Jean Malecha

Therostasin, a Novel Clotting Factor Xa Inhibitor from the Rhynchobdellid Leech, Theromyzon tessulatum

Therostasin is a potent naturally occurring tight-binding inhibitor of mammalian Factor Xa (K i , 34 pm), isolated from the rhynchobdellid leechTheromyzon tessulatum. Therostasin is a cysteine-rich protein (8991 Da) consisting of 82 amino acid residues with 16 cysteine residues. Its amino acid sequence has been determined by a combination of techniques, including Edman degradation, enzymatic cleavage, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) on the native and s-β-pyridylethylated compound. Sequence analysis reveals that it shares no significant homology with other Factor Xa inhibitors except for the putative reactive site. Moreover, it contains a signature pattern for proteins of the endothelin family, potent vasoconstrictors isolated in mammal and snake venom. Therostasin cDNA (825 bp) codes for a polypeptide of 82 amino acid residues preceded by 19 residues, representing a signal peptide sequence. As for the other known inhibitors of Factor Xa, therostasin is expressed and stored in the cells of the leech salivary glands.

Theromin, a novel leech thrombin inhibitor.

We purified the most potent thrombin inhibitor described to date from the rhynchobdellid leech Theromyzon tessulatum. Designated theromin, it was purified to apparent homogeneity by gel permeation and anion exchange chromatography followed by two reverse-phase steps of high performance liquid chromatography. The primary sequence of theromin (a homodimer of 67 amino acid residues including 16 cysteine residues) was determined by a combination of reduction and s-β-pyridylethylation, Edman degradation, trypsin enzymatic digestion, and matrix-assisted laser desorption mass spectrometry measurement. Theromin exhibits no sequence homology with any other thrombin inhibitors. Furthermore, theromin significantly diminishes, in a dose-dependent manner, the level of human granulocyte and monocyte activation induced by lipopolysaccharides. In summary, this potent thrombin inhibitor promises to have high biomedical significance.

Biochemical evidence of angiotensin II-like peptides and proteins in the brain of the rhynchobdellid leech Theromyzon tessulatum

The peptides contained in neurons localized in the brain of the leech Theromyzon tessulatum (Hirudinae, Rhynchobdellida) and showing an immunopositive reaction with an antibody directed against angiotensin II (AII), were purified by reversed-phase HPLC. Three AII-like peptides (P1, P2 and P3) which exhibited the same retention times and chromatographic behaviors as synthetic AVII (fragment 6-8 of AII), AIV (fragment 3-8 of AII) and AII, respectively, were resolved in brain extracts. An identification of the proteins immunoreactive to an anti-AII was performed at the level of both brain extracts and in vitro brain-translated RNA products. The protein detected at the level of the brain extracts (of a molecular mass of approximately 18 kDa) is multipeptidic as it is also recognized by two other antisera, a polyclonal one directed against gamma-MSH and a monoclonal one (Tt159) raised against a leech brain epitope. It could be the pro-AII-like precursor. The protein detected at the level of in vitro brain-translated RNA products (of a molecular mass of approximately 19 kDa) could be the prepro-AII-like precursor.

Isolation and structural characterization of enkephalins in the brain of the Rhynchobdellid leech Theromyzon tessulatum

This paper reports the purification of four peptides related to enkephalins from the brain of the leech Theromyzon tessulatum. After reverse‐phase HPLC purification, the sequence of the enkephalins (YGGFM, YGGFL, FM, FL) was established by a combination of automated Edman degradation, electrospray mass spectrometry measurement, and co‐elution experiments in reverse‐phase HPLC with synthetic peptides. ELISA titrations performed on each purified peptide indicated that the major amount was borne by the leucine‐enkephalin. The ratio of leucine‐enkephalin and methionine‐enkephalin of 2:1 is in line with previous immunocytochemical data obtained on T. tessulatum brains. The presence of enkephalins in T. tessulatum, an animal belonging to the oldest group of coelomate metazoans (the Annelida) establishes the very ancient phylogenetic origin of opioids and their conservation in the course of evolution.

Oxytocin-like peptide : a novel epitope colocalized with the FMRFamide-like peptide in the supernumerary neurons of the sex segmental ganglia of leeches : morphological and biochemical characterization ; putative anti-diuretic function

A large number of oxytocin (OT)-like neurons were detected in the sex segmental ganglia (SG5, SG6) of three species of leeches belonging to different orders: Theromyzon tessulatum, Hirudo medicinalis and Erpobdella octoculata. In this latter species, an epitope close to the vertebrate OT by its C-terminal part (MSH release inhibiting factor: MIF), localized in granules of a size diameter of ca 120 nm and colocalized with FMRFamide(FMRFa)-like material was demonstrated. With reverse phase-high performance liquid chromatography, evidence was given that the two epitopes (OT and FMRFa) colocalized in the same neurons were biochemically different. A titration of OT per SG indicated that the OT-like amount was considerably higher in sex SG than in non-sex SG (ca. 5 pmol vs. ca. 0.5 pmol). Moreover, at the level of sex SG, this amount was ca. 3-fold higher in immature leeches than in mature specimens. Injections of extracts of SG of E. octoculata and of fragments of OT (Tocinoic acid or MIF) to T. tessulatum, indicated that MIF (the epitope found in the sex SG) and sex SG have the same anti-diuretic effect on the leeches injected. These results pointed to an anti-diuretic role of the leech OT-like substance.

Isolation and structural characterization of a novel peptide related to γ-melanocyte stimulating hormone from the brain of the leech Theromyzon tessulatum

This paper reports the purification of a novel pro‐opiomelanocortin derivative peptide (a γ‐melanocyte stimulating hormone‐like (γ‐MSH‐like) molecule) from the brain of the leech Theromyzon tessulatum. After reverse‐phase HPLC purification, the sequence of the γ‐MSH‐like peptide (YVMGHFRWDKFamide) was established by a combination of automated Edman degradation, electrospray mass spectrometry measurement, enzymatic treatment and co‐elution experiments in reverse‐phase HPLC with synthetic peptides.

Cloning and expression analysis of a cDNA that encodes a leech hemerythrin.

We report the cDNA sequence of a leech hemerythrin. A cDNA was isolated from a Theromyzon tessulatum cDNA library and encodes a 120 amino acid protein of about 14 kDa. The predicted protein contains the hemerythrin signature sequence and the iron ligand residues previously identified in crystal structures of hemerythrin and myohemerythrin. The protein displayed the highest identity to myohemerythrin, a non-heme iron-binding protein described in sipunculids. Expression analysis indicated that the mRNA is widely expressed in leech and is stage specific in appearance, being absent after the two first blood meals, appearing after the last blood meal during the period preceding oogenesis and disappearing after egg laying.