Production and characterization of monoclonal antibodies against vaginolysin: Mapping of a region critical for its cytolytic activity

Žvirblienė, Aurelija; Plečkaitytė, Milda; Lasickienė, Rita; Kučinskaitė-Kodzė, Indrė; Zvirblis, Gintautas

doi:10.1016/j.toxicon.2010.03.007

Cited by 31 publications

(73 citation statements)

References 28 publications

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“…This means that the initial state of tBLMs cannot be restored by removing toxin from the system. This type of damage is common for other pore-forming toxins, which attach to and oligomerize on the surface of the phospholipid bilayers [8,22]. Moreover, to confirm the cholesterol-dependent nature of pyolysin we performed pre-experiments where PLO was pre-incubated with different amounts of cholesterol prior to its addition to tethered membranes.…”

Section: Discussionmentioning

confidence: 99%

“…Usually, CDCs activity is determined by in vitro assays using red blood cells or specific cell lines [8], however recently an alternative bioanalytical technique based on the use of tethered bilayer membranes (tBLMs) was proposed [9]. The formation of defects or water-filled pores in A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 3 tBLMs can be easily sensed and followed, in real-time, by electrochemical impedance spectroscopy (EIS) and opens the possibility of tBLMs use in bioanalytical applications [10,11].…”

Section: Introductionmentioning

confidence: 99%

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Tethered bilayer membranes as a complementary tool for functional and structural studies: The pyolysin case

Preta

Jankunec

Heinrich

et al. 2016

Biochimica et Biophysica Acta (BBA) - Biomembranes

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We demonstrate the use of tethered bilayer lipid membranes (tBLMs) as an experimental platform for functional and structural studies of membrane associated proteins by electrochemical techniques. The reconstitution of the cholesterol-dependent cytolysin (CDC) pyolysin (PLO) from Trueperella pyogenes into tBLMs was followed in real-time by electrochemical impedance spectroscopy (EIS). Changes of the EIS parameters of the tBLMs upon exposure to PLO solutions were consistent with the dielectric barrier damage occurring through the formation of water-filled pores in membranes. Parallel experiments involving a mutant version of PLO, which is able to bind to the membranes but does not form oligomer pores, strengthen the reliability of this methodology, since no change in the electrochemical impedance was observed. Complementary atomic force microscopy (AFM) and neutron reflectometry (NR) measurements revealed structural details of the membrane bound PLO, consistent with the structural transformations of the membrane bound toxins found for other cholesterol dependent cytolysins. In this work, using the tBLMs platform we also observed a protective effect of the dynamin inhibitor Dynasore against pyolysin as well as pneumolysin. An effect of Dynasore in tBLMs, which was earlier observed in experiments with live cells, confirms the biological relevance of the tBLMs models, as well as demonstrates the potential of the electrochemical impedance spectroscopy to quantify membrane damage by the pore forming toxins. In conclusion, tBLMs are a reliable and complementary method to explore the activity of CDCs in eukaryotic cells and to develop strategies to limit the toxic effects of CDCs.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Tethered bilayer membranes as a complementary tool for functional and structural studies: The pyolysin case

Preta

Jankunec

Heinrich

et al. 2016

Biochimica et Biophysica Acta (BBA) - Biomembranes

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“…These CDCs have been considered atypical because of their preference for human cells (Gelber et al, 2008; Nagamune et al, 1996; Tabata et al, 2014; Zvirbliene et al, 2010), in contrast to the majority of CDCs that can lyse most eukaryotic cells. ILY is strictly specific for human cells (Nagamune et al, 1996), whereas VLY can bind to non-human cells providing they are cholesterol rich (Zilnyte et al, 2015), and LLY may behave similarly (Tabata et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

Structural Basis for Receptor Recognition by the Human CD59-Responsive Cholesterol-Dependent Cytolysins

et al. 2016

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SUMMARY Cholesterol-dependent cytolysins (CDCs) are a family of pore-forming toxins that punch holes in the outer membrane of eukaryotic cells. Cholesterol serves as the receptor, but a subclass of CDCs first binds to human CD59. Here we describe the crystal structures of vaginolysin and intermedilysin complexed to CD59. These studies, together with small-angle X-ray scattering, reveal that CD59 binds to each at different, though overlapping, sites, consistent with molecular dynamics simulations and binding studies. The CDC consensus undecapeptide motif, which for the CD59-responsive CDCs has a proline instead of a tryptophan in the motif, adopts a strikingly different conformation between the structures; our data suggest that the proline acts as a selectivity switch to ensure CD59-dependent CDCs bind their protein receptor first in preference to cholesterol. The structural data suggest a detailed model of how these water-soluble toxins assemble as prepores on the cell surface.

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“…The apparent dissociation constants (K d ) of the MAbs were determined by an indirect ELISA as described previously 33 . Briefly, the MAbs were prepared in concentrations ranging from 1.9 Â 10 À13 M to 3.3 Â 10 À8 M and incubated in the microtiter plates coated with recombinant CA XII.…”

Section: Determination Of the Apparent Dissociation Constant (K D )mentioning

confidence: 99%

Monoclonal antibodies raised against 167–180 aa sequence of human carbonic anhydrase XII inhibit its enzymatic activity

Dekaminaviciute

Kairys

Zilnyte

et al. 2014

Journal of Enzyme Inhibition and Medicinal Chemistry

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Human carbonic anhydrase XII (CA XII) is a single-pass transmembrane protein with an extracellular catalytic domain. This enzyme is being recognized as a potential biomarker for different tumours. The current study was aimed to generate monoclonal antibodies (MAbs) neutralizing the enzymatic activity of CA XII. Bioinformatics analysis of CA XII structure revealed surface-exposed sequences located in a proximity of its catalytic centre. Two MAbs against the selected antigenic peptide spanning 167-180 aa sequence of CA XII were generated. The MAbs were reactive with recombinant catalytic domain of CA XII expressed either in E. coli or mammalian cells. Inhibitory activity of the MAbs was demonstrated by a stopped flow CO 2 hydration assay. The study provides new data on the surface-exposed linear CA XII epitope that may serve as a target for inhibitory antibodies with a potential immunotherapeutic application. KeywordsHuman carbonic anhydrase XII, inhibition of enzymatic activity, monoclonal antibodies History

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Production and characterization of monoclonal antibodies against vaginolysin: Mapping of a region critical for its cytolytic activity

Cited by 31 publications

References 28 publications

Tethered bilayer membranes as a complementary tool for functional and structural studies: The pyolysin case

Tethered bilayer membranes as a complementary tool for functional and structural studies: The pyolysin case

Structural Basis for Receptor Recognition by the Human CD59-Responsive Cholesterol-Dependent Cytolysins

Monoclonal antibodies raised against 167–180 aa sequence of human carbonic anhydrase XII inhibit its enzymatic activity

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