Prolonged display or rapid internalization of the IgG-binding protein ZZ anchored to the surface of cells using the diphtheria toxin T domain

We have shown previously that the diphtheria toxin transmembranedomain (T) may function as a membrane anchor for soluble proteinsfused at its C-terminus. Binding to membranes is triggered byacidic pH. Here, we further characterized this anchoring device.Soluble proteins may be fused at the N-terminus of the T domainor at both extremities, without modifying its membrane bindingproperties. This allows one to choose the orientation of theprotein to be attached to the membrane. Maximum binding to thecell surface is reached within 1 h. Anchoring occurs on cellspreviously treated with proteinase K, suggesting that T interactswith the lipid phase of the membrane without the help of cellsurface proteins. Binding does not permeabilize cells or affectcell viability, despite the fact that it permeabilizes liposomesand alters their structure. When attached to L929 fibroblasts,the proteins are not internalized and remain displayed at theirsurface for more than 24 h. When bound to K562 myeloid cells,the molecules are internalized and degraded. Thus, dependingon the cell type, soluble proteins may be anchored to the surfaceof cells by the T domain for an extended time or directed towardsan internalization pathway.