Biological activities of recombinant chicken leptin C4S analog compared with unmodified leptins
Biological activities of recombinant chicken leptin C4S analog compared with unmodified leptins
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DOI：
10.1152/ajpendo.2000.279.1.e116
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发表时间：
2000-07-01
影响因子：
5.1
通讯作者：
Taouis, M
中科院分区：
文献类型：
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作者：
Dridi, S;Raver, N;Taouis, M
与哺乳动物瘦素序列不同，鸡瘦素序列在原始cDNA （AF012727）的第3位含有一个未配对的Cys。额外的Cys的存在可能会产生不同的结构并影响瘦素的生物活性。为了解决这个问题，我们在体外和体内研究了野生型和突变型（C4S）鸡瘦素的作用，并将它们与由羊瘦素cDNA制备的哺乳动物瘦素进行了比较。利用诱变试剂盒对编码全尺寸A（-1）鸡瘦素（AF012727）的原核表达载体pMON进行突变，得到C4S类似物。用该载体转化的大肠杆菌细胞在钠二酸诱导下过量表达鸡瘦素C4S。在包体中发现的表达蛋白被重新折叠并在Q-Sepharose柱上纯化至均匀，得到三个电泳纯组分，分别用100、125和150 mM NaCl从柱上洗脱。所有三个组分均显示预期分子质量（16 kDa）的单带，并由>95%的单体蛋白组成。通过将类似物的圆二色光谱与未突变的鸡和羊瘦素的光谱进行比较，证明了其正确的重折叠。C4S类似物的生物活性是通过其能够刺激转染了类似于其未突变对应物的长形式人瘦素受体构建物的瘦素敏感BAF/3细胞的增殖来证明的，这表明Cys4在瘦素活性中没有作用。野生型和突变型鸡瘦素的体外活性都比绵羊瘦素低10倍左右。经静脉或腹腔注射C4S类似物和未突变的鸡、羊瘦素后，饥饿的9日龄肉鸡或5周龄蛋鸡的采食量均降低了11-34%。对食物行为的监测显示，食物摄取量的减少不是因为接近喂食者的次数减少，而是因为每次接近喂食者时平均进食时间的减少。
The chicken leptin sequence, in contrast to mammalian leptins, contains an unpaired Cys at position 3 of the original cDNA (AF012727). The presence of an extra Cys may confer a different structure and affect the leptin's biological activity. To address this, we studied the effects of wild-type and mutated (C4S) chicken leptins in vitro and in vivo and compared them with mammalian leptin prepared from ovine leptin cDNA. The prokaryotic expression vector pMON, encoding full-size A(-1) chicken leptin (AF012727), was mutated using a mutagenesis kit, yielding the C4S analog. Escherichia coli cells transformed with this vector overexpressed large amounts of chicken leptin C4S upon induction with nalidixic acid. The expressed protein, found in the inclusion bodies, was refolded and purified to homogeneity on a Q-Sepharose column, yielding three electrophoretically pure fractions, eluted from the column by 100, 125, and 150 mM NaCl, respectively. All three fractions showed a single band of the expected molecular mass (16 kDa) and were composed of >95% monomeric protein. Proper refolding was evidenced by comparing the circular dichroism spectrum of the analog with spectra of nonmutated chicken and ovine leptins. The biological activity of the C4S analog was evidenced by its ability to stimulate proliferation of leptin-sensitive BAF/3 cells transfected with a long form of human leptin receptor construct similar to its nonmutated counterpart, indicating that Cys4 plays no role in leptin activity. The in vitro activity of both wild-type and mutated chicken leptins was similar to 10-fold lower than that of ovine leptin. After intravenous or intraperitoneal injections, C4S analog and the nonmutated chicken and ovine leptins all lowered the food intake of starved 9-day-old broiler or 5-wk-old layer male chickens by 11-34%. Monitoring food behavior revealed that the attenuated food intake resulted not from a decreased number of approaches to the feeders but from a decrease in the average time spent eating during each approach.