vivo in a wide variety of manners depending on the target protein species and solution conditions.

Tissue transglutaminase modulates α‐synuclein oligomerization

“…At physiological conditions inside neurons, a-synuclein concentration is estimated to be 70 to 140 mM (Lodish 2000;Schults 2006), while the neuronal tTG concentration has been reported to be in the nanomolar range (Konno et al 2005b). Thus intramolecularly cross-linked a-synuclein is likely the main reaction product of tTG cross-linking in vivo, consistent with the observation of highly enriched intramolecularly cross-linked a-synuclein monomers together with a small fraction of oligomers in PD substantia nigra compared to control tissue (Andringa et al 2004).…”

“…In contrast to an earlier report (Konno et al 2005a) using micromolar tTG concentrations, we explored the effect of nanomolar tTG concentrations, consistent with cellular tTG levels, on a-synuclein cross-linking (Murtaugh et al 1984;Konno et al 2005b). SDS-PAGE ( Fig.…”

“…Therefore, we have comprehensively and systematically investigated tTG cross-linking of wild-type and disease-related mutant A30P, E46K, and A53T a-synuclein. We evaluated the effect of physiologically relevant (Murtaugh et al 1984;Konno et al 2005b) nanomolar tTG concentrations on cross-linking kinetics, on affected a-synuclein fraction, and on inhibition of aggregation. Surface plasmon resonance (SPR) measurements revealed high-affinity nanomolar equilibrium dissociation constants of tTG for all a-synuclein variants.…”

Transglutaminase Activity as a Possible Molecular Mechanism in the Etiopathogenesis of Neurodegenerative Diseases

“…However, although all these studies suggest the possible involvement of the TGs in the formation of deposits of protein aggregates in neurodegenerative diseases, they do not indicate whether aberrant TG activity per se directly determines the disease progression. For example, several experimental findings reported that TG2 activity in vitro leads to the formation of soluble aggregates of a-synuclein [47] or polyQ proteins [48,49] . To date, as previously reported, at least ten human CAG-expansion diseases have been described (Table 2) [50][51][52][53][54][55][56][57][58][59] and in at least eight of them their neuropathology is caused by the expansion in the number of residues in the polyglutamine domain to a value beyond [35][36][37][38][39][40].…”

Possible Role of the Transglutaminase Activity in Molecular Mechanisms Responsible for Human Neurodegenerative Diseases

“…However, although all these studies suggest the possible involvement of the TGs in the formation of deposits of protein aggregates in neurodegenerative diseases, they do not indicate whether aberrant TG activity per se directly determines the disease progression. For example, several experimental findings reported that TG2 activity in vitro leads to the formation of soluble aggregates of a-synuclein [47] or polyQ proteins [48,49] . To date, as previously reported, at least ten human CAG-expansion diseases have been described (Table 2) [50][51][52][53][54][55][56][57][58][59] and in at least eight of them their neuropathology is caused by the expansion in the number of residues in the polyglutamine domain to a value beyond 35-40.…”