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Four cationic peptides were purified from mussel (Mytilus galloprovincialis) hemocytes. A combination of Edman degradation and mass spectrometry of plasma revealed (i) a previously characterized molecule, mytilin B (Charlet, M., Chernysh, S., Philippe, H., Hetrut, C., Hoffmann, J., and Bulet, P. (1996) J. Biol. Chem. 271, 21808-21813) and (ii) three new isoforms, mytilin C, D, and G1. The four molecules exhibited complementary antimicrobial properties. The cDNA sequence coding for the mytilin B precursor was obtained from a hemocyte cDNA library. This precursor contains a putative signal peptide of 22 residues, a processing peptide sequence of 34 amino acids, and a C-terminal extension of 48 residues rich in acidic residues. Distribution of mytilin B mRNA and of the corresponding peptide in various mussel tissues revealed that mytilins are synthesized and stored in a specific hemocyte subtype. Furthermore, in an experimental model of infection, we showed (i) a recruitment of hemocytes containing mytilins toward the injection site within hours following bacterial challenge, (ii) that mytilins probably play a prominent role in killing intracellular bacteria after phagocytosis, and (ii) later an increase of mytilin-like material occurred in the plasma suggesting a secondary systemic role.

A soluble 65582.9 Da (in MALDI-TOF) angiotensin converting (ACE)-like enzyme has been purified by a captopril-Sepharose affinity column chromatography from the mollusk Mytilus edulis. This glycosylated peptidyl dipeptidase, with an N-terminal sequence of LDPELSPGCFVANQAGGQLF, hydrolyses the Phe8–His9 bond (at pH 8.4 and 37°C) of angiotensin I with a high catalytic activity i.e. Km: 168 μM and Kcat/Km: 262 s−1mM−1. The hydrolysis of angiotensin I is inhibited by the specific ACE inhibitors captopril and lisonopril (Ic50=50 nM). This activity is increased by Cl−(optimal Cl−concentration 400 mM) and by Zn2+. This zinc metallopeptidase also attacks peptides having a Gly–His, Gly–Phe or a Phe–His bond in their sequence e.g. leucine–enkephalin (Kcat/Km: 1200 s−1mM−1) or bradykinin (Kcat/Km: 2500 s−1mM−1). MytilusACE displays properties of the C-domain of human ACE, indicating a high degree of conservation during evolution. These results are consistent with an ACE activity implicated in metabolism of several neuropeptides in mollusks.

Cells immunoreactive to an antiserum specifically directed against vertebrate alpha-Neo-endorphin (alpha-NE) were detected in the internal wall of anterior and posterior suckers of the rhynchobdellid leech Theromyzon tessulatum. These cells have morphological and ultrastructural characteristics close to the "releasing gland cells" of adhesive organs. The epitope recognized by anti-alpha-NE was contained in granules having a diameter of 0.2-0.3 microm. Previous works involving the brain of this leech demonstrate the existence of approximately 14 neurons immunoreactive to the anti-alpha-NE. Following an extensive purification including high pressure gel permeation and reversed-phase high performance liquid chromatography, epitopes contained in both suckers and central nervous system were isolated. Purity of the isolated peptides was controlled by capillary electrophoresis. Their sequences were determined by a combination of automated Edman degradation, electrospray mass spectrometry measurement, and coelution experiments in reversed-phase high performance liquid chromatography with synthetic alpha-NE. The results demonstrate that epitopes recognized by the anti-alpha-NE in the suckers and the central nervous system are identical to vertebrate alpha-NE (YGGFLRKYPK). This finding constitutes the first biochemical characterization of a prodynorphin-derived peptide in invertebrates. Moreover the isolation of this peptide in the annelida establishes the very ancient phylogenetic origin of alpha-NE as well as its conservation in evolution.

Purification of a material immunoreactive to an antiserum against the C-terminal part of the oxytocin (Pro-Leu-Gly-amide) and present in the central nervous system of the Pharyngobdellid leech Erpobdella octoculata was performed by reversed-phase high performance liquid chromatography combined with both enzyme-linked immunosorbent and dot immunobinding assays for oxytocin. The amino acid sequence of the purified peptide (Ile-Pro-Glu-Pro-Tyr-Val-Trp-Asp) was established by Edman degradation and confirmed by electrospray mass spectrometry measurement. When injected in leeches, purified or synthetic peptides exert an anti-diuretic effect, the most effective ranged between 10 pmol and 1 nmol. They provoked an uptake of water 1-2 h post-injection. Furthermore, electrophysiological experiments conducted in the leech Hirudo medicinalis revealed an inhibition of the potency of Na+ conductances of leech skin by this peptide. Immunocytochemical studies with an antiserum against synthetic oxytocin-like molecule provided the cytological basis for existence of a neuropeptide, since large amounts of immunoreactive neurons were detected in the central nervous systems of E. octoculata. The purified molecule is both different to peptides of the oxytocin/vasopressin family and is a novel neuropeptide in the animal kingdom. It was named the leech osmoregulator factor (LORF). An identification of the proteins immunoreactive to an antiserum against oxytocin performed at the level of both central nervous systems extracts and in vitro central nervous system-translated RNA products indicated that in the two cases, a single protein was detected. These proteins with a molecular masses of, respectively, approximately 34 kDa (homodimer of 17 kDa) for the central nervous systems extracts and approximately 19 kDa for in vitro central nervous system-translated RNA products were not recognized by the antiserum against MSEL- and VLDV-neurophysin (proteins associated to oxytocin and vasopressin), confirming that LORF did not belong to the oxytocin/vasopressin family.