The structural characteristics of nonspecific lipid transfer proteins explain their resistance to gastroduodenal proteolysis.
The structural characteristics of nonspecific lipid transfer proteins explain their resistance to gastroduodenal proteolysis.
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非特异性脂质转运蛋白的结构特征解释了它们对胃十二指肠蛋白水解的抵抗力。
DOI：
10.1021/bi901939z
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发表时间：
2010
期刊：
影响因子：
2.9
通讯作者：
E. Mills
中科院分区：
文献类型：
--
作者：
R. Wijesinha;Yuri Alexeev;Phil Johnson;J. Marsh;A. Sancho;S. U. Abdullah;A. Mackie;P. Shewry;Lorna J. Smith;E. Mills
比较了桃过敏性非特异性脂质转移蛋白（LTP）、大麦LTP 1及其配体LTP 1b的结构和稳定性。所有三种蛋白质都对胃蛋白酶解具有抗性，并且在十二指肠条件下仅在14个潜在胰蛋白酶和胰凝乳蛋白酶切割位点中的1至2个位点处缓慢消化。桃LTP最初在Tyr 79-Lys 80处切割，然后在Arg 39-Thr 40（大麦LTP 1中丢失的位点）处切割。折叠条件下的蛋白质的分子动力学模拟表明，骨干的灵活性是有限的，解释了十二指肠蛋白水解的阻力。Arg 39和Lys 80侧链更灵活的模拟桃相比，大麦LTP 1。这可以解释实验观察到的两种蛋白质的裂解率的差异，并表明，个别氨基酸侧链的灵活性可能是重要的，在确定优选的蛋白水解裂解位点。为了了解对胃蛋白酶水解的抗性，在pH 1.8下通过NMR光谱表征蛋白质。这表明，这两种蛋白质的螺旋区域保持折叠在此pH值。NMR氢交换研究证实了刚性的结构在酸性pH值，与大麦LTP 1显示一些区域具有更大的保护。总的来说，这些数据表明LTP支架的刚性是它们对蛋白水解的抗性的原因。胃十二指肠消化条件不会破坏桃LTP的3D结构，这解释了为什么LTP在消化后保留了结合IgE的能力，从而保留了其致敏潜力。
The structure and stability of the allergenic nonspecific lipid transfer protein (LTP) of peach were compared with the homologous LTP1 of barley and its liganded form LTP1b. All three proteins were resistant to gastric pepsinolysis and were only slowly digested at 1 to 2 out of 14 potential tryptic and chymotryptic cleavage sites under duodenal conditions. Peach LTP was initially cleaved at Tyr79-Lys80 and then at Arg39-Thr40 (a site lost in barley LTP1). Molecular dynamics simulations of the proteins under folded conditions showed that the backbone flexibility is limited, explaining the resistance to duodenal proteolysis. Arg39 and Lys80 side chains were more flexible in simulations of peach compared with barley LTP1. This may explain differences in the rates of cleavage observed experimentally for the two proteins and suggests that the flexibility of individual amino acid side chains could be important in determining preferred proteolytic cleavage sites. In order to understand resistance to pepsinolysis, proteins were characterized by NMR spectroscopy at pH 1.8. This showed that the helical regions of both proteins remain folded at this pH. NMR hydrogen exchange studies confirmed the rigidity of the structures at acidic pH, with barley LTP1 showing some regions with greater protection. Collectively, these data suggest that the rigidity of the LTP scaffold is responsible for their resistance to proteolysis. Gastroduodenal digestion conditions do not disrupt the 3D structure of peach LTP, explaining why LTPs retain their ability to bind IgE after digestion and hence their allergenic potential.