The pathogenic mechanisms of recombinant human αB-crystallin proteins generated by site-directed mutagenesis in Escherichia coli

Cataract diseases are known as the most important cause of global blindness. a-crystallin, the most important chaperone of the lenticular tissues, plays a crucial role in the eye lens transparency throughout human life. Missense mutations in the CRYAB gene are associated with congenital cataract and various myopathies, primarily by disrupting protein folding, oligomerization, and stability, which in turn compromises chaperone function and enhances protein aggregation. Four pathogenic mutations in this gene -R69C, D109H, P20R, and A171T-have been reported to cause cataract and myopathy. In recent studies, after introducing these mutations into the CRYAB gene by site-directed mutagenesis, recombinant protein expression in Escherichia coli and subsequent purification, the structural and functional changes in recombinant proteins were analyzed using various techniques. Substitutions in the N-terminal domain (P20R), a-crystallin domain (R69C, D109H), and C-terminal domain (A171T) in CRYAB alter the secondary, tertiary, and oligomeric structures, diminish in vitro and in vivo chaperone-like activity, and enhance amyloidogenic propensity of human aB-crystallin protein. Together, these findings elucidate the molecular mechanisms underlying CRYAB-related cataract and myopathy. They also highlight the value of recombinant protein models and site-directed mutagenesis in understanding genotype–phenotype relationships. Such insights not only deepen knowledge of disease progression but also provide a framework for developing therapeutic strategies aimed at stabilizing mutant proteins, enhancing chaperone function, and reducing aggregation.