title: Cataract-causing αAG98R-crystallin mutant dissociates into monomers having chaperone activity.
abstract: OBJECTIVE
  The G98R mutation in αA-crystallin is associated with autosomal dominant cataract in humans. We have reported that mutant G98R protein has substrate-dependent chaperone activity. Further studies on this G98R mutant protein revealed that mutant protein shows reduced oligomeric stability and accelerated subunit dissociation at a low protein concentration. The purpose of present study was to investigate the chaperone function of dissociated subunits of αAG98R-crystallin.
  
  
  METHODS
  Substitution of glycine with arginine at position 98 in human αA-crystallin was accomplished by site-directed mutagenesis. The recombinant protein was expressed in E .coli cells and purified by chromatographic techniques. Purified αAG98R-crystallin was diluted to a concentration of 0.1 mg/ml in 50 mM phosphate buffer containing 150 mM NaCl (pH 7.2) and incubated at 37 °C for 24 h. The monomeric subunits were isolated from the oligomers through 50 kDa cutoff filters. The monomers were analyzed by SDS-PAGE, mass spectrometry, and circular dichroism spectroscopy and characterized by multi-angle light-scattering methods. Chaperone activity was tested against four client proteins: citrate synthesis, alcohol dehydrogenate, bovine βB2-crystallin and ovotransferrin.
  
  
  RESULTS
  Gel filtration studies showed that αAG98R-crystallin oligomers dissociate readily into monomers. Subunits of αAG98R-crystallin, isolated either by size exclusion chromatography or filtration showed chaperone activity against heat-denatured alcohol dehydrogenase, citrate synthase, bovine βB2-crystallin, and chemically denatured ovatransferrin. SDS-PAGE analysis of the mutant protein incubated at 37 °C for 12 days showed autolysis, which was confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF MS/MS) analysis of αAG98R-crystallin fragments recovered after SDS-PAGE.
  
  
  CONCLUSIONS
  The present study shows that the G98R mutation in αA-crystallin produces unstable oligomers that dissociate into active chaperone subunits. The chaperone activity of the dissociated subunits against four client proteins suggests that the αA-crystallin subunits are the minimal units of chaperone activity.
authors.affiliation: Department of Ophthalmology, University of Missouri, Columbia, MO 65212, USA.
authors.forename: Murugesan
authors.initials: M
authors.lastname: Raju
authors.forename: Puttur
authors.initials: P
authors.lastname: Santhoshkumar
authors.forename: K Krishna
authors.initials: KK
authors.lastname: Sharma
mesh_terms: D001120:Arginine / Q000737:chemistry; D002386:Cataract / Q000235:genetics*; D002845:Chromatography / Q000379:methods; D002850:Chromatography, Gel; D005799:Genes, Dominant; D005998:Glycine / Q000737:chemistry; D006801:Humans; D008027:Light; D013058:Mass Spectrometry / Q000379:methods; D018832:Molecular Chaperones / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D012542:Scattering, Radiation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D038201:alpha-Crystallins / Q000235:genetics*
chemical_list: D018832:Molecular Chaperones; D038201:alpha-Crystallins; D001120:Arginine; D005998:Glycine