title: Deamidation alters interactions of beta-crystallins in hetero-oligomers.
abstract: OBJECTIVE
  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.affiliation: Department of Integrative Biosciences, School of Dentistry, Oregon Health & Science University, Portland, OR 97239-3098, USA.
authors.forename: Takumi
authors.initials: T
authors.lastname: Takata
authors.forename: Luke G
authors.initials: LG
authors.lastname: Woodbury
authors.forename: Kirsten J
authors.initials: KJ
authors.lastname: Lampi
mesh_terms: D019943:Amino Acid Substitution; D002850:Chromatography, Gel; D002942:Circular Dichroism; D018698:Glutamic Acid / Q000737:chemistry; D005973:Glutamine / Q000737:chemistry; D006801:Humans; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D012542:Scattering, Radiation; D038205:beta-Crystallin A Chain / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D038206:beta-Crystallin B Chain / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: C489108:CRYBA1 protein, human; C105601:CRYBB1 protein, human; D038205:beta-Crystallin A Chain; D038206:beta-Crystallin B Chain; C113634:beta-crystallin B2; D005973:Glutamine; D018698:Glutamic Acid