Qingling Huang

Disruption of α3 Connexin Gene Leads to Proteolysis and Cataractogenesis in Mice

Abstract Gap junction channels formed by α 3 (Cx46) and α 8 (Cx50) connexin provide pathways for communication between the fiber cells in the normal transparent lens. To determine the specific role of α 3 connexin in vivo, the α 3 connexin gene was disrupted in mice. Although the absence of α 3 connexin had no obvious influence on the early stages of lens formation and the differentiation of lens fibers, mice homozygous for the disrupted α 3 gene developed nuclear cataracts that were associated with the proteolysis of crystallins. This study establishes the importance of gap junctions in maintaining normal lens transparency by providing a cell–cell signaling pathway or structural component for the proper organization of lens membrane and cytoplasmic proteins.

LENS ALPHA -CRYSTALLIN : CHAPERONE-LIKE PROPERTIES

Publisher Summary The chapter presents a study on the chaperone-like properties of lens α -crystalline. The most common source for lens α -crystallin has been cow or calf lens. The chapter presents a protocol for preparing calf or cow lens α -crystallin as well as the preparation and purification of recombinant human α B-crystallin. Several assays for the chaperone-like properties of α -crystallin are presented. α -crystallin is one of the abundant structural proteins of the vertebrate eye lens, where it can account for about 40% of the total soluble mass. The a-crystallin family consists of two genes, α A and α B. α B-crystallin has now been found in numerous tissues of the body, such as heart, skeletal muscle, brain, lung, skin, and kidney. α A-crystallin is much less abundant outside the eye lens. α B-crystallin is overexpressed in many degenerative diseases. The biochemical, biophysical, gene regulation, expression, and evolutionary properties of α -crystallin have been studied extensively. Recombinant α -crystallin has properties similar to those of native α -crystallin isolated from eye lens.

Mutation of αB-crystallin: effects on chaperone-like activity

A recent paper by Plater et al. [20], showed that the mutation of a single phenylalanine residue F27R in mouse alpha B completely abolished the chaperone-like property of alpha-crystallin when assayed with insulin at 25 degrees C or with gamma-crystallin at 66 degrees C. We have produced the same mutation as well as some additional mutations in human alpha B-crystallin. Our data suggest that the F27R mutation effected the thermal stability of alpha B-crystallin making it unstable at temperatures > or = 60 degrees C. In agreement with the published work, at these temperatures the F27R human recombinant alpha B-crystallin does not protect the target protein from aggregation. When assayed with insulin or alpha-lactalbumin at 25 or 37 degrees C, however, there were no differences in the protective abilities between the native alpha B-crystallin or the F27R mutated human alpha B-crystallin. Several other multiple mutations involving proline residues were also produced. These mutations did not effect the chaperone-like properties of human alpha B-crystallin, but some of them did effect the native molecular weight size as judged by gel filtration chromatography.

Distribution and activity of glutathione-S-transferase in normal human lenses and in cataractous human epithelia

The distribution of glutathione-S-transferase (GST) activity was determined in frozen normal human lenses. The highest activity of GST was found in the peripheral and equatorial regions, whereas the lowest activity was found in the nucleus. Western blot showed that both mu and pi isoenzymes of GST were present in human lenses. This result is similar to that found in rat lenses. In addition, GST activity was analyzed in 50 lens epithelia which were obtained during cataract surgery. Twenty-seven lens epithelia showed no activity. Statistically significant association was found between cortical and mixed cortical--nuclear cataract and loss of GST activity. No association was found between pure nuclear cataract and loss of epithelial GST activity.

Altered Chaperone-like Activity of α-Crystallins Promotes Cataractogenesis

Despite the enormous number of studies demonstrating changes in the chaperone-like activity of α-crystallins in vitro, little is known about how these changes influence life-long lens transparency in vivo. Using the γB-crystallin I4F mutant protein as a target for αA-crystallins, we examined how cataract phenotypes are modulated by interactions between α-crystallins with altered chaperone-like activities and γB-I4F proteins in vivo. Double heterozygous α-crystallin knock-out αA(+/−) αB(+/−) mice with a decreased amount of α-crystallins were used to simulate reduced total α-crystallin chaperone-like activity in vivo. We found that triple heterozygous αA(+/−) αB(+/−) γB(I4F/+) mice developed more severe whole cataracts than heterozygous γB(I4F/+) mice. Thus, total chaperone-like activity of α-crystallins is important for maintaining lens transparency. We further tested whether mutant αA-crystallin Y118D proteins with increased chaperone-like activity influenced the whole cataract caused by the γB-I4F mutation. Unexpectedly, compound αA(Y118D/+) γB(I4F/+) mutant lenses displayed severe nuclear cataracts, whereas the lens cortex remained unaffected. Thus, the synergistic effect of αA-Y118D and γB-I4F mutant proteins is detrimental to the transparency only in the lens core. α-Crystallins with different chaperone-like activities are likely required in the lens cortex and nucleus for maintaining transparency.