Deamidation alters the structure and decreases the stability of human lens βA3-crystallin

Takumi Takata, Julie T. Oxford, Theodore R. Brandon, Kirsten Lampi

Research output: Contribution to journalArticle

37 Citations (Scopus)

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 that of the 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 quasi-elastic light scattering experiments showed that dimer formation was not disrupted, nor did higher-order 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.

Original languageEnglish (US)
Pages (from-to)8861-8871
Number of pages11
JournalBiochemistry
Volume46
Issue number30
DOIs
StatePublished - Jul 31 2007

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Crystallins
Lenses
Light scattering
Cataract
Dimers
Aging of materials
Circular dichroism spectroscopy
Mutagenesis
Denaturation
Elastic scattering
Light
Crystalline Lens
Mutant Proteins
Glutamine
Ultraviolet spectroscopy
Oligomers
Developing countries
Debris
Blindness
Circular Dichroism

ASJC Scopus subject areas

  • Biochemistry

Cite this

Deamidation alters the structure and decreases the stability of human lens βA3-crystallin. / Takata, Takumi; Oxford, Julie T.; Brandon, Theodore R.; Lampi, Kirsten.

In: Biochemistry, Vol. 46, No. 30, 31.07.2007, p. 8861-8871.

Research output: Contribution to journalArticle

Takata, Takumi ; Oxford, Julie T. ; Brandon, Theodore R. ; Lampi, Kirsten. / Deamidation alters the structure and decreases the stability of human lens βA3-crystallin. In: Biochemistry. 2007 ; Vol. 46, No. 30. pp. 8861-8871.
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