Purification of diphtheria toxin receptor from Vero cells

Diphtheria toxin receptor has been solubilized from Vero cell membranes with octyl beta-D-glucoside. CRM197, the product of a mutated diphtheria toxin gene, was used for the identification of the receptor. The binding activity of the solubilized receptor was assayed by precipitating the receptor with acetone in the presence of phospholipids and carrier proteins. The solubilized receptor was purified by the combination of several chromatographic steps in the presence of the detergent, resulting in about a 10(6)-fold purification of the receptor. The purified receptor showed essentially a single band of 14.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When partially purified receptor fractions were subjected to ligand blotting analysis using 125I-CRM197 as the probe, the 14.5-kDa protein and a few minor protein bands were identified as diphtheria toxin-binding molecules. These results show clearly that the 14.5-kDa protein is the diphtheria toxin receptor, or at least the major diphtheria toxin-binding molecule. When partially purified receptor was applied to a Sephacryl S-300 column in the presence of detergent, the receptor was eluted in the fractions corresponding to the 60-90-kDa size range. This suggests that the protein forms a complex with itself or with another protein.     

Specific chemical cleavage of diphtheria toxin with hydroxylamine. Purification and characterization of the modified proteins

Specific chemical cleavage of diphtheria toxin with hydroxylamine was performed to remove peptides of 10 and 7 kDa from the carboxyl terminus. The resulting modified proteins of 51 and 48 kDa (HA51DT and HA48DT, respectively) were purified and characterized with respect to structural and biological properties. The 51-kDa toxin binds to ATP-agarose, as does intact diphtheria toxin, while HA48DT does not bind to the nucleotide matrix. Neither modified toxin binds to the membranes of diptheria toxin-sensitive cells, and, consequently, neither is toxic. However, when covalently linked to a membrane binding moiety, both HA51DT and HA48DT are toxic. Cell-killing ability during a short exposure time indicated that concanavalin A (Con A) derivatives of diphtheria toxin and HA51DT are equally toxic, ConA HA48DT being somewhat less toxic, while the conjugate of ConA to A-chain kills a small number of cells only at inordinately high concentration (1 microM). We have thus separated the cell membrane binding function of diphtheria toxin from its membrane permeation function by removing specific small peptides from the carboxyl terminus. These modified toxins may have applications in the preparation of highly potent hybrid toxins.     

Crystallization of diphtheria toxin.

Two new crystal forms (forms III and IV) have been grown of diphtheria toxin (DT), which kills susceptible cells by catalyzing the ADP-ribosylation of elongation factor 2, thereby stopping protein synthesis. Forms III and IV diffract to 2.3 A and 2.7 A resolution, respectively. Both forms belong to space group C2; the unit cell parameters for form III are a = 107.3 A, b = 91.7 A, c = 66.3 A and beta = 94.7 degrees and those for form IV are a = 108.3 A, b = 92.3 A, c = 66.1 A and beta = 90.4 degrees. Both forms have one protein chain per asymmetric unit with the dimeric molecule on a twofold axis of symmetry. Form IV is exceptional among all crystal forms of DT in that it can be grown reproducibly. Thus the form IV crystals should yield a crystallographic structure giving insight into the catalytic, receptor-binding and membrane-insertion properties of DT.     

Precursor in cotranslational secretion of diphtheria toxin.

By extracellular labeling of peptides of intact Corynebacterium diphtheriae, followed by fractionation of the cells and chain completion by isolated polysomes, it is shown that diphtheria toxin is formed and secreted cotranslationally by membrane-bound polysomes; free polysomes from none. Moreover, when the chains on these polysomes were completed in vitro, in the absence of membrane they were found to include not only diphtheria toxin of a molecular weight of 62,000, but also a larger precursor of a molecular weight of 68,000. The precursor was identified by several properties: immune precipitation; conversion into toxin fragments A and B; adenosine diphosphate ribosyl-transferase activity after activation with trypsin; and cleavage to 62,000 daltons by membrane enzymes. The precursor yields an N-terminal A fragment with a broadened molecular weight distribution, compared with that from authentic toxin, thus supporting the expectation that the extra segment of the precursor is N-terminal.