Identification of diphtheria toxin receptor and a nonproteinous diphtheria toxin-binding molecule in Vero cell membrane.

Abstract

Abstract. Two substances possessing the ability to bind to diphtheria toxin (DT) were found to be present in a membrane fraction from DT-sensitive Vero cells. One of these substances was found on the basis of its ability to bind DT and inhibit its cytotoxic effect. This inhibitory substance competitively inhibited the binding of DT to Veto cells. However this inhibitor could not bind to CRM197, the product of a missense mutation in the DT gene, and did not inhibit the binding of CRM197 to Vero cells. Moreover, similar levels of the inhibitory activity were observed in membrane fractions from DT-insensitive mouse cells, suggesting the inhibitor is not the DT receptor which is specifically present in DT-sensitive cells. The second DT-binding substance was found in the same Vero cell membrane preparation by assaying the binding of ~25I-labeled CRM197. Such DT-binding activity could not be observed in membrane preparation from mouse L cells. From competition studies using labeled DT and CRM proteins, we conclude that this binding activity is due to the surface receptor for DT. Treatment of these substances with several enzymes revealed that the inhibitor was sensitive to certain RNases but resistant to proteases, whereas the DT receptor was resistant to RNase but sensitive to proteases. The receptor was solubilized and partially purified by chromatography on CM-Sepharose column. Immunoprecipitation and Western blotting analysis of the partially purified receptor revealed that a 14.5-kD protein is the DT receptor, or at least a component of it.D PHTHERIA toxin (DT) ~ is a cytotoxic protein which inhibits cellular protein synthesis (4, 40) in eukaryotes by catalyzing the ADP-ribosylation of EF-2, which results in its inactivation (9, 12). The first step of intoxication by DT is binding of the toxin to a susceptible cell. A specific receptor for DT is believed to be involved in this step (13,43). Cells from a number of mammals including humans and monkeys are sensitive to DT, but mouse and rat cells are insensitive (29). Several lines of evidence show that the difference in sensitivity to DT between species is primarily determined by the presence or absence of a cell surface receptor (18,28,31,48). However, this receptor has not been isolated. The toxins bound to cell surface are then internalized by endocytosis (33), and the toxins, or at least their A fragments, enter the cytoplasm to exert its effects. Like in the case of Semliki Forest virus (SFV) (11), intravesicular low pH is required for the penetration of the toxin into cytoplasm (6,17,20,27,36).Some information has been obtained on the biochemical properties of DT receptor. The treatment of DT-sensitive cells with some proteases or phospholipase C reduces the sensitivity to DT (32), whereas treatment with neuraminidase increases the sensitivity (26). Although these studies 1. Abbreviations used in this paper: CRMs, cross reacting materials related to diphtheria toxin; DT, diphtheria toxin.give a clue to the chemical nature of DT receptor, the possibility that ...

Identification of Mammalian TOM22 as a Subunit of the Preprotein Translocase of the Mitochondrial Outer Membrane

“…Monoclonal Antibody-BALB/c mice were immunized by subcutaneous injection of the alkali-extracted membrane fraction from Vero cells (monkey kidney-derived cells) (37). Spleen cells from the immunized mice were fused with X63-Ag8-653 mouse myeloma cells as described previously (38), and the hybridoma producing an antibody reacting with an ϳ22-kDa mitochondrial protein was selected.…”

“…tor complex using monoclonal antibodies prepared against the membrane fraction of Vero cells as antigens (37,38). We found that one of the monoclonal antibodies reacted with the ϳ22-kDa mitochondrial outer membrane protein 1C9-2.…”

Enhancement of Diphtheria Toxin-induced Apoptosis in Vero Cells by Combination Treatment with Brefeldin A and Okadaic Acid.

“…Diphtheria toxin is a 58 kDa single polypeptide produced by Corynebacterium diphtheriae whereby the carboxyl-terminal B fragment has affinity for a specific cell-surface receptor (Cieplak et al, 1987;Mekada et al, 1988) and acts as a carrier for the amino-terminal A fragment, which possesses the enzymatic activity for protein synthesis inhibition (Reaves and Banting, 1992;Sandvig and Olsnes, 1981;Sandvig and Olsnes, 1982). The diphtheria holotoxin or its A fragment is known to be translocated to the cytosol from endosomes following acidification.…”

Diphtheria Toxin Translocation across Endosome Membranes

“…The T domain consists of nine ␣-helices, whereas the R domain is a ␤-barrel structure. Translocation of fragment A of DT to the cytosol is initiated by binding of DT to a specific receptor on the cell surface (21,22), which is identical to the membrane-anchored form of heparinbinding EGF-like growth factor (pro-HB-EGF) (15).…”