Linlin Ding

Crystal structures of truncated alphaA and alphaB crystallins reveal structural mechanisms of polydispersity important for eye lens function.

Small heat shock proteins alphaA and alphaB crystallin form highly polydisperse oligomers that frustrate protein aggregation, crystallization, and amyloid formation. Here, we present the crystal structures of truncated forms of bovine alphaA crystallin (AAC59–163) and human alphaB crystallin (ABC68–162), both containing the C‐terminal extension that functions in chaperone action and oligomeric assembly. In both structures, the C‐terminal extensions swap into neighboring molecules, creating runaway domain swaps. This interface, termed DS, enables crystallin polydispersity because the C‐terminal extension is palindromic and thereby allows the formation of equivalent residue interactions in both directions. That is, we observe that the extension binds in opposite directions at the DS interfaces of AAC59–163 and ABC68–162. A second dimeric interface, termed AP, also enables polydispersity by forming an antiparallel beta sheet with three distinct registration shifts. These two polymorphic interfaces enforce polydispersity of alpha crystallin. This evolved polydispersity suggests molecular mechanisms for chaperone action and for prevention of crystallization, both necessary for transparency of eye lenses.

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.

Age-dependent changes in the heat-stable crystallin, βBp, of the human lens

The present study examined the effects of aging and cataractogenesis on the biochemical properties of the uniquely heat-stable lens crystallin, beta basic principle polypeptide (βBp). Using the techniques of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blot immunoassay, we analyzed cortical and nuclear lens sections from normal lenses of individuals aged 0 to 91 years for βBp content in the soluble fraction, and retention of heat stability with aging. In addition, we compared the characteristics of βBp in cataractous lenses with those of normal lenses of approximately the same age. While βBp is synthesized in new cortical cells throughout life, the βBp of the nucleus, which had been laid down early in life, decreased significantly in both absolute concentration and in its proportion of the total soluble protein fraction during the normal aging process. In addition, posttranslational changes in the nuclear soluble βBp result in a gradual loss of approximately 3000 d in the apparent mass of...

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.

Mechanism of Cataract Formation in αA-crystallin Y118D Mutation

PURPOSE The aim of this study was to elucidate the molecular mechanisms that lead to a dominant nuclear cataract in a mouse harboring the Y118D mutation in the alphaA-crystallin gene. METHODS The physicochemical properties of alpha-crystallin obtained from mouse lenses with the Y118D mutation as well as a recombinant Y118D alphaA-crystallin were studied using gel filtration, two-dimensional (2D) gel electrophoresis, multi-angle light scattering, circular dichroism, fluorescence, and chaperone activities. RESULTS Both native alpha-crystallin from mutant lens and recombinant alphaA-Y118D displayed higher molecular mass distribution than the wild-type. Circular dichroism spectra indicated changes in the secondary structures of alphaA-Y118D. The alphaA-Y118D protein prevented nonspecific protein aggregation more effectively than wild-type alphaA-crystallin. The gel filtration and 2D gel electrophoresis analysis showed a significant reduction of Y118D mutant protein in comparison with wild-type alphaA protein of heterozygous mutant lenses. Quantitative RT-PCR results confirmed a decrease in alphaA and alphaB transcripts in the homozygous mutant alpha A(Y118D/Y118D) lenses. CONCLUSIONS The alphaA-Y118D mutant protein itself displays an increased chaperone-like activity. However, the dominant nuclear cataract is associated with a significant decrease in the amount of alphaA-crystallin, leading to a reduction in total chaperone capacity needed for maintaining lens transparency.

Vertebrate-like βγ-crystallins in the ocular lenses of a copepod

The diverse crystallins are water-soluble proteins that are responsible for the optical properties of cellular lenses of animal eyes. While all vertebrate lenses contain physiological stress-related α- and βγ-crystallins, some also contain taxon-specific, often enzyme-related crystallins. To date, the α- and βγ-crystallins have been found only in vertebrate lenses. Here we report lenses from an invertebrate, the pontellid copepod Anomalocera ornata, accumulate βγ-crystallin family members as judged by immunocytochemistry, western immunoblotting and microsequencing. Our data provide the first example of βγ-crystallin members in an invertebrate lens, establishing that the use of this protein family as lens crystallins is not confined to vertebrates.

ERRATUM: Vertebrate-like βγ-crystallins in the ocular lenses of a copepod

We previously reported that the ocular lenses of the pontellid copepod Anomalocera ornata possess vertebrate-like β- and γ-crystallins. We cannot repeat our earlier data suggesting that the copepod lens crystallins belong to the β- and γ-crystallin family of proteins. Our new data are consistent with the copepod crystallins being novel proteins.