Objective
Many protein toxins possess the ability to enter the cytosol from the extracellular space. The mechanism of translocation into cells is known in some detail in the case of diphtheria toxin, but in other toxins the knowledge is fragmentary. Toxins of this group are extensively used to form immunotoxins for targeted cytotoxic therapy, and knowledge of the translocation mechanism is essential for development of efficient immunotoxins.
Two toxins have been chosen, Shigella dysenteriae toxin and the related plant toxin viscumin (from mistletoe). The laboratories involved in this application have long experience in work with these toxins and have contributed with important discoveries to their structure and action mechanism. The primary and three-dimensional structure of Shigella toxin is known, viscumin has been cloned and determination of the primary sequence is being completed in Prof. Kozlov's laboratory. Both toxins will be mutagenised to facilitate the study of the entry mechanism of the toxins and to elucidate the reason for the comparatively low activity of the enzymatically active subunit of these two toxins in a cell-free system. From the structure of Shigella toxin it appears that a disulfide-bridged loop of the A-chain protrudes into the enzymatically active site and could be the reason for the lower activity. Appropriate mutations will be carried out to alter the position of this loop to see if that will increase the enzymatic activity.
To follow the intracellular route of viscumin, signals for tyrosine sulfation as well as for glycosylation will be added. If the modified protein becomes labelled upon addition to cells in the presence of labelled sulfate, it is taken as evidence for transport to the Golgi apparatus, if it is glycosylated it is likely that it is transported to the endoplasmic reticulum.
The cloning, expression and mutagenesis will be carried out in Moscow. Studies on cells will be partly carried out in Moscow and partly in Oslo and Copenhagen. The groups can document a long-standing collaboration in the field. The results will be published in international journals of high standard.
Topic(s)
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0310 Oslo
Norway