Figure 4

C. jejuni HtrA is secreted into the supernatant, forms proteolytic active multimers and recombinant C. jejuni HtrA cleaves E-cadherin but not fibronectin. (A) Filtered supernatants of broth-grown wild-type (wt) or htrA mutant C. jejuni (Cj) were subjected to zymography to detect proteolytic active HtrA multimers. (B) In vitro cleavage of full-length (FL) E-cadherin incubated with purified Cj HtrA or H. pylori (Hp) HtrA results in shedding of the extracellular N-terminal fragment (NTF). The anti-HtrA blot serves as loading control. (C) Schematic presentation of the domain structure of full-length human E-cadherin (GenBank accession number CAA78353.1) and the His-tagged NTF domain used for cleavage assays. E-cadherin consists of five extracellular domains (EC1-EC5) comprising the NTF domain, a transmembrane (TM) domain and an intracellular domain called C-terminal fragment (CTF). The cleavage site by some host cell proteases is indicated. (D) In vitro cleavage of the recombinant E-cadherin NTF domain incubated with purified Cj HtrA or Hp HtrA results in cleavage and disappearance of the 90 kDa NTF fragment. (E) In vitro cleavage assay of fibronectin incubated with purified HtrAs under identical conditions shows that fibronectin can be cleaved by Hp HtrA but not Cj HtrA. All reactions were incubated for 16 h at 37°C.