Biochemical Analysis And Molecular Pathogenisis Of Acidic Beta-crystallin 4 G64W Mutant

Cataract refers to the general term for opacity of the lens caused by various causes.Among them congenital cataract refers specifically to the cataract formed during the fetal period.There are about 1.5 million new blind children in the world each year.About 3/4 of them are in developing countries.The economic and social burden caused by child blindness is much higher than that of adult blindness,and 1/3 child blindness is caused by congenital cataracts.The lens is a biconvex,transparent tool used to refract light.Due to the lack of blood vessels in the lens,the high stability of the crystallins is particularly important to maintain high transparency and normal refractive power of the lens.The lens protein can be classified into a-crystallin,?-crystallin,and y-crystallin depending on the molecular weight of the protein under physiological conditions.Among them P-crystallin can be further divided into acidic P-crystallin(?A1,?A2,?A3,?A4)and basic? crystallin(?B1,?B2,?B3)according to the isoelectric point.Studies have shown that congenital cataracts account for about one-third of the total number of cases.Acidic ?-crystallin 4 consists of 196 amino acids and is one of the structural proteins in the lens.Several papers confirmed four acidic ?-crystallin 4 mutants that can cause congenital cataracts,namely(c.281T>C,p.F94S),(c.206T>C,p.L69P),(c.190G>T,p.G64W)and(c.199T>A,p.Y67N).However,the pathogenic mechanisms of the acidic ?-crystallin 4 mutations,whether the protein stability is changed or the structure is affected or not,are still unclear.Here,G64W mutant was chosen as the research object,the biochemical properties including protein folding and interaction with other proteins for both wild-type protein and G64W mutant were detected.The results were further analyzed to verify the differences between wild-type and G64W mutant.Finally,combined with the analysis of previously available crystal structure and reports,the pathogenic mechanisms of G64W mutant at molecular level were better understood.This thesis study has the following results:1)WT and G64W mutant of CRYBA4 proteins were expressed and purified successfully using an N-terminal maltose binding protein tag.2)G64W mutant reduced the thermal stability of acid ?-crystallin 4 and maked the protein more prone to form aggregates as proved by molecular dynamics simulation,circular dichroism,fluorescence correlation spectroscopy,crosslink studies.3)G64W mutant affected the protein folding of acid ?-crystallin 4 in TEM1-?-lactamase screen system and a-crystallin A,which was reported to have a molecular chaperone-like fuction failed to resucue the G64W mutant from misfolding and precipitation.4)Yeast two hybrid and pull-down assay showed that G64W mutant blocked its interaction with basic ?-crystallin 1 or basic ?-crystallin 2 but maintained the interaction with a-crystallin A.