Characterization of Palytoxin Binding to HaCaT Cells Using a Monoclonal Anti-Palytoxin Antibody
Palytoxin (PLTX) is the reference compound for a group of potent marine biotoxins, for which the molecular target is Na+/K+-ATPase. Indeed, ouabain (OUA), a potent blocker of the pump, is used to inhibit some PLTX effects in vitro. However, in an effort to explain incomplete inhibition of PLTX cytot...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2013-02-01
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| Series: | Marine Drugs |
| Subjects: | |
| Online Access: | http://www.mdpi.com/1660-3397/11/3/584 |
| _version_ | 1828370504498544640 |
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| author | Chiara Florio Aurelia Tubaro Silvio Sosa Marco Pelin Mark Poli Sabrina Boscolo |
| author_facet | Chiara Florio Aurelia Tubaro Silvio Sosa Marco Pelin Mark Poli Sabrina Boscolo |
| author_sort | Chiara Florio |
| collection | DOAJ |
| description | Palytoxin (PLTX) is the reference compound for a group of potent marine biotoxins, for which the molecular target is Na+/K+-ATPase. Indeed, ouabain (OUA), a potent blocker of the pump, is used to inhibit some PLTX effects in vitro. However, in an effort to explain incomplete inhibition of PLTX cytotoxicity, some studies suggest the possibility of two different binding sites on Na+/K+-ATPase. Hence, this study was performed to characterize PLTX binding to intact HaCaT keratinocytes and to investigate the ability of OUA to compete for this binding. PLTX binding to HaCaT cells was demonstrated by immunocytochemical analysis after 10 min exposure. An anti-PLTX monoclonal antibody-based ELISA showed that the binding was saturable and reversible, with a Kd of 3 × 10−10 M. However, kinetic experiments revealed that PLTX binding dissociation was incomplete, suggesting an additional, OUA-insensitive, PLTX binding site. Competitive experiments suggested that OUA acts as a negative allosteric modulator against high PLTX concentrations (0.3–1.0 × 10−7 M) and possibly as a non-competitive antagonist against low PLTX concentrations (0.1–3.0 × 10−9 M). Antagonism was supported by PLTX cytotoxicity inhibition at OUA concentrations that displaced PLTX binding (1 × 10−5 M). However, this inhibition was incomplete, supporting the existence of both OUA-sensitive and -insensitive PLTX binding sites. |
| first_indexed | 2024-04-14T06:38:52Z |
| format | Article |
| id | doaj.art-021ea625d1c9425ca52448c137e19bc3 |
| institution | Directory Open Access Journal |
| issn | 1660-3397 |
| language | English |
| last_indexed | 2024-04-14T06:38:52Z |
| publishDate | 2013-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Marine Drugs |
| spelling | doaj.art-021ea625d1c9425ca52448c137e19bc32022-12-22T02:07:23ZengMDPI AGMarine Drugs1660-33972013-02-0111358459810.3390/md11030584Characterization of Palytoxin Binding to HaCaT Cells Using a Monoclonal Anti-Palytoxin AntibodyChiara FlorioAurelia TubaroSilvio SosaMarco PelinMark PoliSabrina BoscoloPalytoxin (PLTX) is the reference compound for a group of potent marine biotoxins, for which the molecular target is Na+/K+-ATPase. Indeed, ouabain (OUA), a potent blocker of the pump, is used to inhibit some PLTX effects in vitro. However, in an effort to explain incomplete inhibition of PLTX cytotoxicity, some studies suggest the possibility of two different binding sites on Na+/K+-ATPase. Hence, this study was performed to characterize PLTX binding to intact HaCaT keratinocytes and to investigate the ability of OUA to compete for this binding. PLTX binding to HaCaT cells was demonstrated by immunocytochemical analysis after 10 min exposure. An anti-PLTX monoclonal antibody-based ELISA showed that the binding was saturable and reversible, with a Kd of 3 × 10−10 M. However, kinetic experiments revealed that PLTX binding dissociation was incomplete, suggesting an additional, OUA-insensitive, PLTX binding site. Competitive experiments suggested that OUA acts as a negative allosteric modulator against high PLTX concentrations (0.3–1.0 × 10−7 M) and possibly as a non-competitive antagonist against low PLTX concentrations (0.1–3.0 × 10−9 M). Antagonism was supported by PLTX cytotoxicity inhibition at OUA concentrations that displaced PLTX binding (1 × 10−5 M). However, this inhibition was incomplete, supporting the existence of both OUA-sensitive and -insensitive PLTX binding sites.http://www.mdpi.com/1660-3397/11/3/584palytoxinouabainbindingELISAHaCaT cells |
| spellingShingle | Chiara Florio Aurelia Tubaro Silvio Sosa Marco Pelin Mark Poli Sabrina Boscolo Characterization of Palytoxin Binding to HaCaT Cells Using a Monoclonal Anti-Palytoxin Antibody Marine Drugs palytoxin ouabain binding ELISA HaCaT cells |
| title | Characterization of Palytoxin Binding to HaCaT Cells Using a Monoclonal Anti-Palytoxin Antibody |
| title_full | Characterization of Palytoxin Binding to HaCaT Cells Using a Monoclonal Anti-Palytoxin Antibody |
| title_fullStr | Characterization of Palytoxin Binding to HaCaT Cells Using a Monoclonal Anti-Palytoxin Antibody |
| title_full_unstemmed | Characterization of Palytoxin Binding to HaCaT Cells Using a Monoclonal Anti-Palytoxin Antibody |
| title_short | Characterization of Palytoxin Binding to HaCaT Cells Using a Monoclonal Anti-Palytoxin Antibody |
| title_sort | characterization of palytoxin binding to hacat cells using a monoclonal anti palytoxin antibody |
| topic | palytoxin ouabain binding ELISA HaCaT cells |
| url | http://www.mdpi.com/1660-3397/11/3/584 |
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