Deamidation, but Not Truncation, Decreases the Urea Stability of a Lens Structural Protein, βB1-Crystallin

Kim, Yung Hae; Kapfer, Deborah M.; Boekhorst, Jos; Lubsen, Nicolette H.; Bächinger, Hans Peter; Shearer, Thomas R.; David, Larry L.; Feix, Jimmy B.; Lampi, Kirsten J.

doi:10.1021/bi026288h

Cited by 72 publications

(89 citation statements)

References 34 publications

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“…These CD spectra are in agreement with the results previously reported by Lampi et al on B1 (19) and Kim et al on B1∆N41 (23). Since it is the peptide backbone that absorbs in the far-UV range, truncation of 41 amino acids is expected to affect the value of the molar ellipticity.…”

Section: Resultssupporting

confidence: 92%

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Oligomerization and Phase Transitions in Aqueous Solutions of Native and Truncated Human βB1-Crystallin

Annunziata

Pande

et al. 2005

Biochemistry

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Human B1-crystallin is a major eye-lens protein that undergoes in vivo truncation at the N-terminus with aging. By studying native B1 and truncated B1∆N41, which mimics an age-related in vivo truncation, we have determined quantitatively the effect of truncation on the oligomerization and phase transition properties of B1 aqueous solutions. The oligomerization studies show that the energy of attraction between the B1∆N41 proteins is about 10% greater than that of the B1 proteins. We have found that B1∆N41 aqueous solutions undergo two distinct types of phase transitions. The first phase transition involves an initial formation of thin rodlike assemblies, which then evolve to form crystals. The induction time for the formation of rodlike assemblies is sensitive to oligomerization. The second phase transition can be described as liquid-liquid phase separation (LLPS) accompanied by gelation within the protein-rich phase. We refer to this process as heterogeneous gelation. These two phase transitions are not observed in the case of B1 aqueous solutions. However, upon the addition of poly(ethylene glycol) (PEG), we observe heterogeneous gelation also for B1. Our PEG experiments allow us to estimate the difference in phase separation temperatures between B1 and B1∆N41. This difference is consistent with the increase in energy of attraction found in our oligomerization studies. Our work suggests that truncation is a cataractogenic modification since it favors protein condensation and the consequent formation of light scattering elements, and highlights the importance of the N-terminus of B1 in maintaining lens transparency.

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Section: Resultssupporting

confidence: 92%

“…However, circular dichroism and fluorescence studies on B1 have shown that truncation does not affect the unfolding/ refolding properties of the protein (23).…”

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confidence: 99%

Oligomerization and Phase Transitions in Aqueous Solutions of Native and Truncated Human βB1-Crystallin

Annunziata

Pande

et al. 2005

Biochemistry

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“…Simulating deamidation in the C-terminal domain at Q204 in βB1 homologous to Q162 in βB2 was associated with a decrease in stability of the N-terminal domain (20).…”

Section: Effects Of Deamidation On βB2 Stabilitymentioning

confidence: 98%

Deamidation in Human Lens βB2-Crystallin Destabilizes the Dimer

et al. 2006

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Two major determinants of the transparency of the lens are protein-protein interactions and stability of the crystallins, the structural proteins in the lens. βB2 is the most abundant β-crystallin in the human lens and is important in formation of the complex interactions of lens crystallins. βB2 readily forms a homodimer in vitro, with interacting residues across the monomer-monomer interface conserved among β-crystallins. Due to their long life spans, crystallins undergo an unusually large number of modifications, with deamidation being a major factor. In this study the effects of two potential deamidation sites at the monomer-monomer interface on dimer formation and stability were determined. Glutamic acid substitutions were constructed to mimic the effects of previously reported deamidations at Q162 in the C-terminal domain and at Q70, its N-terminal homologue. The mutants had a nativelike secondary structure similar to that of wild type βB2 with differences in tertiary structure for the double mutant, Q70E/Q162E. Multiangle light scattering and quasi-elastic light scattering experiments showed that dimer formation was not interrupted. In contrast, equilibrium unfolding and refolding in urea showed destabilization of the mutants, with an inflection in the transition of unfolding for the double mutant suggesting a distinct intermediate. These results suggest that deamidation at critical sites destabilizes βB2 and may disrupt the function of βB2 in the lens.Cataract formation is the leading cause of blindness worldwide and affects an estimated 16 to 20 million people (1). Crystallins are highly soluble structural proteins that comprise 90% of lens proteins (2). The highly ordered, tightly packed crystallins make up the transparent structure of the lens and allow it to focus light onto the retina. These proteins undergo little turnover during the human life span, allowing for accumulation of modifications that may decrease crystallin solubility, alter lens transparency, and diminish vision.During normal aging, crystallins are modified by truncation, oxidation, deamidation, and disulfide bond formation (3-9). Most notable by two-dimensional gel electrophoresis are trains of deamidated species (3). Each of the major crystallin polypeptides in the lens is deamidated during aging, and most are deamidated at multiple residues. Deamidation introduces a negative charge at physiological pH by replacing an amide with a carboxyl group and causes some isomerization. These changes potentially disrupt protein structure.Recently, 34 sites of deamidations have been identified in a single cataractous lens (10). Due to the difficulty of detecting a single mass unit change resulting from deamidation, quantitative data associating deamidation with cataract formation is limited. The large numbers of † Funding provided by the National Institutes of Health, National Eye Institute, Grant Reference No. R01-EY012239 (K.J.L.), Oregon Lions Sight and Hearing Foundation grant (K.J.L.), Shriners Hospitals Shared Facilities Grant, a...

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Section: Effect Of Deamidation On βA3-crystallin Structurementioning

confidence: 99%

“…Thus, Trp emission strongly contributed to the emission at 285 m (Figure 8). The maximum intensity for β B1 was at 336 nm and for β B2 at 227 nm, reflecting the fewer exposed Trp (35,38,47), with PB2 having only one exposed and one partially buried Trp (47).Simulating deamidation in vitro at the predicted domain interface of βA3 did not dramatically alter secondary structure as indicated by only slight differences in the far-UV CD. There were Takata et al…”

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confidence: 97%

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

et al. 2007

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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...

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Deamidation, but Not Truncation, Decreases the Urea Stability of a Lens Structural Protein, βB1-Crystallin

Cited by 72 publications

References 34 publications

Oligomerization and Phase Transitions in Aqueous Solutions of Native and Truncated Human βB1-Crystallin

Oligomerization and Phase Transitions in Aqueous Solutions of Native and Truncated Human βB1-Crystallin

Deamidation in Human Lens βB2-Crystallin Destabilizes the Dimer

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

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