LEECH IMMUNOCYTES CONTAIN PROOPIOMELANOCORTIN NITRIC-OXIDE MEDIATES HEMOLYMPH PROOPIOMELANOCORTIN PROCESSING

This report establishes the presence of mammalian-like proopiomelanoco tropic hormone (POMC), and six of its peptides, including adrenocortic otropic hormone (ACTH) and melanocyte-stimulating hormone (MSH), in th e immune tissues of the leech Theromyzon tessulatum. The 25.4-kDa prot ein 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 electrospra y mass spectrometry. Leech POMC exhibits considerable amino acid seque nce similarity to mammalian POMC, Of the six peptides, three showed hi gh 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 res idues 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 th e Gly-Arg-Lys amidation signal, ACTH and corticotropin-like intermedia ry pituitary peptide were also C-terminally flanked by dibasic amino a cid residues, The coding region of leech POMC. was obtained by reverse transcription-PCR using degenerated oligonucleotide primers, Circulat ing levels of ACTH and MSH were 10 and 1 fmol/mu l hemolymph, respecti vely. Morphine, in a dose-dependent manner, increased the levels of bo th peptides threefold; this effect was blocked by naloxone treatment. Similar results were found with the anandamide. Leech ACTH was process ed 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 demonst rates that POMC is present in invertebrates and its immunoregulatory a ctions have been conserved during evolution.