Abstract
Purpose: :
Bestrophin1 is part of an integral membrane protein complex located in the basolateral membrane of the retinal pigment epithelium. The gene is mutated in Best vitelliform macular dystrophy (VMD), an autosomal dominant macular degeneration with highly variable expressivity and reduced penetrance. Up to now the quaternary structure of the bestrophin-1 protein complexes remains unclear. Sun et al. (PNAS 2002) argued for a tetrameric or heptameric structure whereas Stanton et al (BBA 2006) suggested a dimeric quaternary structure.We sought to extract the bestrophin-1 protein complex under native conditions to investigate whether mutant bestrophin-1 subunits form complexes with wildtype protein subunits to get further information about the function of mutant protein complexes which is not well understood up to now.
Methods: :
MDCK II cells were seeded on transwell filters and cultured in high glucose DMEM supplemented with 10% FCS and 5 times penicillin/streptomycin at 37°C and 5% CO2. Cells were transiently transfected with BEST1 cloned in a pcDNA3(-) vector according to the manufacturers’ instructions at subconfluency. Vectors applied contained wildtype or mutant BEST1 gene. Filters were washed in PBS and lysis buffer was added for 30 min 24 h after transfection. After lysis cells were harvested and protein extraction took place after a modified protocol according to Bordier (JBC 1981). PAGE of the protein extracts was done under native conditions applying different methods (BN-PAGE, CN-PAGE, Na-taurodeoxycholate PAGE).
Results: :
Bestrophin-1 expressed in dimeric complexes in vivo disregard whether mutant or wildtype gene was expressed. Higher molecular weight complexes of unknown composition could be seen in addition to the dimers.
Conclusions: :
Up to now the quaternary structure of the human bestrophin1 protein complex remains unclear. Our results support the structure predicted by Stanton et al. Based on our results we will investigate the preference of mutant forms of bestrophin-1 to form heterodimers or homodimers with the wildtype protein.
Keywords: protein purification and characterization • protein structure/function • retinal pigment epithelium