Okazaki Institute for Integrative Bioscience and Institute for Molecular Science, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan.
Faculty of Advanced Life Science, Hokkaido University, Kita-10 Nishi-8, Kita-ku, Sapporo 060-0810, Japan.
J Biol Chem. 2013 May 17;288(20):14408-14416. doi: 10.1074/jbc.M112.437889. Epub 2013 Apr 11.
Although HAMLET (human α-lactalbumin made lethal to tumor cells), a complex formed by human α-lactalbumin and oleic acid, has a unique apoptotic activity for the selective killing of tumor cells, the molecular mechanisms of expression of the HAMLET activity are not well understood. Therefore, we studied the molecular properties of HAMLET and its goat counterpart, GAMLET (goat α-lactalbumin made lethal to tumor cells), by pulse field gradient NMR and 920-MHz two-dimensional NMR techniques. We also examined the expression of HAMLET-like activities of complexes between oleic acid and other proteins that form a stable molten globule state. We observed that both HAMLET and GAMLET at pH 7.5 were heterogeneous, composed of the native protein, the monomeric molten globule-like state, and the oligomeric species. At pH 2.0 and 50 °C, HAMLET and GAMLET appeared in the monomeric state, and we identified the oleic acid-binding site in the complexes by two-dimensional NMR. Rather surprisingly, the binding site thus identified was markedly different between HAMLET and GAMLET. Furthermore, canine milk lysozyme, apo-myoglobin, and β2-microglobulin all formed the HAMLET-like complex with the anti-tumor activity, when the protein was treated with oleic acid under conditions in which their molten globule states were stable. From these results, we conclude that the protein portion of HAMLET, GAMLET, and the other HAMLET-like protein-oleic acid complexes is not the origin of their cytotoxicity to tumor cells and that the protein portion of these complexes plays a role in the delivery of cytotoxic oleic acid molecules into tumor cells across the cell membrane.
尽管 HAMLET（人乳白蛋白致瘤细胞失活物）是由人乳白蛋白和油酸形成的复合物，对肿瘤细胞具有独特的凋亡活性，但 HAMLET 活性的表达机制尚不清楚。因此，我们使用脉冲场梯度 NMR 和 920MHz 二维 NMR 技术研究了 HAMLET 及其山羊对应物 GAMLET（山羊乳白蛋白致瘤细胞失活物）的分子特性。我们还检查了油酸与其他形成稳定变性球蛋白状态的蛋白质形成的复合物表达 HAMLET 样活性的情况。我们观察到，在 pH 值为 7.5 时，无论是 HAMLET 还是 GAMLET，均为异质的，由天然蛋白质、单体变性球蛋白样状态和寡聚物种组成。在 pH 值为 2.0 和 50°C 时，HAMLET 和 GAMLET 均呈单体状态，我们通过二维 NMR 鉴定了复合物中的油酸结合位点。令人惊讶的是，在 HAMLET 和 GAMLET 之间，鉴定出的结合位点明显不同。此外，犬乳清溶菌酶、脱辅基肌红蛋白和 β2-微球蛋白在其变性球蛋白状态稳定的条件下，用油酸处理时，均形成具有抗肿瘤活性的 HAMLET 样复合物。从这些结果中，我们得出结论，HAMLET、GAMLET 和其他 HAMLET 样蛋白-油酸复合物的蛋白部分不是其对肿瘤细胞毒性的起源，并且这些复合物的蛋白部分在将细胞毒性油酸分子递送至穿过细胞膜的肿瘤细胞中发挥作用。
