Example of Translocated Peptides and Proteins

It has been shown that a number of peptides linked to the N-terminal end of the diphtheria toxin A-fragment are translocated to the cytosol (Stenmark etai, 1991, Stenmark etal., 1992 Ariansen efal., 1993). It is an interesting question to what extent such peptides can subsequently be presented by class I major histocompatibility antigens. If this is the case, fusion proteins of viral or cancer-related peptides and enzymatically inactive mutants of diphtheria toxin could be used for vaccination purposes to expand desired populations of cytotoxic CD T-lymphocytes. 

Dihydrofolate reductase has been used extensively in translocation experiments. A fusion protein with diphtheria toxin A-fragment was shown to be translocated to the cytosol (Klingenberg and Ols-nes, 1996). The translocation was inhibited by methotrexate, which induces a tight folding of the protein. A fusion with a mutated dihydrofolate reductase that does not bind methotrexate tightly was also translocated, and in this case methotrexate was not able to prevent the translocation. This indicates that not only must the toxin A-fragment be unfolded, but the passenger protein must also be able to unfold for translocation to occur. 

The bacterial RNase, barnase, linked to Pseudomonas aeruginosa exotoxin A or to a non-toxic deletion mutant of the toxin lacking the enzymatic domain, was found to be toxic to cells to a greater extent than either component alone (Prior et al., 1991 1992). A C-terminal KDEL sequence was required for toxicity. Cells resistant to the intracellular action of the toxin were also sensitive to the fusion protein. This 

Fusion proteins have been constructed from peptide epitopes from influenza A antigens and the binding and translocation domains of Pseudomonas exotoxin A (Donelly ef al., 1993). When target cells were incubated with these fusion proteins, and subsequently exposed to cytotoxic T lymphocytes (CTLs) specific for the relevant epitopes, a CTL mediate lysis of the target cells was observed. These experiments suggest that the translocation machinery supplied by protein toxins may be useful tools for bringing peptides into cells for presentation via the major histocompatibility class I (MHC I) system. It should be noted that no direct evidence was provided that the translocation occurred by the toxin pathway, and it cannot be excluded that the toxin was only instrumental in accumulating the peptide on the surface of the cells and in the endocytic pathway. 

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