Deamidation Alters the Structure and Decreases the Stability of Human Lens βΑ3-Crystallin

Abstract

According to the World Health Organization, cataracts account for half of the blindness in the world, with the majority occurring in developing countries. A cataract is a clouding of the lens of the eye due to light scattering of precipitated lens proteins or aberrant cellular debris. The major proteins in the lens are crystallins and they are extensively deamidated during aging and cataracts. Deamidation has been detected at the domain and monomer interfaces of several crystallins during aging.The purpose of this study was to determine the effects of two potential deamidation sites at the predicted interface of the βA3-crystallin dimer on its structure and stability. The glutamine residues at the reported in vivo deamidation sites of Q180 in the C-terminal domain and at the homologous site Q85 in the N-terminal domain were substituted with glutamic acid residues by site-directed mutagenesis. Far UV and near UV circular dichroism spectroscopy indicated that there were subtle differences in the secondary structure and more notable differences in the tertiary structure of the mutant proteins compared to wild type βA3-crystallin. The Q85E/Q180E mutant also was more susceptible to enzymatic digestion, suggesting increased solvent accessibility. These structural changes in the deamidated mutants led to decreased stability during unfolding in urea and increased precipitation during heat-denaturation. When simulating deamidation at both residues, there was a further decrease in stability and loss of cooperativity. However, multiangle-light scattering and quasielastic light scattering experiments showed that dimer formation was not disrupted, nor did higherorder oligomers form. These results suggest that introducing charges at the predicted domain interface in the βA3 homodimer may contribute to the insolubilization of lens crystallins or favor other, more stable, crystallin subunit interactions.Cataracts account for half of all blindness according to the World Health Organization (1). The greatest incidence is in developing countries. A cataract is opacity within the lens of the eye due to scattering of light by precipitated proteins or by aberrant cellular debris. The major proteins within the lens belong to the α and β/γ-crystallin families. Protein concentrations can reach 400 mg/mL and above in the center of the lens, and it is their ordered packing that is necessary for transparency (2). There is an extremely low rate of turnover of crystallins in differentiated lens cells. Thus, crystallins accumulate modifications due to environmental and metabolic damage during an individual's entire lifetime. This makes the lens an easily accessible tissue to study the effects of post-translational modifications on protein unfolding and aggregation.The major post-translational modifications in lenses are truncation, methylation, oxidation, disulfide bond formation, advanced glycation end-products and deamidation (3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Of these AUTHOR EMAIL ADDRESS lampik@ohsu.edu. NIH Public Access Author M...

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

“…Reported studies investigated the properties of deamidated properties or considered N to D substitution as a missense mutation. These studies have reported loss of stability, activity of proteins, and also increased aggregation properties as shown in crystallins, 7,8,11,12,23,24,[51][52][53] superoxide dismutase, 49 and immunoglobins. 14,34,54 Reported properties of mutC suggest that this protein has retained the secondary and tertiary structure with a partial loss in activity but with a significant improvement in the ability to withstand multiple heat cycles.…”

“…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.…”

“…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.…”

“…5,6 Deamidation results in an addition of negative charge to the residue resulting in alterations in protein interactions, which could affect the activity or refolding. 7,8 Interest in deamidation has gained importance since the extent of deamidation was associated with aging, 9,10 cataract formation, 9,11,12 immune recognition, 13 shelf-life of protein pharmaceuticals such as antibodies, 9,13,14 and so on. Deamidation of asparagines is initiated by the cyclization reaction, which occurs between the carboxyl group of the asparagine and amide nitrogen of carboxyl side amino acid (n11) followed by deamination resulting in the formation of succinimide intermediate.…”

Ultrastructural analysis of damage to nuclear fiber cell membranes in advanced age-related cataracts from India

“…Third, the observed loss of membrane segments gives the crystallins and fragments access routes to the ECS. Fourth, many of the changes to crystallins result in increased acidity (such as deamidation, Takata et al, 2007;Takemoto and Boyle, 1999); the increased negative charge causes the deposition onto the curved surfaces of AQP0 arrays because these have a net positive charge resulting from four surface positive charges per monomer (Harries et al, 2004). Fifth, the localization of deposits within the embryonic nucleus, where access to non-protein substances is limited, raises the probability that the deposits are derived from the most abundant material in the vicinity.…”