Vimentin-mediated cellular uptake of C3 exoenzyme
Final Report Abstract
The current project focused on the detailed role of the recently by us identified C3bot binding partner vimentin and investigating other additional C3bot surface binding partners. Also, the cellular release mechanisms resulting in the extracellular presentation of vimentin by astrocytes were investigated. By using novel purified glia-free GABAergig and glutamatergic neuronal cultures the role of vimentin for the neurotrophic effects of C3bot was unraveled. Furthermore, C3bot internalization in vimentin knock-out cells and HT22 was examined. In the first part, we could show that knock-out of vimentin resulted in reduced binding of C3bot to different cells. Extracellular application of vimentin to vim-/- cells restored full binding of C3bot. Our amino acid sequence alignment of different C3 isoforms revealed that C3 exoenzymes harbor a RGD-motif. Indeed, competition of C3bot with GRGDNP peptide or with monoclonal antibodies to β1- integrin and to α5-integrin subunit strongly reduced binding of C3bot to intact cells. Additionally, activation of integrins via manganese (II) chloride enhanced binding of C3bot. C3bot-RGD-mutants exhibited significantly reduced binding to different cells. As proof of principle, we demonstrated by use of ELISA a direct binding between C3bot and recombinant α5β1-integrin. In sum, these findings indicate that in addition to vimentin α5β1-integrin is crucial for binding of C3bot. By using an in vitro scratch wound model, we were able to show by a combination of methods for the first time that injured astrocytes release vimentin by exosomal secretion. Extracellular release was also shown in vivo by double-staining with extracellular matrix markers such as CSPGs in the lesion area of the rat spinal cord following injury. Incubation of synaptosomes with recombinant vimentin or exosomes from wild type but not vim-/- astrocytes enhanced binding of C3bot to synaptosomes thereby confirming the positive effects of vimentin for neuronal growth promoting C3bot effects. In the second part, we show that knock-out of vimentin resulted in delayed uptake of C3bot in vim-/- fibroblasts compared to wild type fibroblasts. The subcellular distribution of C3bot in HT22 cells between membrane organelles and cytosol were analyzed. C3bot is degraded by lysosomes and the proteasome. In sum, these findings indicate that C3bot entered cells vimentin/integrin-dependently via membrane organelles within 30 min. Finally, by using a novel NexCre;Ai9xVGAT Venus transgenic mouse line we were able to show that purified GABAergic neurons express higher levels of vimentin than glutamatergic neurons, accompanied by enhanced binding of C3bot and a higher sensitivity to axonontrophic and dendritotrophic effects.
Publications
- Functional role of Arg-Gly-Asp (RGD)-Binding site of C3, P142. Naunyn-Schmiedeberg's Arch Pharmacol 390, 1–101 (2017)
A. Rohrbeck, S. Hagemann, I. Just
(See online at https://doi.org/10.1007/s00210-017-1354-7) - The Rho ADP-ribosylating C3 exoenzyme binds cells via an Arg-Gly-Asp motif. J Biol Chem. 2017 Oct 27; 292(43):17668-17680
Rohrbeck A, Höltje M, Adolf A, Oms E, Hagemann S, Ahnert-Hilger G, Just I
(See online at https://doi.org/10.1074/jbc.m117.798231) - MS-based quantification of RhoA/B and RhoC ADP-ribosylation. J Chromatogr B Analyt Technol Biomed Life Sci. 2018 Aug 15;1092:268-271
Schröder A, Benski A, Oltmanns A, Just I, Rohrbeck A, Pich A
(See online at https://doi.org/10.1016/j.jchromb.2018.06.007) - Role of integrin α5β1 in binding of C3 exoenzyme, P132. Naunyn-Schmiedeberg's Arch Pharmacol 391, 1–93 (2018
N. Schecker, S. Hagemann, I. Just, A. Rohrbeck
(See online at https://doi.org/10.1007/s00210-018-1477-5) - Inhibition of endosomal/lysosomal degradation protects cellular C3, P177. Naunyn-Schmiedeberg's Arch Pharmacol 392, 1–88 (2019)
A. Rohrbeck, S. Hagemann, I. Just
(See online at https://doi.org/10.1007/s00210-019-01621-6) - Release of astroglial vimentin by extracellular vesicles: Modulation of binding and internalization of C3 transferase in astrocytes and neurons. Glia. 2019 Apr; 67(4):703-717
Adolf A, Rohrbeck A, Münster-Wandowski A, Johansson M, Kuhn HG, Kopp MA, Brommer B, Schwab JM, Just I, Ahnert-Hilger G, Höltje M
(See online at https://doi.org/10.1002/glia.23566) - The higher Sensitivity of GABAergic compared to glutamatergic neurons to growth-promoting C3bot treatment is mediated by vimentin. Front Cell Neuroscience. 2020 Nov 3; 14:596072
Adolf A, Turko P, Rohrbeck A, Just I, Vida I, Ahnert-Hilger G, Höltje M
(See online at https://doi.org/10.3389/fncel.2020.596072) - Vimentin and integrin are involved in uptake of Clostridium botulinum C3 exoenzyme, P218. Naunyn-Schmiedeberg's Arch Pharmacol 393, 1–97 (2020)
J. Wehmeyer, S. Hagemann, I. Just, A. Rohrbeck
(See online at https://doi.org/10.1007/s00210-020-01828-y)
