Changes in thermal stability and microunfolding pattern of collagen helix resulting from the loss of α2(I) chain in osteogenesis imperfecta murine
Changes in thermal stability and microunfolding pattern of collagen helix resulting from the loss of α2(I) chain in osteogenesis imperfecta murine
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DOI：
10.1016/s0022-2836(03)00715-0
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发表时间：
2003-08-01
影响因子：
5.6
通讯作者：
Leikin, S
中科院分区：
文献类型：
--
作者：
Kuznetsova, NV;McBride, DJ;Leikin, S
纯合子突变导致形成α 1（I）（3）同源三聚体而不是正常的I型胶原蛋白，导致人类和小鼠轻度至重度成骨障碍（OI）。此前报道了对I型同三聚体热稳定性变化的有限研究，但结果并不完全一致。我们使用野生型（α 1（I）（2）α 2（I）异源三聚体）和oim（α 1（I）（3））小鼠的纯化肌腱胶原蛋白以及通过重折叠大鼠尾肌腱胶原蛋白获得的人工α 1（I）（3）同源三聚体更详细地重新审视了这个问题。我们发现，在相同的加热速率下，同三聚体完全变性的温度比野生型异三聚体高2.5 ℃，这是通过差示扫描量热法测定的。在相同的温度下，同源三聚体的变性比异源三聚体慢100倍，这是由圆二色性测定的。在不同温度下的蛋白水解裂解的详细分析表明，oim同源三聚体和野生型异源三聚体的微解折叠发生在相似的速率，但在一些不同的网站。特别是，oim三螺旋上的最弱点位于溴化氰肽CB 6内从C-末端起100个氨基酸残基附近。野生型胶原蛋白中也存在相同的微去折叠位点，但后者最弱的位点位于靠近CB 8的N末端。氨基酸分析和差异凝胶电泳显示oim小鼠肌腱胶原蛋白几乎没有翻译后过度修饰。此外，人工鼠尾腱同源三聚体的热稳定性和微去折叠与oim同源三聚体相似。因此，观察到的变化与α 1（I）和α 2（I）链的氨基酸组成差异有关，而不是翻译后过度修饰。(C)2003 Elsevier Ltd.保留所有权利。
Homozygous mutations resulting in formation of alpha1(I)(3) homotrimers instead of normal type I collagen cause mild to severe osteogenesis imperfecta (OI) in humans and mice. Limited studies of changes in thermal stability of type I homotrimers were reported previously, but the results were not fully consistent. We revisited this question in more detail using purified tendon collagen from wild-type (alpha1(I)(2)alpha2(I) heterotrimers) and oim (alpha1(I)(3)) mice as well as artificial alpha1(I)(3) homotrimers obtained by refolding of rat-tail-tendon collagen. We found that at the same heating rate oim homotrimers completely denature at similar to2.5 deg.C higher temperature than wild-type heterotrimers, as determined by differential scanning calorimetry. At the same, constant temperature, homotrimers denature similar to100 times slower than heterotrimers, as determined by circular dichroism. Detailed analysis of proteolytic cleavage at different temperatures revealed that microunfolding of oim homotrimers and wild-type heterotrimers occurs at similar rate but within a number of different sites. In particular, the weakest spot on the oim triple helix is located similar to100 amino acid residues from the C-terminal end within the cyanogen bromide peptide CB6. The same microunfolding site is also present in wild-type collagen, but the weakest spot of the latter is located close to the N-terminal end of CB8. Amino acid analysis and differential gel electrophoresis showed virtually no posttranslational overmodification of oim mouse tendon collagen. Moreover, thermal stability and microunfolding of artificial rat-tail-tendon homotrimers were similar to oim homotrimers. Thus, the observed changes are associated with difference in the amino acid composition of alpha1(I) and alpha2(I) chains rather than posttranslational overmodification. (C) 2003 Elsevier Ltd. All rights reserved.