Streptavidin-conjugated C3 protein mediates the delivery of mono-biotinylated RNAse A into macrophages.
The C3 toxin produced by  Clostridium botulinum  (C3bot) catalyzes the mono-ADP-ribosylation of the small GTPases Rho A, B and C, resulting in their inactivation. Recently, a specific endocytotic uptake mechanism of C3bot was identified in macrophages and myeloid leukemia cells. Here, we present a novel delivery system based upon a mutant C3bot devoid of ADP-ribosylation activity (C3Mut) and wild-type streptavidin (Stv). The C3Mut moiety mediates endocytosis into macrophages, whereas Stv functions as an adaptor protein for attaching biotinylated molecules to facilitate their subsequent internalization. First, a bioconjugate consisting of recombinant C3Mut and Stv was generated via a thioether linkage that tightly interacted with biotinylated bovine serum albumin as demonstrated by dot blot analysis. We then showed the internalization of C3Mut-Stv into J774A.1 macrophages by confocal microscopy and observed translocation into the cytosol using cell fractionation. The C3Mut-Stv bioconjugate did not affect cell viability. Next, we prepared mono-biotinylated RNase A, which was attached to the C3Mut-Stv transporter, and demonstrated its C3Mut-Stv-mediated delivery into the cytosol of J774A.1 cells. Finally, C3Mut-Stv also promoted the efficient uptake of mono-biotinylated lysozyme into J774A.1 cells, highlighting its versatility. This study intriguingly demonstrates the use of the novel C3Mut-Stv delivery system for protein transduction and may provide a basis for future applications, in particular, of cytotoxic RNase A mutants.
Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Albert-Einstein-Allee 11, D-89081 Ulm, Germany.
Maren
M
Lillich
Xi
X
Chen
Tanja
T
Weil
Holger
H
Barth
Jörg
J
Fahrer
D000818:Animals; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D019921:Biotinylation; D002460:Cell Line; D003176:Complement C3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D018014:Gene Transfer Techniques; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D015394:Molecular Structure; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019809:Streptavidin / Q000737:chemistry* / Q000378:metabolism
D003176:Complement C3; D001710:Biotin; D019809:Streptavidin; D012259:Ribonuclease, Pancreatic; D009113:Muramidase