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 transmembrane domain (T) may function as a membrane anchor for soluble proteins fused at its C-terminus. Binding to membranes is triggered by acidic pH. Here, we further characterized this anchoring device. Soluble proteins may be fused at the N-terminus of the T domain or at both extremities, without modifying its membrane binding properties. This allows one to choose the orientation of the protein to be attached to the membrane. Maximum binding to the cell surface is reached within 1 h. Anchoring occurs on cells previously treated with proteinase K, suggesting that T interacts with the lipid phase of the membrane without the help of cell surface proteins. Binding does not permeabilize cells or affect cell viability, despite the fact that it permeabilizes liposomes and alters their structure. When attached to L929 fibroblasts, the proteins are not internalized and remain displayed at their surface for more than 24 h. When bound to K562 myeloid cells, the molecules are internalized and degraded. Thus, depending on the cell type, soluble proteins may be anchored to the surface of cells by the T domain for an extended time or directed towards an internalization pathway.
Nizard
P
P
Département d'Ingénierie et d'Etudes des Protéines and Département de Biologie Cellulaire et Moleculaire, CEA-Saclay, 91191 Gif sur Yvette cedex, France.
Chenal
A
A
Beaumelle
B
B
Fourcade
A
A
Gillet
D
D
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004167:Diphtheria Toxin / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis; D006801:Humans; D020014:K562 Cells; D007700:Kinetics; D007739:L Cells; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism / Q000493:pharmacokinetics; D051379:Mice; D018625:Microscopy, Atomic Force; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000493:pharmacokinetics; D013205:Staphylococcal Protein A / Q000235:genetics / Q000378:metabolism*
D004167:Diphtheria Toxin; D008081:Liposomes; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A