Deamidation destabilizes and triggers aggregation of a lens protein, βA3‐crystallin

Abstract

Protein aggregation is a hallmark of several neurodegenerative diseases and also of cataracts. The major proteins in the lens of the eye are crystallins, which accumulate throughout life and are extensively modified. Deamidation is the major modification in the lens during aging and cataracts. Among the crystallins, the bA3-subunit has been found to have multiple sites of deamidation associated with the insoluble proteins in vivo. Several sites were predicted to be exposed on the surface of bA3 and were investigated in this study. Deamidation was mimicked by site-directed mutagenesis at Q42 and N54 on the N-terminal domain, N133 and N155 on the C-terminal domain, and N120 in the peptide connecting the domains. Deamidation altered the tertiary structure without disrupting the secondary structure or the dimer formation of bA3. Deamidations in the C-terminal domain and in the connecting peptide decreased stability to a greater extent than deamidations in the N-terminal domain. Deamidation at N54 and N155 also disrupted the association with the bB1-subunit. Sedimentation velocity experiments integrated with high-resolution analysis detected soluble aggregates at 15%-20% in all deamidated proteins, but not in wild-type bA3. These aggregates had elevated frictional ratios, suggesting that they were elongated. The detection of aggregates in vitro strongly suggests that deamidation may contribute to protein aggregation in the lens. A potential mechanism may include decreased stability and/or altered interactions with other b-subunits. Understanding the role of deamidation in the long-lived crystallins has important implications in other aggregation diseases.Keywords: lens; bA3-crystallin; deamidation; protein aggregation; cataract; sedimentation velocity Cataract is the most common cause of preventable blindness in the world (Resnikoff et al. 2004). Age-related cataract is a protein aggregation disease associated with the insolubization of modified proteins in the lens (Harrington et al. 2004). The lens contains a high concentration of proteins that are mostly structural proteins called crystallins. To enable transparency, crystallins form hetero-oligomers that assemble into ordered structures (Delaye and Tardieu 1983). Modifications that disrupt this order most likely contribute to aggregation and precipitation.The proteins from older lenses contain many modifications including truncation, methylation, oxidation, disulfide bond formation, glycation, racemization, and deamidation (Groenen et

An Internal Disulfide Locks a Misfolded Aggregation-Prone Intermediate in Cataract-Linked Mutants of Human Gamma-D Crystallin

“…In line with the multiple causality of the disease, several distinct biochemical perturbations in vitro have been shown to cause crystallin aggregation (54,(62)(63)(64). Cataract-associated point mutations, especially ones mimicking in vivo chemical modifications, have been highly useful in dissecting the mechanism of aggregation (21,39,51) and developing interventions to modify it (65). Synergistic effects are likely among the various modes of modification or damage.…”

“…Passive chaperones, the α-crystallins, are present in lens, but their capacity declines with age, even as their substrates, the βγ-crystallins, accumulate destabilizing chemical modifications (13) due to a variety of environmental damage (17). The result is generation of partially unfolded intermediate conformational states and progressive increase in light scattering (lens turbidity) due to aggregation (18)(19)(20)(21). No long-range structure, amyloid or otherwise, has been found in the cataractous aggregates (22,23), except in certain rare congenital cases (24,25), but disulfide bonds and other covalent modifications are common (13,(26)(27)(28)(29).…”

Engineering deamidation‐susceptible asparagines leads to improved stability to thermal cycling in a lipase

“…Therefore, it is possible that by selectively replacing only those asparagines that are susceptible to deamidation, the thermotolerance of a protein could be improved. Although substantial information is available on deamidation-mediated loss in protein properties, that is, aggregation, stability, activity, most of the mutational studies were limited to site-directed mutagenesis with aspartate 7,23,24 and glutamate 7,25 and rarely glycine, alanine, and serine. 8,26 These studies have focused on explaining functional consequences of deamidation.…”

“…In the published reports, the susceptible asparagines were mostly replaced with aspartic acid. [23][24][25]39 To identify the suitable amino acid replacement at these five positions, a SSM at these five positions was performed. The mutant library generated by SSM was screened by exposing the culture supernatants to 25 C, 85 C, and 95…”

“…Deamidation in bA3 crystallins initiates aggregation and precipitation leading to the formation of cataracts. 7,23,42 Deamidation of long-lived self-proteins may qualitatively or quantitatively affect the spectrum of self-displayed peptides to T cells and thus contributing to exacerbation of auto immune diseases. 43,44 It has also been suggested that in vivo deamidation of proteins could serve as a molecular timer of many important biological events, such as aging.…”