Bojnik Engin, Kleczkowska Patrycja, Marron Fernandez de Velasco Ezequiel, Corbani Maïthé, Babos Fruzsina, Lipkowski Andrzej W, Magyar Anna, Benyhe Sandor
Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, 6726 Szeged, Temesvari krt 62, Hungary.
Mossakowski Medical Research Centre, Polish Academy of Sciences, Department of Neuropeptides, Warsaw, Poland.
Comp Biochem Physiol B Biochem Mol Biol. 2014 Aug;174:29-35. doi: 10.1016/j.cbpb.2014.06.002. Epub 2014 Jun 16.
Endogenous opioids are derived from four related polypeptide precursors: proenkephalin (PENK), prodynorphin (PDYN), pronociceptin (PNOC) and proopiomelanocortin (POMC). In mammals PENK encodes for four copy of Met-enkephalin, one octapeptide Met-enkephalin-Arg-Gly-Leu, one heptapeptide Met-enkephalin-Arg-Phe and a single copy of Leu-enkephalin. Our detailed bioinformatic search on the existing PENK sequences revealed several atypical hexapeptide Met-enkephalins in different vertebrate animals. They are located either in the second enkephalin unit or in the seventh enkephalin core position at the C-terminus. Altogether four different hexapeptide sequences were obtained representing eleven animal species: Met-enkephalin-Arg(6) (YGGFMR) in the bird zebra finch, Met-enkephalin-Asp(6) (YGGFMD), Met-enkephalin-Ile(6) (YGGFMI) in zebrafish; and Met-enkephalin-Ser(6) (YGGFMS) in two pufferfish species. All novel peptides were chemically synthesized and studied in receptor binding and G-protein activation assays performed on rat brain membranes. The four novel enkephalins were equipotent in stimulating G-proteins. Affinities of the peptides determined by equilibrium competition assays in receptor binding experiments were statistically different. At the MOP receptors the highest affinity (Ki 4nM) was obtained with the zebra finch peptide Met-enkephalin-Arg(6). The pufferfish Met-enkephalin-Ser(6) exhibited the highest affinity (Ki 6.7nM) at the DOP receptor. Phylogenetic neuropeptide libraries, defined here as a collection of mutationally different species variants of orthologous and paralogous peptide sequences, represent the natural molecular diversity of the neuropeptides. Such libraries can provide a wide range of structural information establishing comparative functional analyses. Since DNA sequencing data are rapidly increasing, more development in the natural peptide library concept is expected.
前脑啡肽原（PENK）、前强啡肽原（PDYN）、前痛敏肽原（PNOC）和阿黑皮素原（POMC）。在哺乳动物中，PENK编码四个拷贝的甲硫氨酸脑啡肽、一个八肽甲硫氨酸脑啡肽-精氨酸-甘氨酸-亮氨酸、一个七肽甲硫氨酸脑啡肽-精氨酸-苯丙氨酸以及一个亮氨酸脑啡肽拷贝。我们对现有PENK序列进行的详细生物信息学搜索揭示了不同脊椎动物中几种非典型的六肽甲硫氨酸脑啡肽。它们位于C端第二个脑啡肽单元或第七个脑啡肽核心位置。总共获得了代表11个动物物种的四种不同六肽序列：鸟类斑胸草雀中的甲硫氨酸脑啡肽-精氨酸(6)（YGGFMR）、甲硫氨酸脑啡肽-天冬氨酸(6)（YGGFMD）、斑马鱼中的甲硫氨酸脑啡肽-异亮氨酸(6)（YGGFMI）；以及两种河豚中的甲硫氨酸脑啡肽-丝氨酸(6)（YGGFMS）。所有新肽均经化学合成，并在大鼠脑膜上进行的受体结合和G蛋白激活试验中进行了研究。这四种新的脑啡肽在刺激G蛋白方面具有同等效力。在受体结合实验中通过平衡竞争试验测定的肽的亲和力在统计学上存在差异。在MOP受体上，斑胸草雀肽甲硫氨酸脑啡肽-精氨酸(6)具有最高亲和力（Ki 4nM）。河豚甲硫氨酸脑啡肽-丝氨酸(6)在DOP受体上表现出最高亲和力（Ki 6.7nM）。系统发育神经肽文库，在此定义为直系同源和旁系同源肽序列的突变不同物种变体的集合，代表了神经肽的天然分子多样性。这样的文库可以提供广泛的结构信息以进行比较功能分析。由于DNA测序数据正在迅速增加，预计天然肽文库概念将有更多发展。
