Ž . Molecular Brain Research 63 1999 340–350 Research report Mytilus edulis hemolymph contains pro-opiomelanocortin: LPS and morphine stimulate differential processing George B. Stefano a,b , Beatrice Salzet-Raveillon b , Michel Salzet a,b, ) a Multidisciplinary Center for the study of Aging, Neuroscience Research Institute, State UniÕersity of New York, College at Old Westbury, Old Westbury, NY 11568-0210, USA b Centre de Biologie Cellulaire, Laboratoire d’Endocrinologie des Annelides, Groupe de Neuroimmunite des Hirudinees, UPRES A CNRS, UniÕersite des ´ ´ ´ ´ Sciences et Technologies de Lille, 59655 VilleneuÕe d’Ascq Cedex, France ´ Accepted 8 September 1998 Abstract Ž . Mytilus edulis hemolymph contains mammalian-like proopiomelanocortin POMC . The 20 kDa protein was purified by high pressure Ž . gel permeation chromatography, anti-adrenocorticotropin ACTH -affinity column and reverse-phase HPLC. The amino acid sequence determination was by Edman degradation, enzymatic treatments and Western blot analysis. Of the six peptides found in this opioid Ž . precursor, methionine-enkephalin, g-melanocyte stimulating hormone MSH , a-MSH and ACTH exhibited 100, 80, 85 and 74% sequence identity, respectively, with the mammalian counterparts. b-Endorphin and g-LPH exhibited only 25 and 10% sequence identity. Dibasic amino acid residues were found at the C-terminus of MSH and ACTH, indicating cleavage sites. The a-MSH is flanked at the Ž . C-terminus by Gly–Lys–Lys, representing an amidation signal. ACTH and CLIP 80% sequence identity are also C-terminally flanked by dibasic amino acid residues. Furthermore, morphine, in a dose-dependent manner, increased the hemolymph levels of a-MSH and Ž . Ž . ACTH 1–39 in a naloxone and phosphoramidon antagonizable manner, indicating a neutral endopeptidase 24.11; NEP mediated Ž . Ž . Ž . cleavage. Lipopolysaccharide 10 mgranimal stimulated the processing of ACTH 1–39 yielding ACTH 1–24 in a cleavage that is Ž . independent of NEP, but dependant on aspartyl proteases, demonstrating differential enzymatic cleavage of ACTH 1–39 . Taken together, POMC is present in invertebrates and its processing can be altered depending on the signal. q 1999 Elsevier Science B.V. All rights reserved. Keywords: ACTH; Leech; MSH; Anandamide; Morphine; POMC; Immunocyte; Nitric oxide 1. Introduction The first biochemical demonstration of a mammalian- Ž . like pro-opiomelanocortin POMC peptide in inverte- wx brates is that of Duvaux-Miret et al. 3 . This report found an extremely high homology between the genes of Schisto- soma mansoni and vertebrates. As such, the authors con- cluded that this may have resulted from the transfer of Abbreviations: ACTH, adrenocorticotropic hormone; a-ACTH, anti- ACTH; DIA, dot immunobinding assay; ELISA, enzyme-linked immuno- sorbent assay; ESMS, electropray mass spectrometry; HPLC, high pres- sure liquid chromatography; HPGPC, high pressure gel permeation chro- matography; LPH, lipotropin hormone; FPLC, fast protein liquid chro- matography; MALDI-TOF, matrix assisted laser desorption-time of flight; MSH, melanocyte stimulating hormorne; PTH, phenylthiohydantoin; POMC, proopiomelanocotropin; TBS, Tris-buffered saline ) Corresponding author. Fax: q33-03-2004-1130; E-mail: salzet@pop.univ-lille1.fr genetic material from the host, probably by a viral mecha- nism. The other possibility was that evolutionary conserva- tion was responsible for the high homology between evolu- tionarily divergent organisms. Recently, another POMC- like molecule has been isolated and sequenced in the fresh w x water leech Theromyzon tessulatum 26 . This leech opioid precursor also contained highly conserved mammalian-like POMC derived peptides, i.e., a-melanocyte stimulating Ž . hormone MSH , flanked by dibasic amino acids for enzy- matic processing. Given the fact that both S. mansoni and T. tessulatum are vertebrate parasites the question of contamination, as noted above, may still emerge. Thus, the aim of the present study was to determine if a free-living invertebrate also w x expresses POMC. In this regard, we demonstrate as 22 that the marine bivalve Mytilus edulis contains a POMC- like molecule with highly conserved peptides, i.e., a-MSH. Additionally, the present report demonstrates that its pro- 0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. Ž . PII: S0169-328X 98 00252-6