Mytilus edulis hemolymph contains pro-opiomelanocortin: LPS and morphine stimulate differential processing

Abstract

Mytilus edulis hemolymph contains mammalian-like proopiomelanocortin (POMC). The 20 kDa protein was purified by high pressure 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 precursor, methionine-enkephalin, γ-melanocyte stimulating hormone (MSH), α-MSH and ACTH exhibited 100, 80, 85 and 74% sequence identity, respectively, with the mammalian counterparts. β-Endorphin and γ-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 α-MSH is flanked at the 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 α-MSH and ACTH (1–39) in a naloxone and phosphoramidon antagonizable manner, indicating a neutral endopeptidase (24.11; NEP) mediated cleavage. Lipopolysaccharide (10 μg/animal) stimulated the processing of ACTH (1–39) yielding ACTH (1–24) in a cleavage that is 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.