ABSTRACT
Studies have established that congenital cataract is the major cause of blindness in children across the globe. The ß-crystallin protein family is the richest and most soluble structural protein in the lens. Their solubility and stability are essential in maintaining lens transparency. In this study, we identified a novel ßB2 mutation W151R in a rare progressive cortical congenital cataract family and explored its pathogenesis using purified protein and mutant related cataract-cell models. Due to its low solubility and poor structural stability, the ßB2 W151R mutation was prone to aggregation. Moreover, the W151R mutation enhanced the exposure of the hydrophobic side chains in the fourth Greek Key motif, which were readily degraded by trypsin. However, upon the administration of lanosterol, the negative effect of the W151R mutation was reversed. Therefore, lanosterol is a potential therapeutic option for cataracts.
Subject(s)
Cataract/congenital , Lanosterol/therapeutic use , Lens, Crystalline/pathology , Protein Aggregation, Pathological/genetics , beta-Crystallin B Chain/genetics , Cataract/drug therapy , Cataract/genetics , Cataract/pathology , Child, Preschool , DNA Mutational Analysis , Female , HEK293 Cells , HeLa Cells , Humans , Hydrophobic and Hydrophilic Interactions/drug effects , Lanosterol/pharmacology , Lens, Crystalline/drug effects , Male , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Mutation , Pedigree , Protein Aggregation, Pathological/congenital , Protein Aggregation, Pathological/drug therapy , Protein Conformation, beta-Strand/drug effects , Protein Conformation, beta-Strand/genetics , Proteolysis/drug effects , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Trypsin/metabolism , beta-Crystallin B Chain/chemistry , beta-Crystallin B Chain/isolation & purification , beta-Crystallin B Chain/metabolismABSTRACT
BetaB2-crystallin, the major subunit of beta-crystallins, is difficult to purify either from lens homogenate or from betaH-or betaL-crystallins. It has been prepared by heterologous expression in Escherichia coli. Most often, the methods used for purifying a recombinant globular protein employ the combination of ion-exchange with gel filtration chromatography. In the case of betaB2-crystallin too, different approaches have been used to obtain the purified protein, majority of which use a combination of ion-exchange and gel filtration chromatography. We present a new approach to purify betaB2-crystallin using hydrophobic interaction chromatography. In this method, the protein is bound to the hydrophobic matrix in the presence of high concentration of a non-chaotropic salt and eluted by decreasing the salt concentration. The method that we have used for the purification of this globular protein has definite advantages over the earlier methods in its simplicity and efficiency. The most noted advantage of this procedure is the rapid purification with a relatively purified product and a comparatively high yield (>20 mg/L of culture). Over all, the present protocol provides a rapid, efficient and simplified procedure for the preparation of betaB2-crystallin in large yield, sufficient for structural and functional studies.
