Recombinant Destabilase from Hirudo medicinalis Is Able to Dissolve Human Blood Clots In Vitro.

Leeches are amazing animals that can be classified as conditionally poisonous animals since the salivary cocktail they produce is injected directly into the victim, and its components have strictly defined biological purposes, such as preventing blood clot formation. Thrombolytic drugs are mainly aimed at treating newly formed blood clots. Aged clots are stabilized by a large number of isopeptide bonds that prevent the action of thrombolytics. These bonds are destroyed by destabilase, an enzyme of the leech's salivary glands. Here, we conducted a pilot study to evaluate the feasibility and effectiveness of the use of destabilase in relation to blood clots formed during real pathological processes. We evaluated the isopeptidase activity of destabilase during the formation of a stabilized fibrin clot. We showed that destabilase does not affect the internal and external coagulation cascades. We calculated the dose-response curve and tested the ability of destabilase to destroy isopeptide bonds in natural blood clots. The effect of aged and fresh clots dissolving ability after treatment with destabilase coincided with the morphological characteristics of clots during surgery. Thus, recombinant destabilase can be considered as a potential drug for the treatment of aged clots, which are difficult to treat with known thrombolytics.

Destabilase-lysozyme-2 - original recombinant thrombolytic preparation of medicinal leech inhibits horse platelets aggregation.

The purpose. Identifying the capacity of the medicinal leech novel original recombinant thrombolytic preparation Destabilase-Lysozyme-2 to inhibit the blood platelet aggregation. Methods: Gene of destabilase-lysozyme. ds2 (mlDL-Ds2 ), was cloned in E.coli cells. Recombinant protein was isolated in denaturing conditions using metal-chelate chromatography followed by denaturation of the polypeptide by rapid dilution in exact accordance with the procedure described by Kurdyumov A.S. et al. ( 2016, Russian Journal of Bioorganic Chemistry, v.42, s. 42-52). Blood was collected from the jugular vein of 18 horses. The functional status of platelets in the presence of different destabilase-lysozyme concentrations were evaluated for their aggregation in Platelet Rich Plasma ( PRP) and in Washed Platelet suspension (WP) using aggregometers Chrono-Log-700 and Сhrono-Log-560, USA560, США. As used aggregation inducers of ADP, collagen type III and human thrombin. Results: First demonstrated the ability of newly synthesized (Kurdyumov A.S. et al. 2016, Russian Journal of Bioorganic Chemistry, v42, s. 42-52) thrombolytic recombinant enzyme destabilase-lyzosyme to inhibit more than 40% of ADP-stimulated PRP aggregation and ADP- stimulated aggregation of horse blood washed platelets. Conclusion: The ability of destabilase-lyzosyme -2 to inhibit platelets aggregation extends biological properties of recombinant thrombolytic enzyme, pre-clinical trials which resulted in the end of 2015.

A comparison of the enzymatic properties of three recombinant isoforms of thrombolytic and antibacterial protein--Destabilase-Lysozyme from medicinal leech.

BACKGROUND: Destabilase-Lysozyme (mlDL) is a multifunctional i-type enzyme that has been found in the secretions from the salivary glands of medicinal leeches. mlDL has been shown to exhibit isopeptidase, muramidase and antibacterial activity. This enzyme attracts interest because it expresses thrombolytic activity through isopeptidolysis of the ε-(γ-Glu)-Lys bonds that cross-link polypeptide chains in stabilised fibrin. To date, three isoforms of mlDL have been identified. The enzymatic properties of pure mlDL isoforms have not yet been described because only destabilase complexes containing other proteins could be isolated from the salivary gland secretion and because low product yield from the generation of recombinant proteins has made comprehensive testing difficult. RESULTS: In the present study, we optimised the procedures related to the expression, isolation and purification of active mlDL isoforms (mlDL-Ds1, mlDL-Ds2, mlDL-Ds3) using an Escherichia coli expression system, and we detected and compared their muramidase, lytic, isopeptidase and antimicrobial activities. After optimisation, the product yield was 30 mg per litre of culture. The data obtained in our study led to the suggestion that the recombinant mlDL isoforms isolated from inclusion bodies form stable oligomeric complexes. Analyses of the tested activities revealed that all isoforms exhibited almost identical patterns of pH and ionic strength effects on the activities. We determined that mlDL-Ds1, 2, 3 possessed non-enzymatic antibacterial activity independent of their muramidase activity. For the first time, we demonstrated the fibrinolytic activity of the recombinant mlDL and showed that only intact proteins possessed this activity, suggesting their enzymatic nature. CONCLUSIONS: The recombinant Destabilase-Lysozyme isoforms obtained in our study may be considered potential thrombolytic agents that act through a mechanism different from that of common thrombolytics.

Verification and refinement of cellular glycosphingolipid profiles using HPLC.

Glycosphingolipids (GSLs) are hybrid molecules consisting of the sphingolipid ceramide linked to a mono- or oligo-saccharide. In comparison to other membrane lipids, the family of GSLs stands out because of the extensive variation in the carbohydrate headgroup. GSLs are cell surface binding partners, in cis with growth factor receptors, and in trans with bacterial toxins and viruses, and are among the host-derived membrane components of viral particles, including those of HIV. In spite of their biological relevance, GSL profiles of commonly used cell lines have been analyzed to different degrees. Here, we directly compare the GSL complements from CHO-K1, COS-7, HeLa, HEK-293, HEPG2, Jurkat, and SH-SY5Y cells using an HPLC-based method requiring modest amounts of material. Compared to previous studies, the HPLC-based analyses provided more detailed information on the complexity of the cellular GSL complement, qualitatively as well as quantitatively. In particular for cells expressing multiple GSLs, we found higher numbers of GSL species, and different levels of abundance. Our study thus extends our knowledge of biologically relevant lipids in widely used cell lines.

Generation of recombinant destabilase-lysozyme from medicinal leeches in three different expression systems.

Destabilase-lysozyme (mlDL) is an enzyme secreted by the salivary gland cells of medicinal leeches. Destabilase-lysozyme possesses lysozyme and isopeptidase activities. We generated recombinant destabilase-lysozyme isoform 2 in three expression systems, i.e., in the bacteria Escherichia coli, in the yeast Pichia pastoris, and in the human cell line Expi293F. In E. coli, we generated both polypeptide in inclusion bodies that was later undergone to the refolding and soluble protein that had been fused with the chaperone SlyD. The chaperone was later cleaved by a specific TEV-protease. In cultures of the yeast P. pastoris and the human cell line Expi293F, the soluble form of destabilase-lysozyme was accumulated in the culture media. For the generated enzymes, we determined the lysozyme, isopeptidase and fibrinolytic activities and tested their general antimicrobial effects. The comparisons of the enzymes generated in the different expression systems revealed that all of the destabilase-lysozymes obtained in the soluble forms possessed equal levels of lysozyme, isopeptidase and fibrinolytic activities that exceeded several to ten times the levels of the same activities of the destabilase-lysozyme renaturated from the inclusion bodies. A similar pattern of the differences in the levels of the general antimicrobial effects was observed for the destabilase-lysozymes generated in the soluble form and as inclusion bodies.

[Highly active fractions of the medicinal leech recombinant destabilase-lysozyme].

From the highly purified but lowly active recombinant protein Destabilas-Lysozyme (Dest-Lys) by use cation-exchange column TSK CM 3-SW chromatography, it was separated non-active fraction IV, contained 90% of protein. Fractions I, II and III, represented proteins with lysozyme and isopeptidase activities. Their lysozyme activity correlates with the activity of natural Des-Lys. The ratio of the activities in fractions I - III is such, that maximal lysozyme activity is concentrated in fraction III, isopeptidase - in fraction I. It is discussed the possibility of Dest-Lys different functions regulation is depended on the formation of protein complex forms.

[Catalytic sites of medicinal leech enzyme destabilase-lysozyme (Mldl). Structure-functional correlation].

Based on three-dimensional model of the bifunctional enzyme Destabilase-Lysozyme (mlDL-Ds2) in complex with trimer of N-acetylglucosoamine (NAG)3 the functional role of the stereochemically based group of amino acids (Glu14, Asp26, Ser 29, Ser31, Lys38, His92), in manifestation of glycosidase and isopeptidase activities has been elucidated. By method of site-directed mutagenesis it has been shown that mlDL glycosidase active site includes catalytic Glu14 and Asp26, and isopeptidase site functions as Ser/Lys dyad presented by catalytic residues Lys38 and Ser29. Thus, among the invertebrate lysozymes mlDL presents first example of the bifunctional enzyme with identified position of the isopeptidase active site and localization of the corresponding catalytic residues.

Small bite, large impact-saliva and salivary molecules in the medicinal leech, Hirudo medicinalis.

Blood-sucking leeches have been used for medical purposes in humans for hundreds of years. Accordingly, one of the most prominent species has been named Hirudo medicinalis by Carl Linne in 1758. Feeding on vertebrate blood poses some serious problems to blood-sucking ectoparasites, as they have to penetrate the body surface of the host and to suppress the normal reactions of the host to such injuries (swelling, pain, inflammation) to remain undetected during the feeding period. Furthermore, the parasites have to take measures to inhibit the normal reactions in host tissues to blood vessel damage, namely hemostasis and blood coagulation (platelet aggregation and activation, activation of thrombin and formation of fibrin clots). During evolution, leeches have acquired the ability to control these processes in their hosts by transferring various bioactive substances to the host. These substances are supposedly produced in unicellular salivary gland cells and injected into the wound at the feeding site through tiny salivary ductule openings in the jaws that the leech uses to slice open the host body surface and to cut blood vessels in the depth of the wound. This review summarizes current knowledge about the salivary gland cells and the biological effects of individual saliva components as well as hints to the potential usefulness of some of these compounds for medical purposes.

[Lysozyme activity of the salivary gland secretion of the medicinal leech H. verbana, H. medicinalis and H. orientalis].

Salivary gland secretions of three species of the medicinal leech differ in the level of lysozyme peptidoglycan-lysing activity. Using the synthetic fluorogenic substrate, 4-methyl-umbelliferyl tetra N-acetyl-beta-chitotetraosid, the glycosidase activity (as one of peptidoglycan-lysing activities) of salivary gland secretion of three species of the medicinal leech was quantitatively evaluated in comparison with egg lysozyme. It is supposed, that lysozyme activity of the leech secretions is determined not only by 5 isoforms of destabilase-lysozyme, but by some other enzymes which can utilize this substrate. These may be lysozymes other than i- (invertebrate) lysozymes (such as destabilase-lysozyme, or related enzymes).

[Medicinal leeches]. / Les sangsues.

Leeches are hermaphroditic and hematophagous annelids. One important medical species, Hirudo medicinalis, comes from hirudiniculture of fresh water pools. Thanks to their three mandibles with some 300 teeth on their anterior muscular sucker, they easily grab to tissues and by secreting their saliva containing numerous powerful enzymes, such as hyaluronidase, collagenase and inhibitors of platelet aggregation and coagulation, like hirudin, allow blood sucking. Once they are full of blood (up to 15 g of blood), they detach themselves from their prey. Used ever since the 18th Egyptian Dynasty, leeches became famous during the first part of the XIXth century, thanks to a French physician, François Joseph Victor Broussais, known to his adversaries as the "vampire of medicine" for treating various conditions such as phlebotomy, laryngitis, ocular problems, obesity, mental disorders, etc. Overfishing, therapeutic failures and most particularly, the emergence of hygiene, brought the decline of living leeches. In 1884, an extract of leeches was obtained--hirudin and henceforth used. Nowadays, leeches are still used in microsurgery to enhance the venous circulation in finger reimplantation or skin flap transposition. Hirudin is synthesized through recombinant DNA technology and molecules such as lepirudin and desirudin are available on the market as anticoagulant.

[Polyfunctionality of destabilase, a lysozyme from a medicinal leech].

Experimental data indicating the polyfunctionality of destabilase, a lysozyme from the salivary gland secretion of the medicinal leech, a unique representative of invertebrate lysozymes, were analyzed. The destabilase combines the properties of endo-s-lysyl-y-glutamyl isopeptidase (D-dimer monomerase), lysozyme, and chitinase and simultaneously is a nonenzymatic antimicrobial agent. The polypeptide sequence of lysozyme destabilase is encoded by a family of three genes (Ds1, Ds2, and Ds3). The ability of the enzyme to hydrolyze endoisopeptide bonds formed by transglutaminases, which are detected in many pathological conditions, including thrombosis, is considered from the viewpoint of its further application in practice.

Antibacterial non-glycosidase activity of invertebrate destabilase-lysozyme and of its helical amphipathic peptides.

BACKGROUND: Since bactericidal properties of some lysozymes are independent of their glycosidase activity, we have investigated this phenomenon for destabilase-lysozyme (DL) from medicinal leech (Hirudo medicinalis). METHODS: Glycosidase activity was determined on Micrococcus luteus, non-enzymatic antibacterial activity of heat-treated DL and of synthetic peptides alpha1, alpha2 and alpha3 (fragments of its primary structure) on M. luteus, Escherichia coli, Bacillus brevis and Streptomyces chrysomallus. RESULTS: Glycosidase activity disappeared after the heating of native DL at 100 degrees C for 40 min. Antibacterial activity of heat-treated DL for M. luteus MDMSU128 and MDMSU140 expressed as minimal inhibitory concentration was 9.8.10(-8) and 12.10(-8) M, respectively, and to E. coli MDMSU52 11.10(-8) M. Antibacterial activity of synthetic peptide alpha1 for M. luteus MDMSU128 and for E. coli MDMSU52 was 8.3.10(-5) and 4.9.10(-5) M, respectively. CONCLUSION: DL is the first invertebrate lysozyme with combined enzymatic and non-enzymatic antibacterial action.

Sodium-dependent potassium channels in leech P neurons.

In leech P neurons the inhibition of the Na(+)-K(+) pump by ouabain or omission of bath K(+) leaves the membrane potential unaffected for a prolonged period or even induces a marked membrane hyperpolarization, although the concentration gradients for K(+) and Na(+) are attenuated substantially. As shown previously, this stabilization of the membrane potential is caused by an increase in the K(+) conductance of the plasma membrane, which compensates for the reduction of the K(+) gradient. The data presented here strongly suggest that the increased K(+) conductance is due to Na(+)-activated K(+) (K(Na)) channels. Specifically, an increase in the cytosolic Na(+) concentration ([Na(+)](i)) was paralleled by a membrane hyperpolarization, a decrease in the input resistance (R(in)) of the cells, and by the occurrence of an outwardly directed membrane current. The relationship between R(in) and [Na(+)](i) followed a simple model in which the R(in) decrease was attributed to K(+) channels that are activated by the binding of three Na(+) ions, with half-maximal activation at [Na(+)](i) between 45 and 70 mM. At maximum channel activation, R(in) was reduced by more than 90%, suggesting a significant contribution of the K(Na) channels to the physiological functioning of the cells, although evidence for such a contribution is still lacking. Injection experiments showed that the K(Na) channels in leech P neurons are also activated by Li(+).

Recombinant destabilase-lysozyme: synthesis de novo in E. coli and action mechanism of the enzyme expressed in Spodoptera frugiperda.

Destabilase-lysozyme (DL) from salivary gland secretion of the medicinal leech (Hirudo medicinalis) is as a member of the invertebrate lysozyme family, which sharply differs from other lysozyme families. In this study, DL lysozyme function was confirmed during expression of a gene encoding DL in Escherichia coli. Several constructs of the expression vectors pKK OmpA and pET-3A with or without bacterial, leech, or yeast signal peptides (SP) were used. The use of a construct without signal peptide genes resulted in normal growth of the transformed cells. Transformation of E. coli cells with the constructs containing SP was accompanied by the disruption of the forming cells. The use of the expression vector pET-32 LTC-System for production of DL as a fusion protein with thioredoxin also resulted in normal cell growth. However, specific activity of DL isolated from such cells was significantly lower than that of enzyme purified from extracts of Spodoptera frugiperda cells, which were infected with the baculovirus vector carrying DL cDNA. It is shown that the action mechanism of invertebrate lysozyme does not differ from that of other families: recombinant DL from S. frugiperda extracts catalyzed cleavage of synthetic substrate, hexamer of N-acetylglucosamine, to di- and tetramers, which is typical for enzymatic function of other lysozyme families.