Molecular Mechanism Of Congenital Hereditary Cataract Caused By Mutations In The C-terminal Domain Of ?/?-crystallin

Congenital cataract is one of the leading causes of childhood blindness worldwide, and about one-third of the cases have a genetic origin. Among the familiar cataract, about one-third cases are caused by mutations in crystallin genes. About 50 mutations have been reported in ?/?-crystallins,but only a few mutations have been investigated in detail. ?/?-Crystallins are the predominant structural proteins in the lens, and are highly homologous in their tertiary structures composing four Greek key motif divided into two domains (the N- and C-terminal domains). The major difference between ?- and ?-crystallins is their oligomeric states: P-crystallins are homomers or heteromers,while y-crystallins are monomers. In this research, we studied the molecular mechanisms of several cataract-linked mutations located at the C-terminal domain of ?/?-crystallins via biophysical and structural methods.?B1 has been found to have a role in regulating the size distributions of ?-crystallins in the lens. The most striking feature in ?B1 primary sequence is the long N- and C-terminal extensions.R233H is the only inherited mutation occurred at the C-terminal extension of ?B1. The results herein indicated that the mutation did not affect ?B1 structural stability under neutral conditions, but decreased ?B1 stability under mild acidic conditions. Furthermore, we found that the mutation significantly decreased the stability of ?B1/?A3 hereomer. These results suggested that R233 might be involved in heteromer formation, and play a role in the structural stability of ?B1/?A3 hereomer.PB2 is the most abundant among various crystallins in the lens. Our previous results have indicated that the mutations V187M and R188H decrease ?B2 stability and promote PB2 aggregation. However, it remains elusive whether these two mutations are involved in the core of PB2 aggregates. In this research, acid denaturation was used to separate the effects of mutations on?B2 structure, stability, amorphous aggregation and fibrilization. Our results indicated that V187M did not affect the aggregation mechanism of ?B2, but was more likely to cause cataract by decreasing protein stability. On the contrary, R188H not only decreased protein stability, but also promoted aggregation/fibrilization by regulating the core of aggregates/fibrils.Collaborated with doctors in hospitals, we have identified a novel G129C mutation in ?C-crystallin, which is linked to autosomal dominant congenital nuclear cataract. Our results indicated that the mutation significantly decreased protein stability. Meanwhile, the mutation promotes the accumulation of unfolding intermediates under low guanidine hydrochloride concentrations, which further accelerate the aggregation processes. Cell assays indicated that the aggregates formed under low guanidine hydrochloride concentrations were toxic,which might associate with the progression of congenital cataract.In summary,our results suggested that the C-terminal domain plays a crucial role in the oligomeric equilibrium and structural stability of ?/?-crystallins. The C-terminus might have important roles in regulating the core of ?/?-crystallin aggregates.