Department of Ophthalmology, University of Missouri, Columbia, MO 65212, USA.
Free Radic Biol Med. 2011 May 15;50(10):1429-36. doi: 10.1016/j.freeradbiomed.2011.01.036. Epub 2011 Feb 23.
Previous studies have shown that both αA- and αB-crystallins bind Cu2+, suppress the formation of Cu2+-mediated active oxygen species, and protect ascorbic acid from oxidation by Cu2+. αA- and αB-crystallins are small heat shock proteins with molecular chaperone activity. In this study we show that the mini-αA-crystallin, a peptide consisting of residues 71-88 of αA-crystallin, prevents copper-induced oxidation of ascorbic acid. Evaluation of binding of copper to mini-αA-crystallin showed that each molecule of mini-αA-crystallin binds one copper molecule. Isothermal titration calorimetry and nanospray mass spectrometry revealed dissociation constants of 10.72 and 9.9 μM, respectively. 1,1'-Bis(4-anilino)naphthalene-5,5'-disulfonic acid interaction with mini-αA-crystallin was reduced after binding of Cu2+, suggesting that the same amino acids interact with these two ligands. Circular dichroism spectrometry showed that copper binding to mini-αA-crystallin peptide affects its secondary structure. Substitution of the His residue in mini-αA-crystallin with Ala abolished the redox-suppression activity of the peptide. During the Cu2+-induced ascorbic acid oxidation assay, a deletion mutant, αAΔ70-77, showed about 75% loss of ascorbic acid protection compared to the wild-type αA-crystallin. This difference indicates that the 70-77 region is the primary Cu2+-binding site(s) in human native full-size αA-crystallin. The role of the chaperone site in Cu2+ binding in native αA-crystallin was confirmed by the significant loss of chaperone activity by the peptide after Cu2+ binding.
先前的研究表明，αA-和αB-晶体蛋白都能结合 Cu2+，抑制 Cu2+介导的活性氧物质的形成，并保护抗坏血酸免受 Cu2+的氧化。αA-和αB-晶体蛋白是具有分子伴侣活性的小热休克蛋白。在这项研究中，我们发现由αA-晶体蛋白的 71-88 个残基组成的 mini-αA-晶体蛋白肽可防止铜诱导的抗坏血酸氧化。评估铜与 mini-αA-晶体蛋白的结合表明，每个 mini-αA-晶体蛋白分子结合一个铜分子。等温滴定量热法和纳米喷雾质谱法分别揭示了 10.72 和 9.9 μM 的离解常数。1,1'-双(4-苯胺基)萘-5,5'-二磺酸与 mini-αA-晶体蛋白的相互作用在结合 Cu2+后减少，表明相同的氨基酸与这两种配体相互作用。圆二色性光谱表明，铜与 mini-αA-晶体蛋白肽结合会影响其二级结构。用丙氨酸取代 mini-αA-晶体蛋白中的 His 残基，可使该肽的氧化还原抑制活性丧失。在 Cu2+-诱导的抗坏血酸氧化测定中，与野生型αA-晶体蛋白相比，缺失突变体αAΔ70-77 导致抗坏血酸保护丧失约 75%。这种差异表明，70-77 区域是人源全长αA-晶体蛋白中 Cu2+的主要结合位点。通过 Cu2+结合后肽的伴侣活性显著丧失，证实了伴侣结合位点在天然αA-晶体蛋白中 Cu2+结合中的作用。
