Intracellular trafficking of diphtheria toxin and its mutated form, CRM197, in the endocytic pathway.

OBJECTIVE: Diphtheria toxin (DTx) is a well-characterized bacterial toxin. However, the endocytic pathway of the mutant of DTx, CRM197, which is used as an immunological adjuvant, has not yet been fully explained. The aim of this study was to investigate the intracellular trafficking of CRM197-loaded endosomes. METHODS: Human umbilical vein endothelial cells (HUVECs) were used in a cell culture. The effective incubation time was determined by transmission electron microscopy in toxin-treated cells. Density gradient centrifugation and ADP-ribosylation assay were used to isolate and detect toxin-loaded endosomal fractions. Endosomal fractions from CRM197-treated cells were elicited after 15 minutes of incubation and the presence of fragment A was demonstrated using Western blot. Immunofluorescence microscopy was used to identify endosomes in CRM197-treated endothelial cells. RESULTS: DTx-loaded endosomes were detected as enlarged vesicles in the perinuclear area with 15 minutes of toxin treatment. DTx-loaded endosomal fractions were determined by ADP-ribosyltransferase activity test and Western blot analysis. Enzymatic activity of the toxin-loaded endosomal fraction increased by 20% in actin cytoskeletal-damaged cells treated with cytochalasin D. The steps for the toxin treatment of HUVECs with DTx and obtaining endosomal fractions were repeated for CRM197. In the CRM197-loaded endosomal fraction, actin and Hsp90 were identified in addition to fragment A. Fluorescent images revealed that CRM197-loaded endosomes were co-localized with actin filaments and that Rab11, which signals the return to the plasma membrane, was more prominent than Rab7, the lysosomal pathway indicator. CONCLUSION: These results suggest that CRM197-loaded endosomes participate in the recycling pathway.

Cross-reacting material 197 (CRM197) affects actin cytoskeleton of endothelial cells.

CRM197, cross-reacting material 197, is a mutant of diphtheria toxin (DTx). CRM197 is used in pharmacology as a carrier protein. It has been recently shown that CRM197 causes breakdown in actin filaments. In order to show intracellular localization of CRM197 and visualize cell structure via actin cytoskeleton, endothelial cells were cultured and subjected to CRM197 in vitro. To address the interaction between CRM197 and actin both experimental and theoretical studies were carried out. Colocalization of CRM197 with actin filaments was determined by immunofluorescence microscopy. Following 24-hour incubation, the loss of cell-cell contact between cells was prominent. CRM197 was shown to bind to G-actin by gel filtration chromatography, and this binding was confirmed by Western blot analysis of eluted samples obtained following chromatography. Based on crystal structure, docked model of CRM197-actin complex was generated. Molecular dynamics simulation revealed that Lys42, Cys218, Cys233 of CRM197 interacts with Gly197, Arg62 and Ser60 of G-actin, respectively. CRM197 binding to G-actin, colocalization of CRM197 with actin filament, and actin cytoskeleton rearrangement resulting in the loss of cell-cell contact show that actin comes into sight as target molecule for CRM197.

Interleukin-1ß effect on the endogenous ADP-ribosylation and phosphorylation of eukaryotic elongation factor 2.

Eukaryotic elongation factor 2 (eEF2) plays an important role in eukaryotic polypeptide chain elongation. Adenosine diphosphate (ADP)-ribosylation is a post-translational modification reaction that catalyzes the transfer of ADP-ribose group to eEF2 and this causes the inhibition of protein synthesis. Indeed, in the absence of diptheria toxin, endogenous ADP-ribosylation can occur. eEF2 is phosphorylated by eEF2 kinase which prevents binding to ribosomes thus inhibiting its activity. Increase in endogenous ADP-ribosylation level approximately 70-75 % was observed in IL-1ß treated HUVECs. Moreover, a 70 % rise of phosphorylation of eEF2 was measured. Alteration of endogenous ADP-ribosylation of eEF2 activity was related with cellular mono-ADP-ribosyltransferases (ADPrT). Increment of endogenous ADP-ribosylation on eEF2 did not seem to occur as a direct effect of IL-1ß; it arises from the activation of ADPrT. This 2.5 fold increase was abolished by ADPrT inhibitors. Due to these post-translational modifications, global protein synthesis is inhibited. After dephosphorylation of phospho-eEF2, around 20 % increase in protein synthesis was observed. In conclusion, systemic IL-1ß has an important role in the regulation of global protein synthesis.

Clinical significance of serum ADP-ribosylation and NAD glycohydrolase activity in patients with colorectal cancer.

The objective of this study was to evaluate the clinical significance of serum ADP-ribosylation and NAD glycohydrolase activity in patients with colorectal cancer (CRC). A total of 108 patients with CRC who underwent curative surgery and 20 healthy volunteers were enrolled in this study. ADP-ribosylation and NAD glycohydrolase activity levels were determined. The association of ADP-ribosylation and NAD glycohydrolase with clinical and laboratory factors and their impact on overall survival (OS) and disease free survival (DFS) were shown. The preoperative ADP-ribosylation and NAD glycohydrolase activity levels were significantly higher in patients with CRC than in the control group (p<0.001). ADP-ribosylation and NAD glycohydrolase activity levels were correlated with tumor stage (p=0.05, p=0.001), stage of disease (p<0.001, p<0.001), serum CEA level (p<0.001, p<0.001), and site of lesion (p<0.001, p<0.001), respectively. Patients with high ADP-ribosylation had significantly unfavorable OS and DFS compared with those with lower levels (p<0.001, p<0.001), respectively. Moreover, the patients with high NAD glycohydrolase activity showed significantly worse OS and DFS rates, similar to ADP-ribosylation. Serum levels of ADP-ribosylation and NAD glycohydrolase activity correlate well with tumor stage, stage of disease, serum CEA level, and site of lesion. In conclusion, elevated levels of preoperative ADP-ribosylation and NAD glycohydrolase levels in serum are associated with poor prognosis in patients with CRC.

The cytotoxic effect of diphtheria toxin on the actin cytoskeleton.

Diphtheria toxin (DT) and its N-terminal fragment A (FA) catalyse the transfer of the ADP-ribose moiety of nicotinamide adenine dinucleotide (NAD) into a covalent linkage with eukaryotic elongation factor 2 (eEF2). DT-induced cytotoxicity is versatile, and it includes DNA cleavage and the depolymerisation of actin filaments. The inhibition of the ADP-ribosyltransferase (ADPrT) activity of FA did not affect the deoxyribonuclease activity of FA or its interaction with actin. The toxin entry rate into cells (HUVEC) was determined by measuring the ADP-ribosyltransferase activity. DT uptake was nearly 80% after 30 min. The efficiency was determined as K(m) = 2.2 nM; V(max) = 0.25 pmol.min(-1). The nuclease activity was tested with hyperchromicity experiments, and it was concluded that G-actin has an inhibitory effect on DT nuclease activity. In the presence of DT and mutant of diphtheria toxin (CRM197), F-actin depolymerisation was determined with gel filtration, WB and fluorescence techniques. In the presence of DT and CRM197, 60-65% F-actin depolymerisation was observed. An in vitro FA-actin interaction and F-actin depolymerisation were reported in our previous paper. The present study thus confirms the depolymerisation of actin cytoskeleton in vivo.

On diphtheria toxin fragment A release into the cytosol--cytochalasin D effect and involvement of actin filaments and eukaryotic elongation factor 2.

Diphtheria toxin has been well characterized in terms of its receptor binding and receptor mediated endocytosis. However, the precise mechanism of the cytosolic release of diphtheria toxin fragment A from early endosomes is still unclear. Various reports differ regarding the requirement for cytosolic factors in this process. Here, we present data indicating that the distribution of actin filaments due to cytochalasin D action enhances the retention of diphtheria toxin in early endosomes. Treating cells with cytochalasin D reduces the cytosolic fragment A activity and leads to changes in the intracellular distribution and size of early endosomes with toxin cargo. F-actin and eukaryotic elongation factor 2 can promote fragment A release from toxin-loaded early endosomes in an in vitro translocation system. Moreover, these proteins bind to toxin-loaded early endosomes in vitro and promote each other's binding. They are thus thought to be involved in the cytosolic release of fragment A. Finally, ADP-ribosylation of eukaryotic elongation factor 2 is shown to inhibit fragment A release and, via a feed-back mechanism, to account for the minute amounts of fragment A normally found in the cytosol.

Interaction of diphtheria toxin (fragment A) with actin.

It was shown by gel filtration and viscosity measurements that N-terminal fragment (FA) of diphtheria toxin (DT) can interact with both G- and F-actin (filamentous actin). Elution profiles on Sephadex G-100 indicated the formation of a binary complex of fragment A (FA) with globular actin monomer (G-actin), which was inhibited by gelsolin. Deoxyribonuclease I (DNase I) in turn appeared to interact with this complex. Tritiated FA was found to bind to F-actin stoichiometrically. This binding was inhibited again by gelsolin and G-actin, but not by DNase I. The binding of FA inhibited polymerization of G-actin and induced a time-dependent breakdown of F-actin under polymerization conditions. Inhibition of its ADP-ribosyltransferase activity did not have any effect on the interactions of FA with actin. FA interacted with actin also in the cell. After treatment of human umbilical vein endothelial cells (HUVEC) with biotin-labeled DT, Western blot analysis revealed predominantly the presence of actin in affinity-isolated complexes of the labeled FA. Similarly, FA was found in immunoaffinity-isolated complexes of actin.