Fujii Noriko, Awakura Minori, Takemoto Larry, Inomata Mitsushi, Takata Takumi, Fujii Norihiko, Saito Takeshi
Research Reactor Institute, Kyoto University, Osaka, Japan.
Mol Vis. 2003 Jul 7;9:315-22.
Lens alpha-crystallins, composed of two subunits of alphaA- and alphaB-crystallin, form low molecular weight (LMW) water soluble aggregates with an average molecular mass of approximately 800 kDa. In the intact lens, some of the alpha-crystallins are associated with even larger high-molecular-weight (HMW) aggregates which are thought to be precursors of components found in the water insoluble fraction. Although the mechanism of HMW aggregation and insolubilization are not known, the process is considered to be related to cataract formation. The purpose of the present study is to compare alphaA-crystallins from HMW and LMW forms in order to help understand the mechanism of insolubilization.
HMW and LMW alphaA-crystallins were isolated from lenses of 50 year old and 2 year old human donors and compared with respect to chaperone activity and fluorescence. We also analyzed isomerization and racemization of Asp-58 and Asp-151 residues in alphaA-crystallin from HMW and LMW fractions.
The chaperone activity of HMW alphaA-crystallin was lower than that of LMW alphaA-crystallin. Fluorescence spectra indicated that HMW alphaA-crystallin was more hydrophobic than that of LMW. Isomerization of normal alpha-linkage to abnormal beta-linkage at both Asp-58 and Asp-151 residues markedly increased in HMW alphaA-crystallin compared with that of LMW alphaA-crystallin. The D/L ratio of beta-Asp-58 of either HMW or LMW forms were higher than 1.0, showing that inversion occurred in this site. In addition, the D/L ratio of the Asp-151 residue from HMW alphaA-crystallin was significantly lower than that of the LMW form.
These results were qualitatively the same as those previously found in alphaA-crystallin from total proteins of cataractous versus normal lenses, suggesting that changes in the native structure of alphaA-crystallin associated with the HMW fraction of normal lenses reflect the same changes that occur to a greater degree in total proteins of human cataractous lens.
晶状体α-晶体蛋白由αA-晶体蛋白和αB-晶体蛋白两个亚基组成，形成平均分子量约为800 kDa的低分子量（LMW）水溶性聚集体。在完整的晶状体中，一些α-晶体蛋白与更大的高分子量（HMW）聚集体相关联，这些聚集体被认为是水不溶性部分中成分的前体。尽管HMW聚集和不溶性化的机制尚不清楚，但该过程被认为与白内障形成有关。本研究的目的是比较HMW和LMW形式的αA-晶体蛋白，以帮助理解不溶性化的机制。
从50岁和2岁人类供体的晶状体中分离出HMW和LMWαA-晶体蛋白，并比较其伴侣活性和荧光。我们还分析了HMW和LMW组分中αA-晶体蛋白中Asp-58和Asp-151残基的异构化和消旋化。
HMWαA-晶体蛋白的伴侣活性低于LMWαA-晶体蛋白。荧光光谱表明，HMWαA-晶体蛋白比LMWαA-晶体蛋白更疏水。与LMWαA-晶体蛋白相比，HMWαA-晶体蛋白中Asp-58和Asp-151残基处正常α-键向异常β-键的异构化明显增加。HMW或LMW形式的β-Asp-58的D/L比值均高于1.0，表明该位点发生了反转。此外，HMWαA-晶体蛋白中Asp-151残基的D/L比值明显低于LMW形式。
这些结果在质量上与先前在白内障晶状体与正常晶状体总蛋白中的αA-晶体蛋白中发现的结果相同，表明与正常晶状体HMW部分相关的αA-晶体蛋白天然结构的变化反映了人类白内障晶状体总蛋白中更大程度发生的相同变化。
