Diphtheria toxin mutant (CRM197)
From DrugPedia: A Wikipedia for Drug discovery
Diphtheria toxin mutant (CRM197)
Diphtheria toxin is an exotoxin secreted by Corynebacterium diphtheriae, the pathogen bacterium that causes diphtheria. Diphtheria toxin is a single polypeptide chain of 535 amino acids consisting of two subunits linked by disulfide bridges. Binding to the cell surface of the less stable of these two subunits allows the more stable part of the protein to penetrate the host cell. It catalyzes the ADP-ribosylation of eukaryotic elongation factor-2 (eEF2 using NAD as a substrate., inactivating this protein. It does so by ADP-ribosylating the unusual amino acid diphthamide. In this way, it acts as a RNA translational inhibitor. The exotoxin A of Pseudomonas aeruginosa uses a similar mechanism of action. Diphtheria toxin is extraordinarily potent. The lethal dose for humans is about 0.1 μg of toxin per kg of bodyweight. A massive release of toxin into the body will likely cause lethal necrosis of the heart and liver.
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[edit] Structure
Diphtheria toxin is synthesized and excreted as a proenzyme, composed of a single polypeptide chain having an approximate molecular weight of 63,000 daltons.6,7,8,9 For its enzymic activity to be expressed, the toxin must undergo two covalent alterations in structure. First, mild proteolysis results in the formation of “nicked toxin,” which is enzymically inactive and consists of two major fragments, A and B, linked by a disulfide bond. Reduction of the nicked toxin with thiols releases the N-terminal A fragment (molecular weight 24,000 daltons) which is enzymically active. The Cterminal B fragment (molecular weight 39,000 daltons) has no apparent enzymic activity, but is required for toxicity. Evidence suggests that the B fragment is responsible for recognizing and binding the toxin to cell surface receptors.
[edit] Diphtheria toxin receptor
Diphtheria toxin receptor has been shown to be a cell-surface expressed heparin-binding epidermal growth factor-like growth factor (HB-EGF)precursor. In monkey cells, another membrane protein, CD-9 increases the affinity of DT for HBEGF, indicating that two proteins may function together as a receptor.14,15 Sensitivity of mammalian cell lines to diphtheria toxin varies with the presence of receptors. Monkey Vero cells are highly sensitive,1 6 hamster cells moderately sensitive, where rat and mouse cells without receptors are resistant.
[edit] Diphtheria toxin mutants
Mutant forms of diphtheria toxin (DT), isolated in the early 1970's, contributed to an understanding of the AB structure of DT. Uchida et al described the isolation and properties of several mutants including cross-reactive material (CRM) 197. CRM 197 is a non-toxic DT mutant containing a lesion in the A chain blocking ADP-ribosylation. CRM results from a single base change in the structural gene resulting in the substitution of glutamic acid for glycine. While CRM shows no enzymatic activity, it is immunologically indistinguishable from diphtheria toxin. In its applications, CRM 197 is similar to diphtheria toxoid. CRM has the advantage of being a well defined protein in contrast to formaldehyde treated toxin (toxoid) which is non-specifically cross linked and subject to rearrangement. 25,26 CRM functions as a carrier for polysaccharides and haptens making them immunogenic. On SDS gels, this protein migrates as a single major band of approximate molecular weight 63,000 daltons.
