A recombinant fusion toxin based on enzymatic inactive C3bot1 selectively targets macrophages.
BACKGROUND: The C3bot1 protein (~23 kDa) from Clostridium botulinum ADP-ribosylates and thereby inactivates Rho. C3bot1 is selectively taken up into the cytosol of monocytes/macrophages but not of other cell types such as epithelial cells or fibroblasts. Most likely, the internalization occurs by a...
Main Authors: | , , , , , , |
---|---|
Format: | Article |
Language: | English |
Published: |
Public Library of Science (PLoS)
2013-01-01
|
Series: | PLoS ONE |
Online Access: | http://europepmc.org/articles/PMC3549961?pdf=render |
id |
doaj-5e295bbd9bbf47baa028eda685f33b3a |
---|---|
record_format |
Article |
spelling |
doaj-5e295bbd9bbf47baa028eda685f33b3a2020-11-25T01:48:09ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-0181e5451710.1371/journal.pone.0054517A recombinant fusion toxin based on enzymatic inactive C3bot1 selectively targets macrophages.Lydia DmochewitzChristina FörtschChristian ZwergerMartin VaethEdward FelderMarkus Huber-LangHolger BarthBACKGROUND: The C3bot1 protein (~23 kDa) from Clostridium botulinum ADP-ribosylates and thereby inactivates Rho. C3bot1 is selectively taken up into the cytosol of monocytes/macrophages but not of other cell types such as epithelial cells or fibroblasts. Most likely, the internalization occurs by a specific endocytotic pathway via acidified endosomes. METHODOLOGY/PRINCIPAL FINDINGS: Here, we tested whether enzymatic inactive C3bot1E174Q serves as a macrophage-selective transport system for delivery of enzymatic active proteins into the cytosol of such cells. Having confirmed that C3bot1E174Q does not induce macrophage activation, we used the actin ADP-ribosylating C2I (∼50 kDa) from Clostridium botulinum as a reporter enzyme for C3bot1E174Q-mediated delivery into macrophages. The recombinant C3bot1E174Q-C2I fusion toxin was cloned and expressed as GST-protein in Escherichia coli. Purified C3bot1E174Q-C2I was recognized by antibodies against C2I and C3bot and showed C2I-specific enzyme activity in vitro. When applied to cultured cells C3bot1E174Q-C2I ADP-ribosylated actin in the cytosol of macrophages including J774A.1 and RAW264.7 cell lines as well as primary cultured human macrophages but not of epithelial cells. Together with confocal fluorescence microscopy experiments, the biochemical data indicate the selective uptake of a recombinant C3-fusion toxin into the cytosol of macrophages. CONCLUSIONS/SIGNIFICANCE: In summary, we demonstrated that C3bot1E174Q can be used as a delivery system for fast, selective and specific transport of enzymes into the cytosol of living macrophages. Therefore, C3-based fusion toxins can represent valuable molecular tools in experimental macrophage pharmacology and cell biology as well as attractive candidates to develop new therapeutic approaches against macrophage-associated diseases.http://europepmc.org/articles/PMC3549961?pdf=render |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Lydia Dmochewitz Christina Förtsch Christian Zwerger Martin Vaeth Edward Felder Markus Huber-Lang Holger Barth |
spellingShingle |
Lydia Dmochewitz Christina Förtsch Christian Zwerger Martin Vaeth Edward Felder Markus Huber-Lang Holger Barth A recombinant fusion toxin based on enzymatic inactive C3bot1 selectively targets macrophages. PLoS ONE |
author_facet |
Lydia Dmochewitz Christina Förtsch Christian Zwerger Martin Vaeth Edward Felder Markus Huber-Lang Holger Barth |
author_sort |
Lydia Dmochewitz |
title |
A recombinant fusion toxin based on enzymatic inactive C3bot1 selectively targets macrophages. |
title_short |
A recombinant fusion toxin based on enzymatic inactive C3bot1 selectively targets macrophages. |
title_full |
A recombinant fusion toxin based on enzymatic inactive C3bot1 selectively targets macrophages. |
title_fullStr |
A recombinant fusion toxin based on enzymatic inactive C3bot1 selectively targets macrophages. |
title_full_unstemmed |
A recombinant fusion toxin based on enzymatic inactive C3bot1 selectively targets macrophages. |
title_sort |
recombinant fusion toxin based on enzymatic inactive c3bot1 selectively targets macrophages. |
publisher |
Public Library of Science (PLoS) |
series |
PLoS ONE |
issn |
1932-6203 |
publishDate |
2013-01-01 |
description |
BACKGROUND: The C3bot1 protein (~23 kDa) from Clostridium botulinum ADP-ribosylates and thereby inactivates Rho. C3bot1 is selectively taken up into the cytosol of monocytes/macrophages but not of other cell types such as epithelial cells or fibroblasts. Most likely, the internalization occurs by a specific endocytotic pathway via acidified endosomes. METHODOLOGY/PRINCIPAL FINDINGS: Here, we tested whether enzymatic inactive C3bot1E174Q serves as a macrophage-selective transport system for delivery of enzymatic active proteins into the cytosol of such cells. Having confirmed that C3bot1E174Q does not induce macrophage activation, we used the actin ADP-ribosylating C2I (∼50 kDa) from Clostridium botulinum as a reporter enzyme for C3bot1E174Q-mediated delivery into macrophages. The recombinant C3bot1E174Q-C2I fusion toxin was cloned and expressed as GST-protein in Escherichia coli. Purified C3bot1E174Q-C2I was recognized by antibodies against C2I and C3bot and showed C2I-specific enzyme activity in vitro. When applied to cultured cells C3bot1E174Q-C2I ADP-ribosylated actin in the cytosol of macrophages including J774A.1 and RAW264.7 cell lines as well as primary cultured human macrophages but not of epithelial cells. Together with confocal fluorescence microscopy experiments, the biochemical data indicate the selective uptake of a recombinant C3-fusion toxin into the cytosol of macrophages. CONCLUSIONS/SIGNIFICANCE: In summary, we demonstrated that C3bot1E174Q can be used as a delivery system for fast, selective and specific transport of enzymes into the cytosol of living macrophages. Therefore, C3-based fusion toxins can represent valuable molecular tools in experimental macrophage pharmacology and cell biology as well as attractive candidates to develop new therapeutic approaches against macrophage-associated diseases. |
url |
http://europepmc.org/articles/PMC3549961?pdf=render |
work_keys_str_mv |
AT lydiadmochewitz arecombinantfusiontoxinbasedonenzymaticinactivec3bot1selectivelytargetsmacrophages AT christinafortsch arecombinantfusiontoxinbasedonenzymaticinactivec3bot1selectivelytargetsmacrophages AT christianzwerger arecombinantfusiontoxinbasedonenzymaticinactivec3bot1selectivelytargetsmacrophages AT martinvaeth arecombinantfusiontoxinbasedonenzymaticinactivec3bot1selectivelytargetsmacrophages AT edwardfelder arecombinantfusiontoxinbasedonenzymaticinactivec3bot1selectivelytargetsmacrophages AT markushuberlang arecombinantfusiontoxinbasedonenzymaticinactivec3bot1selectivelytargetsmacrophages AT holgerbarth arecombinantfusiontoxinbasedonenzymaticinactivec3bot1selectivelytargetsmacrophages AT lydiadmochewitz recombinantfusiontoxinbasedonenzymaticinactivec3bot1selectivelytargetsmacrophages AT christinafortsch recombinantfusiontoxinbasedonenzymaticinactivec3bot1selectivelytargetsmacrophages AT christianzwerger recombinantfusiontoxinbasedonenzymaticinactivec3bot1selectivelytargetsmacrophages AT martinvaeth recombinantfusiontoxinbasedonenzymaticinactivec3bot1selectivelytargetsmacrophages AT edwardfelder recombinantfusiontoxinbasedonenzymaticinactivec3bot1selectivelytargetsmacrophages AT markushuberlang recombinantfusiontoxinbasedonenzymaticinactivec3bot1selectivelytargetsmacrophages AT holgerbarth recombinantfusiontoxinbasedonenzymaticinactivec3bot1selectivelytargetsmacrophages |
_version_ |
1725012644980588544 |