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D-Crystallin–Associated Protein Aggregation and Lens Fiber Cell Denucleation

¹From the Vision Science Program and School of Optometry, and the ⁴UC Berkeley/UCSF Joint Graduate Program in Bioengineering, University of California, Berkeley, Berkeley, California; the ²Eye Center at Affiliated Second Hospital, Zhejiang University College of Medicine, Hangzhou, China; the ⁵Jules Stein Eye Institute, University of California, Los Angeles, Los Angeles, California; and the ⁶Department of Molecular Biology, The Scripps Research Institute, La Jolla, California.

PURPOSE. To understand the underlying molecular mechanism for a dominant cataract caused by a point mutation in the D-crystallin gene.

METHODS. A dominant cataractous mouse line was identified from chemically induced mouse mutations by phenotypic screening with slit lamp examination. Genomewide linkage analysis and DNA sequencing were used to determine the causative gene mutation. Histology, immunohistochemistry, Western blotting, and in vitro transfection studies were used to characterize mutant lenses.

RESULTS. Cataracts in mutant mice were caused by a point mutation in the D-crystallin gene (D-V76D). Intranuclear -crystallin aggregates, incomplete denucleation, and decreased connexins were observed in mutant lens fiber cells. Mutant D-V76D proteins became less soluble in the lens, and structural modeling suggested that the substituted aspartic acid residue (D) altered hydrogen bond formation and surface electrostatic potential of the protein. Unexpectedly, the formation of cold cataracts, which occurred in wild-type lenses at low temperature, was abolished in D-V76D mutant lenses. In vitro transfection studies revealed that wild-type D proteins were uniformly distributed in the cytosol and nucleus of transfected cells, whereas D-V76D proteins formed cytosolic and nuclear aggregates.

CONCLUSIONS. Mutant D-V76D reduces protein solubility in the lens and forms substantial intranuclear aggregates that disrupt the denucleation process of inner lens fiber cells. Sustained fiber cell nuclei and nuclear remnants scatter light, whereas other downstream events, such as decreased connexins, presumably disrupt gap junction communication and lens homeostasis, further contributing to the cataract phenotype in mutant lenses. This work also suggests that D-crystallin is one of the crucial components for the formation of cold cataracts in vivo.

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