Proteinase inhibitors from the european medicinal leech Hirudo medicinalis: Structural, functional and biomedical aspects

Ascenzi, Paolo; Amiconi, Gino; Bode, Wolfram; Bolognesi, Martino; Coletta, Massimo; Menegatti, Enea

doi:10.1016/0098-2997(95)00002-x

Cited by 39 publications

(39 citation statements)

References 255 publications

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“…Several proteinaceous inhibitors of serine proteases have been isolated from leeches (12). Leech-derived inhibitors include anticoagulants such as the thrombin-specific hirudins and the factor Xa-specific antistasin (13), and other serine protease inhibitors with a biological function not fully understood.…”

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confidence: 99%

“…A variety of structurally diverse inhibitors of serine proteinases have previously been purified from leeches (12), most prominently among them the thrombin-specific hirudins and the factor Xa-specific antistasins, anticoagulants that are required to maintain the liquid state of the blood during feeding and inside the leech. Potentially, LCI may participate in the elimination of blood clots by inhibiting plasma carboxypeptidase B, an enzyme recently been shown to retard fibrinolysis (38,39).…”

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confidence: 99%

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A Carboxypeptidase Inhibitor from the Medical Leech Hirudo medicinalis

Reverter¹,

Vendrell²,

Canals³

et al. 1998

Journal of Biological Chemistry

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A novel metallocarboxypeptidase inhibitor was isolated from the medical leech Hirudo medicinalis. Amino acid sequence analysis provided a nearly complete primary structure. which was subsequently verified and completed by cDNA cloning using reverse transcriptasepolymerase chain reaction/rapid amplification of cDNA end techniques. The inhibitor, called LCI (leech carboxypeptidase inhibitor), is a cysteine-rich polypeptide composed of 66 amino acid residues. It does not show sequence similarity to any other protein except at its C-terminal end. In this region, the inhibitor shares the amino acid sequence -Thr-Cys-X-Pro-Tyr-Val-X with Solanacea carboxypeptidase inhibitors, suggesting a similar mechanism of inhibition where the C-terminal tail of the inhibitor interacts with the active center of metallocarboxypeptidases in a substrate-like manner. This hypothesis is supported by the hydrolytic release of the C-terminal glutamic acid residue of LCI after binding to the enzyme. Heterologous overexpression of LCI in Escherichia coli, either into the medium or as an intracellular thioredoxin fusion protein, yields a protein with full inhibitory activity. Both in the natural and recombinant forms, LCI is a tightly binding, competitive inhibitor of different types of pancreatic-like carboxypeptidases, with equilibrium dissociation constants K i of 0.2-0.4 ؋ 10 ؊9 M for the complexes with the pancreatic enzymes A1, A2, and B and plasma carboxypeptidase B. Circular dichroism and nuclear magnetic resonance spectroscopy analysis indicate that recombinant LCI is a compactly folded globular protein, stable to a wide range of pH and denaturing conditions. In recent years, there has been an increasing interest in the role of proteases in various biological processes such as peptide processing, defense mechanisms, fertilization, carcinogenesis, trauma, inflammation, and coagulation/fibrinolysis, among others (1-3). The biological actions of most proteases is controlled by the interaction with specific proteinaceous inhibitors. In contrast to the wide variety of structurally diverse inhibitors regulating the activity of endoproteinases (4), only a few specific inhibitors that form tight complexes with carboxypeptidases have been identified (5). The only metallocarboxypeptidase inhibitors described so far are the 38 -39-residue proteins from Solanacea (potato and tomato) (6), a 65-residue protein from Ascaris suum (7), and, more recently, a 223-residue protein from rat brain (8). The proteins from Solanacea and Ascaris inhibit carboxypeptidases by a common mechanism where their C-terminal tail interacts with the target enzymes in a substrate-like manner. The C terminus of the mammalian inhibitor from rat brain, however, does not seem to be a suitable substrate for carboxypeptidases (8); this protein is thought to interact with carboxypeptidases by a small region that shows sequence similarity to the inhibitory loop in the propeptides of these enzymes. This inhibitor region may bind in a fashion similar to the long (94 -96 residues) ...

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confidence: 99%

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confidence: 99%

A Carboxypeptidase Inhibitor from the Medical Leech Hirudo medicinalis

Reverter¹,

Vendrell²,

Canals³

et al. 1998

Journal of Biological Chemistry

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“…These bioactive substances are produced and stored in unicellular salivary gland cells in the anterior body part of the leech and secreted during feeding (Ascenzi et al, 1995;Baskova et al, 1992;Baskova and Zavalova, 2001;Hildebrandt and Lemke, 2011;Kvist et al, 2013;Müller et al, 2015). We have recently shown (Hildebrandt and Lemke, 2011), and confirmed in this study (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…During feeding, leeches release bioactive salivary proteins and peptides (Baskova et al, 2004;Baskova and Zavalova, 2001;Hildebrandt and Lemke, 2011), produced in unicellular salivary glands in the anterior segments of the leech body, into the wound (Hildebrandt and Lemke, 2011). Only a handful of these substances out of up to 100 that may be present in leech saliva (Baskova et al, 2004) are known so far, among them enzymes, anti-inflammatory or antimicrobial agents and inhibitors like anti-coagulants (Ascenzi et al, 1995;Baskova et al, 1992Baskova et al, , 2008Baskova and Zavalova, 2001;Deckmyn et al, 1995;Greinacher and Warkentin, 2008;Gronwald et al, 2008;Hildebrandt and Lemke, 2011;Kvist et al, 2013;Müller et al, 2015;Rigbi et al, 1996;Vilahur et al, 2004). During one blood meal that lasts at least 20-30 min (Lent et al, 1988), up to 1 mg of salivary protein is secreted into the wound (Lemke et al, 2013), resulting in partial or complete emptying of the approximately 40,000 salivary gland cells (Lemke et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

Be ready at any time: postprandial synthesis of salivary proteins in salivary gland cells of the haematophagous leech Hirudo verbana

Lemke

Müller

Hildebrandt

2016

Journal of Experimental Biology

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Sanguivorous leeches are ectoparasites having access to body fluids of potential hosts only infrequently. During feeding, salivary proteins are released from unicellular salivary glands into the wound. These substances, among them anti-coagulants, anti-inflammatory or antimicrobial agents, allow these animals proper feeding and long-term storage of host blood in their crops for several months. Using histological, protein biochemical and molecular techniques, we investigated whether synthesis of salivary proteins and refilling of salivary gland cells occur immediately after feeding or later when stored nutrients in the crop are getting scarce. The results of the histological analyses showed that gland cell area was significantly smaller right after feeding when compared with those in unfed animals. This parameter recovered quickly and reached the control level at 1 week after feeding. 2D gel electrophoresis and analysis of the abundance of individual proteins in extracts of leech tissues revealed that a subset of proteins that had been present in extracts of unfed animals virtually disappeared during feeding, but re-appeared within 1 week of feeding (most probably secretory proteins) while another subset did not change during the experimental period (most probably housekeeping proteins). Semi-quantitative PCR analysis of hirudin cDNA prepared from leech RNA samples revealed that the amount of hirudin transcripts increased immediately after feeding, peaked at 5 days after feeding and declined to control values thereafter. Our results indicate that bloodsucking leeches synthesize salivary proteins and refill their salivary gland cell reservoirs within a week of a blood meal to be prepared for another feeding opportunity.

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“…Taking into account the structural similarity between p-aminobenzamidine, the prototype of trypsin-like serine protease inhibitors (12)(13)(14), and the I 1 -R ligands agmatine and of N-amidino-2-hydroxypyrrolidine (11) (Fig. 1), p-aminobenzamidine binding to I 1 -R in rat heart membranes has been investigated.…”

Section: Introductionmentioning

confidence: 99%

Clonidine Displacement from Type 1 Imidazoline Receptor by p ‐Aminobenzamidine, the Prototype of Trypsin‐Like Serine Protease Inhibitors

2002

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Summaryp-Aminobenzamidine inhibits competitively the catalytic activity of enzymes that recognize preferentially the L-arginyl side chain and related structures. Notably, p-aminobenzamidine is considered as the prototype of trypsin-like serine protease inhibitors. Furthermore, p-aminobenzamidine inhibits the catalytic activity of nitric oxide synthase type I and type II as well as copper amine oxidase. Taking into account the structural similarity between p-aminobenzamidine, agmatine (the putative endogenous ligand of the membrane type 1 imidazoline receptor (I 1 -R)), and N-amidino-2-hydroxypyrrolidine (the product of agmatine oxidation by copper amine oxidase), the [ 3 H]clonidine displacement from I 1 -R in rat heart membranes by p-aminobenzamidine was investigated. p-Aminobenzamidine is as effective as agmatine and N-amidino-2-hydroxypyrrolidine and more effective than the antihypertensive drug clonidine to displace [ 3 H]clonidine from I 1 -R. Therefore, trypsin-like serine protease inhibitors structurally related to p-aminobenzamidine should be administrated under careful control.IUBMB Life, 54: 301-304, 2002

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Proteinase inhibitors from the european medicinal leech Hirudo medicinalis: Structural, functional and biomedical aspects

Cited by 39 publications

References 255 publications

A Carboxypeptidase Inhibitor from the Medical Leech Hirudo medicinalis

A Carboxypeptidase Inhibitor from the Medical Leech Hirudo medicinalis

Be ready at any time: postprandial synthesis of salivary proteins in salivary gland cells of the haematophagous leech Hirudo verbana

Clonidine Displacement from Type 1 Imidazoline Receptor by p ‐Aminobenzamidine, the Prototype of Trypsin‐Like Serine Protease Inhibitors

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