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

Manuvera, Valentin A.; Kurdyumov, Alexey S.; Filonova, Kseniya A.; Лазарев, В. Н.

doi:10.1016/j.pep.2015.08.012

Cited by 5 publications

(5 citation statements)

References 19 publications

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“…6 in Ref. 37 ) that the latter approach results in a protein with extremely low isopeptidase activity and with circular dichroism (CD) spectra of denatured protein, while the protein from Expi293F shows high activity and reasonable CD spectra. Therefore, we suggest that the contradiction originates from low functional expression in E.coli and mutagenesis experiments should be reproduced with the protein obtained from Expi293F cell line.…”

Section: Resultsmentioning

confidence: 99%

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Structural insights into thrombolytic activity of destabilase from medicinal leech

Marin

Kornilov

Bukhdruker

et al. 2023

Sci Rep

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Destabilase from the medical leech Hirudo medicinalis belongs to the family of i-type lysozymes. It has two different enzymatic activities: microbial cell walls destruction (muramidase activity), and dissolution of the stabilized fibrin (isopeptidase activity). Both activities are known to be inhibited by sodium chloride at near physiological concentrations, but the structural basis remains unknown. Here we present two crystal structures of destabilase, including a 1.1 Å-resolution structure in complex with sodium ion. Our structures reveal the location of sodium ion between Glu34/Asp46 residues, which were previously recognized as a glycosidase active site. While sodium coordination with these amino acids may explain inhibition of the muramidase activity, its influence on previously suggested Ser49/Lys58 isopeptidase activity dyad is unclear. We revise the Ser49/Lys58 hypothesis and compare sequences of i-type lysozymes with confirmed destabilase activity. We suggest that the general base for the isopeptidase activity is His112 rather than Lys58. pKa calculations of these amino acids, assessed through the 1 μs molecular dynamics simulation, confirm the hypothesis. Our findings highlight the ambiguity of destabilase catalytic residues identification and build foundations for further research of structure–activity relationship of isopeptidase activity as well as structure-based protein design for potential anticoagulant drug development.

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Section: Resultsmentioning

confidence: 99%

“…The preparation of recombinant destabilase was described in detail earlier 37 . Briefly, DNA fragments encoding destabilase isoform Ds2 (UniProt ID Q25091) were synthesized from oligonucleotides.…”

Section: Methodsmentioning

confidence: 99%

Structural insights into thrombolytic activity of destabilase from medicinal leech

Marin

Kornilov

Bukhdruker

et al. 2023

Sci Rep

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“…In C. elegans , genes encoding SCPs have also been shown to be upregulated during infections by several bacterial pathogens [55] . Finally, destabilases exert antibacterial activity by enzymatically cleaving a glycosidic linkage within the peptidoglycan of bacterial cell walls [68] . Further optimisation of experimental conditions, such as culture temperature, methanol concentration, and vector feeding strategy, is required to achieve expression of Tc his, T scp, and T des [69] .…”

Section: Discussionmentioning

confidence: 99%

Structural and functional analyses of nematode-derived antimicrobial peptides support the occurrence of direct mechanisms of worm-microbiota interactions

Rooney,

Rivera-de-Torre,

et al. 2024

Computational and Structural Biotechnology Journal

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“…The isolation and purification of recombinant destabilase were performed as described previously [33]. Briefly, for protein production, the Expi293F cell line was transfected with a plasmid (pcDNA3.4-Dest2), expressing recombinant destabilase in the culture medium.…”

Section: Recombinant Destabilase Isolationmentioning

confidence: 99%

“…Furthermore, all three known isoforms of destabilase from medicinal leeches were expressed in E. coli in the form of recombinant proteins [32]. Then, the second isoform of recombinant destabilase was obtained in human cells and characterized [33]. We described the effect of destabilase on stabilized fibrin and the product of its limited proteolysis, the D-dimer, the monomers of which are linked by isopeptide bonds [34].…”

Section: Introductionmentioning

confidence: 99%

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

Bobrovsky

Manuvera

Baskova

et al. 2021

CIMB

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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.

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

Cited by 5 publications

References 19 publications

Structural insights into thrombolytic activity of destabilase from medicinal leech

Structural insights into thrombolytic activity of destabilase from medicinal leech

Structural and functional analyses of nematode-derived antimicrobial peptides support the occurrence of direct mechanisms of worm-microbiota interactions

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

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