Institute of Biochemistry, Food Science and Nutrition, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel.
Mol Genet Metab. 2010 Jun;100(2):193-7. doi: 10.1016/j.ymgme.2010.02.015. Epub 2010 Feb 25.
A novel homozygous mutation of the leptin gene was recently reported in an Egyptian child and his sister with severe early onset obesity. This mutation results from the substitution of asparagine (AAC) by lysine (AAA) at codon 103 of a non-mature (signal peptide-containing) leptin and corresponds to the N82K mutation in the mature protein. The patient had very low serum leptin levels, raising the question of whether the obese phenotype resulted from low leptin levels or from its lower intrinsic activity. To answer this question, we characterized the functional consequences of the N82K mutation. Wild-type (WT) human leptin was mutated accordingly, expressed in Escherichia coli at high yield, purified to homogeneity as a monomer and compared to WT human leptin prepared by the same methodology. Circular dichroism analysis of the mutated leptin indicated proper refolding and a secondary structure identical to that of the WT human leptin. In contrast to WT human leptin, the N82K mutant did not form a detectable complex with human leptin-binding domain (hLBD) and its binding capacity to hLBD assessed in a nonradioactive receptor-binding assay was at least 500-fold lower than that of WT human leptin. The biological activity of the N82K mutant, tested in two cell bioassays, was reduced by more than three orders of magnitude relative to WT human leptin. Therefore, though the present report does not explain the reason for the low circulating leptin levels it definitely documents that the reported obese phenotype originates not only from low serum leptin levels but also from the N82K mutant's almost total lack of intrinsic leptin activity.
最近在一名埃及儿童及其姐姐中报道了一种瘦素基因的新型纯合突变，他们患有严重的早发性肥胖症。这种突变是由于在非成熟（含信号肽）瘦素的第 103 位密码子处天冬酰胺（AAC）被赖氨酸（AAA）取代而导致的，与成熟蛋白中的 N82K 突变相对应。患者血清瘦素水平非常低，这引发了一个问题，即肥胖表型是否是由于低水平的瘦素引起的，还是由于其内在活性较低引起的。为了回答这个问题，我们对 N82K 突变的功能后果进行了表征。野生型（WT）人瘦素相应发生突变，在大肠杆菌中高产表达，以单体形式高度纯化，并与通过相同方法制备的 WT 人瘦素进行比较。突变瘦素的圆二色性分析表明其正确折叠，并且二级结构与 WT 人瘦素相同。与 WT 人瘦素相反，N82K 突变体不能与人类瘦素结合域（hLBD）形成可检测的复合物，并且在非放射性受体结合测定中其与 hLBD 的结合能力至少比 WT 人瘦素低 500 倍。通过两种细胞生物测定法测试 N82K 突变体的生物学活性，相对于 WT 人瘦素降低了三个数量级以上。因此，尽管本报告并未解释循环瘦素水平低的原因，但它明确证明了所报道的肥胖表型不仅源于血清瘦素水平低，而且还源于 N82K 突变体几乎完全缺乏内在瘦素活性。
