Internalization of biotinylated compounds into cancer cells is promoted by a molecular Trojan horse based upon core streptavidin and clostridial C2 toxin.
The C2 toxin produced by Clostridium botulinum is a binary AB-type exotoxin composed of the enzyme subunit C2I and the binding/translocation moiety C2II. After proteolytic activation, C2IIa mediates the subsequent internalization of C2I into the cytosol of mammalian target cells. The N-terminal domain of C2I (C2IN) is necessary for C2IIa-dependent uptake, but lacks the enzyme domain that is responsible for cytotoxicity. In the present study, we generated a delivery system building on C2IN and a truncated core streptavidin (Stv13) with enhanced solubility for the C2IIa-dependent internalization of biotinylated cargo molecules into mammalian cells. C2IN-Stv13 fusion protein expressed in Escherichia coli was obtained in high yields and purity. The affinity-purified protein formed tetramers and a defined higher order species in solution as shown by gel filtration and retained its biotin-binding properties, however with an obvious reduction in affinity. Uptake of C2IN-Stv13 into the cytosol of HeLa and other cancer cell lines was observed by immunoblot analysis, which was corroborated by confocal microscopy. In addition, the fusion protein was not cytotoxic and did not inhibit cell proliferation as determined by MTS assay. Finally, we demonstrated the C2IN-Stv13/C2IIa-mediated uptake of biocytin-Alexa 488 as cargo into HeLa cells, underscoring the functionality of the generated transport system.
Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Albert-Einstein-Allee 11, 89081 Ulm, Germany.
Jörg
J
Fahrer
Joschua
J
Funk
Maren
M
Lillich
Holger
H
Barth
D000818:Animals; D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D001905:Botulinum Toxins / Q000235:genetics*; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D003600:Cytosol / Q000378:metabolism; D016503:Drug Delivery Systems / Q000379:methods*; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D019169:Jurkat Cells; D008239:Lysine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics; D011485:Protein Binding / Q000502:physiology; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary / Q000235:genetics; D021122:Protein Subunits / Q000378:metabolism / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D019809:Streptavidin / Q000235:genetics*; D014709:Vero Cells
D005456:Fluorescent Dyes; D010446:Peptide Fragments; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D001710:Biotin; D019809:Streptavidin; D001905:Botulinum Toxins; C096324:botulinum toxin type C; C013411:biocytin; D008239:Lysine