Siezen R J, Wu E, Kaplan E D, Thomson J A, Benedek G B
Department of Physics, Massachusetts Institute of Technology, Cambridge 02139.
J Mol Biol. 1988 Feb 5;199(3):475-90. doi: 10.1016/0022-2836(88)90619-5.
We have isolated, purified and characterized six individual gamma-crystallin polypeptides present in the rat lens. Comparison of their amino acid compositions with the known structure of the six gamma-crystallin genes permits a one-to-one correspondence to be made between each protein synthesized and the encoding gene. This demonstrates that each of the six genes is actually expressed in vivo. Two classes of three gamma-crystallins each, which we have designated classes gamma ABC and gamma DEF, are known to exist, on the basis of internal sequence homology. We have measured the temperature-dependent phase-separation characteristics of solutions of the six purified gamma-crystallins, and find that the three members of the gamma DEF class (gamma 2-2, gamma 3-1 and gamma 4-1) are all cryo-proteins with relatively high phase-separation temperatures, whereas the three gamma ABC crystallins (gamma 1-1, gamma 1-2 and gamma 2-1) do not show phase separation above -7 degrees C. We have measured the spatial distribution in rat lens of each of the alpha-, beta- and gamma-crystallins as a function of age from 1 to 420 days, using size-exclusion and ion-exchange high-pressure liquid chromatography (HPLC). Our findings in the cortical layer permit us to establish the differential synthesis of each of the crystallins during lens development. Particular attention has been devoted to the spatial and temporal distribution of the six individual gamma-crystallins. Up to birth, synthesis of the three components of the gamma DEF class predominates, and in particular that of gamma 2-2. In subsequent development the three components of the gamma ABC class assume a greater proportion of monomeric crystallins synthesized, while beta s-crystallin synthesis predominates in late development. Our analysis of different layers within single lenses provides novel information on spatial gradients of the water-soluble and water-insoluble protein fractions as a function of age. We consider the consequences of these findings for lens transparency and opacity in both rat and mouse lens. We show that the high concentrations of gamma DEF-crystallins appear to be responsible for the opacity known to occur in young rat lenses. We conclude from these observations that close control of the differential synthesis of gamma-crystallins plays an important role in maintaining lens transparency during development.
我们已经分离、纯化并鉴定了大鼠晶状体中存在的六种γ-晶状体蛋白多肽。将它们的氨基酸组成与六个γ-晶状体蛋白基因的已知结构进行比较，可以在每个合成的蛋白质与编码基因之间建立一对一的对应关系。这表明六个基因中的每一个实际上都在体内表达。基于内部序列同源性，已知存在两类γ-晶状体蛋白，每类各有三种，我们将其命名为γABC类和γDEF类。我们测量了六种纯化的γ-晶状体蛋白溶液的温度依赖性相分离特性，发现γDEF类的三个成员（γ2-2、γ3-1和γ4-1）都是具有相对较高相分离温度的低温保护蛋白，而γABC类的三种晶状体蛋白（γ1-1、γ1-2和γ2-1）在-7℃以上不显示相分离。我们使用尺寸排阻和离子交换高压液相色谱（HPLC）测量了1至420天龄大鼠晶状体中α-、β-和γ-晶状体蛋白各自的空间分布。我们在皮质层的研究结果使我们能够确定晶状体发育过程中每种晶状体蛋白的差异合成情况。我们特别关注了六种单个γ-晶状体蛋白的空间和时间分布。在出生前，γDEF类的三种成分的合成占主导，尤其是γ2-2的合成。在随后的发育过程中，γABC类的三种成分在合成的单体晶状体蛋白中所占比例更大，而βs-晶状体蛋白的合成在发育后期占主导。我们对单个晶状体不同层的分析提供了关于水溶性和水不溶性蛋白质组分随年龄变化的空间梯度的新信息。我们考虑了这些发现对大鼠和小鼠晶状体透明度和不透明度的影响。我们表明，γDEF-晶状体蛋白的高浓度似乎是幼鼠晶状体中已知出现的不透明度的原因。我们从这些观察结果得出结论，γ-晶状体蛋白差异合成的严格控制在发育过程中维持晶状体透明度方面起着重要作用。
