Aggregation of lens crystallins in an in vivo hyperbaric oxygen guinea pig model of nuclear cataract: Dynamic light-scattering and HPLC analysis
Aggregation of lens crystallins in an in vivo hyperbaric oxygen guinea pig model of nuclear cataract: Dynamic light-scattering and HPLC analysis
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DOI：
10.1167/iovs.05-0843
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发表时间：
2005-12-01
影响因子：
4.4
通讯作者：
Giblin, FJ
中科院分区：
文献类型：
--
作者：
Simpanya, MF;Ansari, RR;Giblin, FJ
目的.在人类核性白内障的发展过程中，氧在透镜高分子量（HMW）蛋白聚集体形成中的作用还不清楚。本研究旨在通过体内和体外方法研究高压氧（HBO）处理后豚鼠透镜晶体蛋白聚集体的形成。用HBO每周3次连续7个月处理豚鼠，并使用动态光散射（DLS）和水不溶性（WI）蛋白的HPLC分析在体内和体外研究透镜晶体蛋白聚集。在豚鼠晶状体的4.5- 5.0-mm光轴上，每隔0.1 mm进行一次DLS测量。HBO处理动物晶状体核（中心区域）中蛋白质的平均表观直径几乎是对照动物的两倍（P < 0.001）。在将蛋白质分为小直径和大直径组后，在核和皮质（透镜的外周）中的一个选定点进行的尺寸分布分析显示，与相同的对照组相比，在O-2处理的核中，大直径聚集蛋白质的强度增加了三倍（P < 0.001），表观尺寸增加了一倍（P = 0.03）。与对照组相比，在O-2处理的皮层中未检测到表观蛋白质直径的显著变化。在O-2处理的细胞核中的单个选定位置处的蛋白质聚集体的平均直径估计为150 nm，该尺寸能够散射光并且与在人类核性白内障中发现的聚集体的尺寸相似。HPLC分析表明，一半的实验核WI蛋白级分（已溶解在胍中）由二硫键交联的150- 1000-kDa聚集体组成，不存在于对照中。HPLC分离的聚集体含有α A-、β-、γ-和ζ-晶状体蛋白，但不含α B-晶状体蛋白，其缺乏- SH基团，因此不参与二硫键交联。所有存在于核WI组分中的ζ-晶体蛋白似乎是二硫键交联的结果。结果表明，在体内分子氧可以诱导豚鼠透镜核晶体蛋白交联成大的二硫键聚集体，能够散射光。人类核性白内障的形成也可能涉及类似的过程。
PURPOSE. The role of oxygen in the formation of lens high-molecular-weight (HMW) protein aggregates during the development of human nuclear cataract is not well understood. The purpose of this study was to investigate lens crystallin aggregate formation in hyperbaric oxygen (HBO)- treated guinea pigs by using in vivo and in vitro methods.METHODS. Guinea pigs were treated three times weekly for 7 months with HBO, and lens crystallin aggregation was investigated in vivo with the use of dynamic light-scattering (DLS) and in vitro by HPLC analysis of water-insoluble (WI) proteins. DLS measurements were made every 0.1 mm across the 4.5- to 5.0-mm optical axis of the guinea pig lens.RESULTS. The average apparent diameter of proteins in the nucleus ( the central region) of lenses of HBO-treated animals was nearly twice that of the control animals ( P < 0.001). Size distribution analysis conducted at one selected point in the nucleus and cortex ( the outer periphery of the lens) after dividing the proteins into small-diameter and large-diameter groups, showed in the O-2-treated nucleus a threefold increase in intensity ( P < 0.001) and a doubling in apparent size ( P = 0.03) of large-diameter aggregate proteins, compared with the same control group. No significant changes in apparent protein diameter were detected in the O-2-treated cortex, compared with the control. The average diameter of protein aggregates at the single selected location in the O-2-treated nucleus was estimated to be 150 nm, a size capable of scattering light and similar to the size of aggregates found in human nuclear cataracts. HPLC analysis indicated that one half of the experimental nuclear WI protein fraction ( that had been dissolved in guanidine) consisted of disulfide cross-linked 150- to 1000-kDa aggregates, not present in the control. HPLC-isolated aggregates contained alpha A-, beta-, gamma-, and zeta-crystallins, but not alpha B-crystallin, which is devoid of - SH groups and thus does not participate in disulfide cross-linking. All zeta-crystallin present in the nuclear WI fraction appeared to be there as a result of disulfide cross-linking.CONCLUSIONS. The results indicate that molecular oxygen in vivo can induce the cross-linking of guinea pig lens nuclear crystallins into large disulfide-bonded aggregates capable of scattering light. A similar process may be involved in the formation of human nuclear cataract.