Deamidation alters interactions of beta-crystallins in hetero-oligomers.

Abstract

PURPOSE:

Cataracts are a major cause of blindness worldwide. A potential mechanism for loss of visual acuity may be due to light scattering from disruption of normal protein-protein interactions. During aging, the lens accumulates extensively deamidated crystallins. We have previously reported that deamidation in the betaA3-crystallin (betaA3) dimer decreased the stability of the dimer in vitro. The purpose of the present study was to investigate if deamidation altered the interaction of betaA3 with other beta-crystallin subunits.

METHODS:

Deamidation was mimicked by replacing glutamines, Q85 and Q180, at the predicted interacting interface between betaA3 domains with glutamic acids by site-directed mutagenesis. Human recombinant wild type betaA3 or the doubly deamidated mutant betaA3 Q85E/Q180E (DM betaA3) were mixed with either betaB1- or betaB2-crystallin (betaB1 or betaB2) subunits. After incubation at increasing temperatures, hetero-oligomers were resolved from individual subunits and their molar masses determined by size exclusion chromatography with in line multiangle laser light scattering. Structural changes of hetero-oligomers were analyzed with fluorescence spectroscopy and blue-native PAGE.

RESULTS:

Molar masses of the hetero-oligomer complexes indicated betaA3 formed a polydispersed hetero-tetramer with betaB1 and a mondispersed hetero-dimer with betaB2. Deamidation at the interface in the betaA3 dimer decreased formation of the hetero-oligomer with betaB1 and further decreased formation of the hetero-dimer with betaB2. During thermal-induced denaturation of the deamidated betaA3 dimer, betaB1 but not betaB2 was able to prevent precipitation of betaA3.

CONCLUSIONS:

Deamidation decreased formation of hetero-oligomers between beta-crystallin subunits. An excess accumulation of deamidated beta-crystallins in vivo may disrupt normal protein-protein interactions and diminish the stabilizing effects between them, thus, contributing to the accumulation of insoluble beta-crystallins during aging and cataracts.

• Authors: Takumi Takata, Luke G Woodbury, Kirsten J Lampi
• Journal: Molecular vision (Mol Vis) Vol. 15 Pg. 241-9 ( 2009) ISSN: 1090-0535 [Electronic] United States
• PMID: 19190732 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• CRYBA1 protein, human
• CRYBB1 protein, human
• beta-Crystallin A Chain
• beta-Crystallin B Chain
• beta-crystallin B2
• Glutamine
• Glutamic Acid

Topics

• Amino Acid Substitution
• Chromatography, Gel
• Circular Dichroism
• Glutamic Acid (chemistry)
• Glutamine (chemistry)
• Humans
• Protein Multimerization
• Protein Structure, Quaternary
• Scattering, Radiation
• beta-Crystallin A Chain (chemistry, genetics, metabolism)
• beta-Crystallin B Chain (chemistry, genetics, metabolism)