Salzet M, Salzet-Raveillon B, Cocquerelle C, Verger-Bocquet M, Pryor S C, Rialas C M, Laurent V, Stefano G B
Centre de Biologie Cellulaire, Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France.
J Immunol. 1997 Dec 1;159(11):5400-11.
This report establishes the presence of mammalian-like proopiomelanocotropic hormone (POMC), and six of its peptides, including adrenocorticotropic hormone (ACTH) and melanocyte-stimulating hormone (MSH), in the immune tissues of the leech Theromyzon tessulatum. The 25.4-kDa protein was purified by high pressure gel permeation chromatography, anti-ACTH-affinity column, and reverse-phase HPLC. Its characterization was performed by Edman degradation, enzymatic treatments, and electrospray mass spectrometry. Leech POMC exhibits considerable amino acid sequence similarity to mammalian POMC. Of the six peptides, three showed high sequence similarity to their vertebrate counterparts met-enkephalin, alpha-MSH, and ACTH: 100, 84.6, and 70%, respectively; whereas gamma-MSH, beta-endorphin, and gamma-lipotropin hormone exhibited only 45, 20, and 10% sequence identity, respectively. No dibasic amino acid residues were found at the C terminus of the gamma- and beta-MSH peptides. In contrast, the leech alpha-MSH was flanked at its C-terminal by the Gly-Arg-Lys amidation signal. ACTH and corticotropin-like intermediary pituitary peptide were also C-terminally flanked by dibasic amino acid residues. The coding region of leech POMC was obtained by reverse transcription-PCR using degenerated oligonucleotide primers. Circulating levels of ACTH and MSH were 10 and 1 fmol/microl hemolymph, respectively. Morphine, in a dose-dependent manner, increased the levels of both peptides threefold; this effect was blocked by naloxone treatment. Similar results were found with the anandamide. Leech ACTH was processed to MSH by the enzymes neutral endopeptidase (24.11) and angiotensin-converting enzyme. Leech alpha-MSH had the same activity as authentic alpha-MSH in two bioasssay systems. Taken together, the study demonstrates that POMC is present in invertebrates and its immunoregulatory actions have been conserved during evolution.
本报告证实了在欧洲医蛭（Theromyzon tessulatum）的免疫组织中存在类哺乳动物促阿片黑素皮质激素（POMC）及其六种肽，包括促肾上腺皮质激素（ACTH）和促黑素细胞激素（MSH）。通过高压凝胶渗透色谱、抗ACTH亲和柱和反相高效液相色谱法纯化了25.4 kDa的蛋白质。通过埃德曼降解、酶处理和电喷雾质谱对其进行了表征。水蛭POMC与哺乳动物POMC表现出相当大的氨基酸序列相似性。在这六种肽中，三种与它们的脊椎动物对应物甲硫氨酸脑啡肽、α-MSH和ACTH表现出高度的序列相似性，分别为100%、84.6%和70%；而γ-MSH、β-内啡肽和γ-促脂素激素的序列同一性分别仅为45%、20%和10%。在γ-和β-MSH肽的C末端未发现双碱性氨基酸残基。相比之下，水蛭α-MSH在其C末端两侧有Gly-Arg-Lys酰胺化信号。ACTH和促肾上腺皮质激素样中间垂体肽在C末端也有双碱性氨基酸残基。使用简并寡核苷酸引物通过逆转录PCR获得了水蛭POMC的编码区。ACTH和MSH的循环水平分别为10和1 fmol/μl血淋巴。吗啡以剂量依赖的方式使两种肽的水平增加了三倍；纳洛酮处理可阻断这种作用。花生四烯酸乙醇胺也得到了类似的结果。水蛭ACTH被中性内肽酶（24.11）和血管紧张素转换酶加工成MSH。在两个生物测定系统中，水蛭α-MSH与天然α-MSH具有相同的活性。综上所述，该研究表明POMC存在于无脊椎动物中，并且其免疫调节作用在进化过程中得以保留。
