Vimentin Mediates Uptake of C3 Exoenzyme

<div><p><i>Clostridium botulinum</i> C3 exoenzyme (C3) selectively inactivates RhoA/B/C GTPases by ADP-ribosylation. Based on this substrate specificity C3 is a well-established tool in cell biology. C3 is taken up by eukaryotic cells although lacking an uptake and translocation domain. Based on different approaches vimentin was identified as membranous C3-interaction partner by mass spectrometry. Vimentin in fact was partly localized at the outer surface of hippocampal HT22 cells and J744A.1 macrophages. Domain analysis identified the rod domain as binding partner of C3. Vimentin was also involved in uptake of C3 as shown by knock down of vimentin in HT22 and J774A.1 cells. The involvement of vimentin in uptake of C3 was further supported by the findings that the vimentin disruptor acrylamide blocked uptake of C3. Vimentin is not only a major organizing element of the intermediate filament network but is also involved in both binding and uptake of C3 exoenzyme.</p></div

Uptake of C3 in HT22 and J744A.1 cells is dependent on vimentin distribution and integrity.

<p>A) Influence of Vim-siRNA knock down (for 48 h) on the uptake of C3 into HT22 cells detected as RhoA degradation (induced by C3-catalysed ADP-ribosylation). In a pulse-chase experiment, HT22 cells were incubated with C3 (500 nM) at 4°C for 60 min. Afterwards unbound C3 was removed by washing the cells three times with PBS and fresh medium was added. Cells were then cultivated for further 48 h. Cell lysates were generated and separated by SDS-PAGE followed by Western blot analysis probing RhoA and β-actin. One representative experiment is shown (n = 3 independent experiments). B) Cellular levels of RhoA proteins were quantified by densitometric evaluation of RhoA (from A) and adjusted to the corresponding actin band. C) HT22 cells were pre-treated with acrylamide (5 mM) for 30 min followed by incubation with C3 (500 nM) for 24 h. Cells were lysed and submitted to Western blot analysis probing RhoA and β-actin. C3 alone causes a complete mol weight shift of RhoA in SDS-PAGE. Western blot analysis of one representative experiment is shown (n = 3 independent experiments). D) RhoA shift (indicative of Rho-ADP-ribosylation) by quantified by densitometric evaluation of RhoA (from C) and adjusted to the corresponding β-actin signal. E) Influence of Vim-siRNA knock down (for 48 h) on the uptake of C3 into J774A.1 cells detected as incomplete RhoA ADP-ribosylation. J774A.1 macrophages were incubated with C3 (500 nM) at 37°C for 4 h. Cell lysates were generated and separated by SDS-PAGE followed by Western blot analysis probing RhoA and β-actin. One representative experiment is shown (n = 3 independent experiments). F) J774A.1 cells were pre-treated with acrylamide (5 mM) for 30 min followed by incubation with C3 (500 nM) for 4 h. Cells were lysed and submitted to Western blot analysis probing RhoA and β-actin.</p

Analysis of vimentin distribution in analyzed cells.

<p>A) Vimentin was detected by anti-vimentin at the cell surface of HT22 cells transfected with siRNA for 48 h at 37°C by FACS analysis. B) Confocal microscopy of vimentin in HT22 cells transfected for 48 h at 37°C. The green (oregon green 488) anti-vimentin, DNA staining in blue (Dapi), rhodamine red-staining for actin and a merge image are shown for each panel. In the enlarged images the cell boundaries are shown. Significant difference between the vimentin distribution was detected for the transfected cells (lower panel) in comparison to the control (upper panel). Scale bar = 20 µM. C) Detection of vimentin at the cell surface of J774A.1 cells transfected with siRNA. D) Confocal microscopy of vimentin distribution in J774A.1 cells transfected for 48 h.</p

Binding of C3 to HT22 cells after pronase treatment.

<p>A) Pronase pre-incubated HT22 cells were exposed to 100 or 500 nM of C3 for 1 h at 4°C. Subsequently, β-actin and bound C3 were detected by Western blot. NC = negative control without C3, PC = positive control lysate with 10 ng C3. One representative experiment is shown (n = 3 independent experiments). B) Pronase-treated HT22 cells were exposed to 500 nM of C3-E174Q-FITC for 1 h at 4°C and bound C3- E174Q-FITC was analyzed by FACS.</p

C3-overlay (binding of C3 to HT22 proteins).

<p>A) Whole cell lysate, cytosolic fraction or particulate fraction from HT22 cells were generated as described in material and methods followed by separation through SDS-PAGE and transfer onto nitrocellulose. Nitrocellulose was incubated with 10 µg/ml of C3 for 60 min at 4°C. After washing bound C3 was detected by anti-C3. Arrows indicate the protein of interest (55 kDa). B) The right panel shows the anti-C3 Western blot without C3-overlay. M = molecular mass marker, WCL = whole cell lysate, PF = particulate fraction, CF = cytosolic fraction, WCL +10 ng C3 = C3 was added to whole cells lysate prior to SDS-PAGE and blotting to generate a positive C3 signal.</p

Image_2_Brain blood vessel autoantibodies in patients with NMDA and GABAA receptor encephalitis: identification of unconventional Myosin-X as target antigen.TIF

Introduction: The antibody repertoire from CSF-derived antibody-secreting cells and memory B-cells in patients with encephalitis contains a considerable number of antibodies that do not target the disease-defining autoantigen such as the GABA or NMDA receptors. This study focuses on the functional relevance of autoantibodies to brain blood vessels in patients with GABAA and NMDA receptor encephalitis.Methods: We tested 149 human monoclonal IgG antibodies from the cerebrospinal fluid of six patients with different forms of autoimmune encephalitis on murine brain sections for reactivity to blood vessels using immunohistochemistry. Positive candidates were tested for reactivity with purified brain blood vessels, effects on transendothelial electrical resistance (TEER), and expression of tight junction proteins as well as gene regulation using human brain microvascular endothelial hCMEC/D3 cells as in vitro blood-brain barrier model. One blood-vessel reactive antibody was infused intrathecally by pump injection in mice to study in vivo binding and effects on tight junction proteins such as Occludin. Target protein identification was addressed using transfected HEK293 cells.Results: Six antibodies reacted with brain blood vessels, three were from the same patient with GABAAR encephalitis, and the other three were from different patients with NMDAR encephalitis. One antibody from an NMDAR encephalitis patient, mAb 011-138, also reacted with cerebellar Purkinje cells. In this case, treatment of hCMEC/D3 cells resulted in decreased TEER, reduced Occludin expression, and mRNA levels. Functional relevance in vivo was confirmed as Occludin downregulation was observed in mAb 011-138-infused animals. Unconventional Myosin-X was identified as a novel autoimmune target for this antibody.Discussion: We conclude that autoantibodies to blood vessels occur in autoimmune encephalitis patients and might contribute to a disruption of the blood-brain barrier thereby suggesting a potential pathophysiological relevance of these antibodies.</p