Kumar M Satish, Reddy P Yadagiri, Kumar P Anil, Surolia Ira, Reddy G Bhanuprakash
National Institute of Nutrition, Hyderabad 500 007, India.
Biochem J. 2004 Apr 15;379(Pt 2):273-82. doi: 10.1042/BJ20031633.
Alpha-crystallin is a member of the small heat-shock protein family and functions like a molecular chaperone, and may thus help in maintaining the transparency of the eye lens by protecting the lens proteins from various stress conditions. Non-enzymic glycation of long-lived proteins has been implicated in several age- and diabetes-related complications, including cataract. Dicarbonyl compounds such as methylglyoxal and glyoxal have been identified as the predominant source for the formation of advanced glycation end-products in various tissues including the lens. We have investigated the effect of non-enzymic browning of alpha-crystallin by reactive dicarbonyls on its molecular chaperone-like function. Non-enzymic browning of bovine alpha-crystallin in vitro caused, along with altered secondary and tertiary structures, cross-linking and high-molecular-mass aggregation. Notwithstanding these structural changes, methylglyoxal- and glyoxal-modified alpha-crystallin showed enhanced anti-aggregation activity in various in vitro aggregation assays. Paradoxically, increased chaperone-like activity of modified alpha-crystallin was not associated with increased surface hydrophobicity and rather showed less 8-anilinonaphthalene-l-sulphonic acid binding. In contrast, the chaperone-like function of modified alpha-crystallin was found to be reduced in assays that monitor the prevention of enzyme inactivation by UV-B and heat. Moreover, incubation of bovine lens with methylglyoxal in organ culture resulted in cataract formation with accumulation of advanced glycation end-products and recovery of alpha-crystallin in high proportions in the insoluble fraction. Furthermore, soluble alpha-crystallin from methylglyoxal-treated lenses showed decreased chaperone-like activity. Thus, in addition to describing the effects of methylglyoxal and glyoxal on structure and chaperone-like activity, our studies also bring out an important caveat of aggregation assays in the context of the chaperone function of alpha-crystallin.
α-晶状体蛋白是小热休克蛋白家族的成员，具有分子伴侣的功能，因此可能通过保护晶状体蛋白免受各种应激条件的影响，有助于维持晶状体的透明度。长寿蛋白的非酶糖基化与包括白内障在内的几种与年龄和糖尿病相关的并发症有关。二羰基化合物如甲基乙二醛和乙二醛已被确定为包括晶状体在内的各种组织中晚期糖基化终产物形成的主要来源。我们研究了反应性二羰基化合物对α-晶状体蛋白的非酶褐变对其分子伴侣样功能的影响。体外牛α-晶状体蛋白的非酶褐变除了导致二级和三级结构改变外，还引起交联和高分子量聚集。尽管有这些结构变化，甲基乙二醛和乙二醛修饰的α-晶状体蛋白在各种体外聚集试验中显示出增强的抗聚集活性。矛盾的是，修饰的α-晶状体蛋白的伴侣样活性增加与表面疏水性增加无关，反而显示出较少的8-苯胺基萘-1-磺酸结合。相反，在监测紫外线B和热对酶失活的预防作用的试验中，发现修饰的α-晶状体蛋白的伴侣样功能降低。此外，在器官培养中用甲基乙二醛孵育牛晶状体导致白内障形成，同时晚期糖基化终产物积累，并且α-晶状体蛋白在不溶性部分中大量回收。此外，来自甲基乙二醛处理的晶状体的可溶性α-晶状体蛋白显示出降低的伴侣样活性。因此，除了描述甲基乙二醛和乙二醛对结构和伴侣样活性的影响外，我们的研究还揭示了在α-晶状体蛋白伴侣功能背景下聚集试验的一个重要警告。
