The congenital cataract‐causing mutations P20R and A171T are associated with important changes in the amyloidogenic feature, structure and chaperone‐like activity of human αB‐crystallin

Abstract

Cataract is the major reason for human blindness worldwide. α-Crystallin, as a key chaperone of eye lenses, keeps the lenticular tissues in its transparent state over time.In this study, cataract-causing familial mutations, P20R and A171T, were introduced in CRYАB gene. After successful expression in Escherichia coli and subsequent purification, the recombinant proteins were subjected to extensive structural and functional analyses using various spectroscopic techniques, gel electrophoresis, and electron microscopy.The results of fluorescence and Raman assessments suggest important but discreet conformational changes in human αB-Cry upon these cataractogenic mutations.Furthermore, the mutant proteins exhibited significant secondary structural alteration as revealed by FTIR and Raman spectroscopy. An increase in conformational stability was seen in the human αB-Cry bearing these congenital cataractogenic mutations. The oligomeric size distribution and chaperone-like activity of human αB-Cry were significantly altered by these mutations. The P20R mutant protein was observed to loose most of the chaperone-like activity. Finally, these cataractogenic mutant proteins exhibited an increased propensity to form the amyloid fibrils when incubated under environmental stress. Overall, the structural and functional changes in mutated human αB-Cry proteins can shed light on the pathogenic development of congenital cataracts. K E Y W O R D Scataract, chaperone activity, human αB-Cry, mutation, structure 1 | INTRODUCTION α-Crystallin (α-Cry) is a stable β-sheet rich protein with relatively low turnover. It accounts for 40% of the total soluble proteins of the lenticular tissues. This guardian protein of the eye lenses is a member of the small heat shock protein (sHSP) family that binds selectively to the unfolded proteins. α-Cry thus suppresses protein aggregation and keeps the lenticular tissues in its transparent state during aging. [1][2][3][4][5][6][7] In the lenticular tissues, 30 to 40 subunits of α-Cry (αA and αB) assemble in the ratio of 3:1, respectively, and form polydisperse hetero-oligomeric ensembles. [8][9][10] In humans, αA (173 residues) and αB (175 residues) are coded by CRYAA and CRYАB genes which are located on chromosomes 21 and 11, respectively. Human αA and αB share 57% sequence homology [4,11] and both contain three domains. In each subunit the highly conserved central α-Cry domain (ACD) (90-100 residues), characteristic of the sHSP family, links the conformationally flexible N-terminal domain (NTD) to a C-terminal domain (CTD) rich in charged residues. [12,13] While the ACD with its chaperone activity prevents the selfassembly of different protein aggregates, [14] the NTD and CTD maintain the oligomerization and solubility of the client proteins. [15]

Effect of Arginine on Chaperone-Like Activity of HspB6 and Monomeric 14-3-3ζ

“…where I lim is the limiting value of I at t→∞ and D is a constant. Integration of Equation (A1) gives the following expression for the dependence of the light scattering intensity on time [58,59]:…”

“…In the case of more complex mechanisms of aggregation (for example, in the case of the aggregation process involving the stage of protein unfolding proceeding via the dissociative mechanism [61]) parameter n can accept noninteger values. Therefore in the general case we propose to use parameter m instead of parameter n [58,59]:…”