Tissue transglutaminase catalyzes the deamidation of glutamines in lens βB₂- and βB₃-crystallins

Abstract

Tissue transglutaminase (tTG) is a Ca²⁺-dependent enzyme catalyzing the formation of covalent crosslinks between peptide-bound glutamine and lysine residues. Lens crystallins, including αB-crystallin and several β-crystallins, are in vitro substrates for tTG. In both human and bovine fetal lens extracts treated with commercially available guinea pig liver tTG we detected the formation of high molecular weight (HMW) aggregates containing crosslinked βB₂- and βA₃-crystallin. More interestingly, 2D-gel electrophoresis combined with mass spectrometry analysis revealed that glutamines present in the N-terminal arms of βB₂- and βB₃-crystallins deamidate readily in the presence of tTG. We found that both tTG-catalyzed crosslinking and deamidation disrupt the β-crystallin complex, suggesting that these tTG-catalyzed modifications can influence the macromolecular assembly of lens crystallins. These data together suggest that tTG can contribute to the age-related deamidation of glutamine residues of lens crystallins.

Introduction

Due to aging and life long exposure to multiple environmental insults, such as toxic compounds or UV-light, the eye lens with time loses its transparency and refractivity needed to focus light onto the retina, and in turn, cortical cataract may develop (Soderberg, 1990). The refractivity is provided by its three major structural proteins, the α-, β-, and γ-crystallins. The six human β-crystallin gene products βA₁/βA₃, βA₂, βA₄, βB₁, βB₂ and βB₃ (Miesbauer et al., 1993, David et al., 1996, Lampi et al., 1997) make up over a third of the proteins in human eye lens (Lampi et al., 1997, Robinson et al., 2006). Lens proteins do not turn over with age and, in time, undergo extensive posttranslational modifications such as backbone cleavage, oxidation, deamidation and protein crosslinking. Crystallins can be oxidized in a non-enzymatic reaction by UV light generated free radicals (Berry and Truscott, 2001, Korlimbinis et al., 2006), and deamidation of asparagines and glutamines may also take place in spontaneous reactions (Wright, 1991, Robinson and Robinson, 2001, Takemoto et al., 2001). In contrast to asparagines, glutamines are much more resistant to such non-enzymatic deamidation (Wright, 1991, Takemoto et al., 2001). Remarkably though, many crystallin deamidation sites have been found at glutamine residues (Takemoto and Boyle, 1998, Zhang et al., 2003, Lapko et al., 2002, Wilmarth et al., 2006). These modifications may disrupt the native protein structure, which causes light scattering and, in turn, contribute to age-dependent cataract development (Lapko et al., 2002, Lampi et al., 2001, Flaugh et al., 2006, Lampi et al., 2006).

Tissue transglutaminase (tTG, also known as TGase2) (EC 2.3.2.13) has been demonstrated to contribute to cataractogenesis by crosslinking of predominantly β-crystallins in the eye lens (Lorand et al., 1981). The isodipeptide crosslink is catalyzed in a two step Ca²⁺-dependent reaction between the ε-amino group of polypeptide-bound lysines and the γ-carboxamide group of polypeptide-bound glutamines (Fesus and Piacentini, 2002, Lorand and Graham, 2003). A thiolester formation between the active site cysteine and the target glutamine initiates the reaction, while ammonia is released (the acylation step) (Folk, 1983). In the next and rate-limiting transamidation step, the acyl group is transferred to the acyl-acceptor amine (lysine), forming an isopeptide bond (also known as deacylation or crosslinking) (Folk, 1983). However, in the absence of the lysine, at a much slower rate, deamidation of the glutamine can also take place (Folk, 1983, Gorman and Folk, 1984). The substrate preference is not yet fully understood, but a common notion is that tTG is much less selective towards lysine than to glutamine residues.

The crosslinking activity of tTG has been implicated in several physiological processes, including cell motility (Gentile et al., 1992, Akimov et al., 2000), wound healing, extracellular matrix remodeling, differentiation and apoptosis (Griffin and Verderio, 2000, Griffin et al., 2002, Piacentini et al., 2002). As a result of increased tTG activity crosslinked proteins have been found in the detergent-insoluble protein particles in Alzheimer's disease and sporadic inclusion body myositis patients (Choi et al., 2000, Kim et al., 1999). Interestingly, tTG-mediated deamidation of glutamines in gliadin has been suggested to play an important role in celiac disease (van de Wal et al., 1998, Vader et al., 2002), although such deamidation of gliadin has not yet been detected in vivo. More importantly, we have recently shown that tTG deamidates the small heat shock protein (sHsp) Hsp20 with high efficiency at a glutamine site specifically available for deamidation (Boros et al., 2006). In lens, β-crystallins together with the intermediate filament vimentin are the primary targets for tTG-catalyzed crosslinking (Lorand et al., 1987, Groenen et al., 1993, Groenen et al., 1994, Clement et al., 1998). βB₂-, βB₃- and βA₃-crystallins have been identified as potent glutamine substrates for tTG (Berbers et al., 1983, Berbers et al., 1984, Lorand et al., 1985), but βA₃-, together with βB₁- and αB-crystallin also expose lysine-donor sites for tTG (Mulders et al., 1987, Groenen et al., 1992, Lorand et al., 1992, Groenen et al., 1994).

In this study we examined the transglutaminase reactivity of lens proteins. Our findings suggest that guinea pig liver tTG could selectively deamidate βB₂- and βB₃-crystallin in human and bovine fetal lens extracts, whereas βA₃- and βB₁-crystallins are mainly targets for crosslinking. Moreover, these modifications result in the disruption of the β-crystallin complex. In line with these results, we show that upon aging, β-crystallins undergo similar modifications in aging lens.