HT-29 treated with mature fragilysin LC-MSMS

Fragilysin is a protein secreted by toxigenic Bacteroides fragilis strains. Fragilysin has a motive HEXXHXXGXXH, which is a zinc-binding motif found in metalloproteinases clan termed metzincins. In this study we obtained all three known recombinant fragilysin isoforms in Escherichia coli and tested its activity. We detected no cleavage of gelatin and chromogenic substrates (such as azocoll and azocasein) by all isoforms. We demonstrated that treatment of HT-29 cells with fragilysins caused E-cadherin cleavage, nevertheless cleavage of recombinant E-cadherins was not observe as well as E-cadherin in isolated cell fractions. It was shown that the native structure of active site characteristic for metalloproteinases is necessary for fragilysin biologic activity. We detected proteins that are released in cultural medium after HT-29 cells treatment with fragilysin. These proteins are potential substrates for fragilysin.