Deamidation alters interactions of β-crystallins in hetero-oligomers

Takumi Takata, Luke G. Woodbury, Kirsten Lampi

Research output: Contribution to journalArticle

27 Citations (Scopus)

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 βA3-crystallin (βA3) dimer decreased the stability of the dimer in vitro. The purpose of the present study was to investigate if deamidation altered the interaction of βA3 with other β-crystallin subunits. Methods: Deamidation was mimicked by replacing glutamines, Q85 and Q180, at the predicted interacting interface between βA3 domains with glutamic acids by site-directed mutagenesis. Human recombinant wild type βA3 or the doubly deamidated mutant βA3 Q85E/Q180E (DM βA3) were mixed with either βB1- or βB2-crystallin (βB1 or βB2) 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 βA3 formed a polydispersed hetero-tetramer with βB1 and a mondispersed hetero-dimer with βB2. Deamidation at the interface in the βA3 dimer decreased formation of the hetero-oligomer with βB1 and further decreased formation of the hetero-dimer with βB2. During thermal-induced denaturation of the deamidated βA3 dimer, βB1 but not βB2 was able to prevent precipitation of βA3. Conclusions: Deamidation decreased formation of hetero-oligomers between β-crystallin subunits. An excess accumulation of deamidated β-crystallins in vivo may disrupt normal protein-protein interactions and diminish the stabilizing effects between them, thus, contributing to the accumulation of insoluble β-crystallins during aging and cataracts.

Original languageEnglish (US)
Pages (from-to)241-249
Number of pages9
JournalMolecular Vision
Volume15
StatePublished - Jan 28 2009

Fingerprint

Crystallins
Cataract
Proteins
Glutamates
Native Polyacrylamide Gel Electrophoresis
Light
Fluorescence Spectrometry
Blindness
Site-Directed Mutagenesis
Glutamine
Lenses
Visual Acuity
Gel Chromatography
Lasers
Hot Temperature
Temperature

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Deamidation alters interactions of β-crystallins in hetero-oligomers. / Takata, Takumi; Woodbury, Luke G.; Lampi, Kirsten.

In: Molecular Vision, Vol. 15, 28.01.2009, p. 241-249.

Research output: Contribution to journalArticle

Takata, Takumi ; Woodbury, Luke G. ; Lampi, Kirsten. / Deamidation alters interactions of β-crystallins in hetero-oligomers. In: Molecular Vision. 2009 ; Vol. 15. pp. 241-249.
@article{7371af5d861246d2bb98d2f8a04991f3,
title = "Deamidation alters interactions of β-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 βA3-crystallin (βA3) dimer decreased the stability of the dimer in vitro. The purpose of the present study was to investigate if deamidation altered the interaction of βA3 with other β-crystallin subunits. Methods: Deamidation was mimicked by replacing glutamines, Q85 and Q180, at the predicted interacting interface between βA3 domains with glutamic acids by site-directed mutagenesis. Human recombinant wild type βA3 or the doubly deamidated mutant βA3 Q85E/Q180E (DM βA3) were mixed with either βB1- or βB2-crystallin (βB1 or βB2) 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 βA3 formed a polydispersed hetero-tetramer with βB1 and a mondispersed hetero-dimer with βB2. Deamidation at the interface in the βA3 dimer decreased formation of the hetero-oligomer with βB1 and further decreased formation of the hetero-dimer with βB2. During thermal-induced denaturation of the deamidated βA3 dimer, βB1 but not βB2 was able to prevent precipitation of βA3. Conclusions: Deamidation decreased formation of hetero-oligomers between β-crystallin subunits. An excess accumulation of deamidated β-crystallins in vivo may disrupt normal protein-protein interactions and diminish the stabilizing effects between them, thus, contributing to the accumulation of insoluble β-crystallins during aging and cataracts.",
author = "Takumi Takata and Woodbury, {Luke G.} and Kirsten Lampi",
year = "2009",
month = "1",
day = "28",
language = "English (US)",
volume = "15",
pages = "241--249",
journal = "Molecular Vision",
issn = "1090-0535",

}

TY - JOUR

T1 - Deamidation alters interactions of β-crystallins in hetero-oligomers

AU - Takata, Takumi

AU - Woodbury, Luke G.

AU - Lampi, Kirsten

PY - 2009/1/28

Y1 - 2009/1/28

N2 - 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 βA3-crystallin (βA3) dimer decreased the stability of the dimer in vitro. The purpose of the present study was to investigate if deamidation altered the interaction of βA3 with other β-crystallin subunits. Methods: Deamidation was mimicked by replacing glutamines, Q85 and Q180, at the predicted interacting interface between βA3 domains with glutamic acids by site-directed mutagenesis. Human recombinant wild type βA3 or the doubly deamidated mutant βA3 Q85E/Q180E (DM βA3) were mixed with either βB1- or βB2-crystallin (βB1 or βB2) 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 βA3 formed a polydispersed hetero-tetramer with βB1 and a mondispersed hetero-dimer with βB2. Deamidation at the interface in the βA3 dimer decreased formation of the hetero-oligomer with βB1 and further decreased formation of the hetero-dimer with βB2. During thermal-induced denaturation of the deamidated βA3 dimer, βB1 but not βB2 was able to prevent precipitation of βA3. Conclusions: Deamidation decreased formation of hetero-oligomers between β-crystallin subunits. An excess accumulation of deamidated β-crystallins in vivo may disrupt normal protein-protein interactions and diminish the stabilizing effects between them, thus, contributing to the accumulation of insoluble β-crystallins during aging and cataracts.

AB - 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 βA3-crystallin (βA3) dimer decreased the stability of the dimer in vitro. The purpose of the present study was to investigate if deamidation altered the interaction of βA3 with other β-crystallin subunits. Methods: Deamidation was mimicked by replacing glutamines, Q85 and Q180, at the predicted interacting interface between βA3 domains with glutamic acids by site-directed mutagenesis. Human recombinant wild type βA3 or the doubly deamidated mutant βA3 Q85E/Q180E (DM βA3) were mixed with either βB1- or βB2-crystallin (βB1 or βB2) 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 βA3 formed a polydispersed hetero-tetramer with βB1 and a mondispersed hetero-dimer with βB2. Deamidation at the interface in the βA3 dimer decreased formation of the hetero-oligomer with βB1 and further decreased formation of the hetero-dimer with βB2. During thermal-induced denaturation of the deamidated βA3 dimer, βB1 but not βB2 was able to prevent precipitation of βA3. Conclusions: Deamidation decreased formation of hetero-oligomers between β-crystallin subunits. An excess accumulation of deamidated β-crystallins in vivo may disrupt normal protein-protein interactions and diminish the stabilizing effects between them, thus, contributing to the accumulation of insoluble β-crystallins during aging and cataracts.

UR - http://www.scopus.com/inward/record.url?scp=59349092370&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=59349092370&partnerID=8YFLogxK

M3 - Article

VL - 15

SP - 241

EP - 249

JO - Molecular Vision

JF - Molecular Vision

SN - 1090-0535

ER -