A verotoxin 1 B subunit-lambda CRO chimeric protein specifically binds both DNA and globotriaosylceramide (Gb(3)) to effect nuclear targeting of exogenous DNA in Gb(3) positive cells.
Inefficient nuclear incorporation of foreign DNA remains a critical roadblock in the development of effective nonviral gene delivery systems. DNA delivered by traditional protocols remains within endosomal/lysosomal vesicles, or is rapidly degraded in the cytoplasm. Verotoxin I (VT), an AB(5) subunit toxin produced by enterohaemorrhagic Escherichia coli, binds to the cell surface glycolipid, globotriaosylceramide (Gb(3)) and is internalized into preendosomes. VT is then retrograde transported to the Golgi, endoplasmic reticulum (ER), and nucleus of highly VT-sensitive cells. We have utilized this nuclear targeting of VT to design a unique delivery system which transports exogenous DNA via vesicular traffic to the nucleus. The nontoxic VT binding subunit (VTB) was fused to the lambda Cro DNA-binding repressor, generating a 14-kDa VTB-Cro chimera. VTB-Cro binds specifically via the Cro domain to a 25-bp DNA fragment containing the consensus Cro operator. VTB-Cro demonstrates simultaneous specific binding to Gb(3). Treatment of Vero cells with fluorescent-labeled Cro operator DNA in the presence of VTB-Cro, results in DNA internalization to the Golgi, ER, and nucleus, whereas fluorescent DNA alone is incorporated poorly and randomly within the cytoplasm. VTB-Cro mediated nuclear DNA transport is prevented by brefeldin A, consistent with Golgi/ER intracellular routing. Pretreatment with filipin had no effect, indicating that caveoli are not involved. This novel VTB-Cro shuttle protein may find practical applications in the fields of intracellular targeting, gene delivery, and gene therapy.
Facchini
L M
LM
Division of Infection, Immunity, Injury and Repair, Research Institute, University of Toronto, Toronto, Canada.
Lingwood
C A
CA
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D020126:Brefeldin A / Q000494:pharmacology; D002467:Cell Nucleus / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D002522:Chlorocebus aethiops; D004247:DNA / Q000187:drug effects / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D005372:Filipin / Q000494:pharmacology; D005822:Genetic Vectors / Q000378:metabolism*; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D000922:Immunotoxins / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D022622:Shiga Toxin 1 / Q000378:metabolism*; D022161:Transport Vesicles / Q000187:drug effects / Q000378:metabolism; D014281:Trihexosylceramides / Q000378:metabolism*; D014709:Vero Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D014760:Viral Fusion Proteins / Q000138:chemical synthesis / Q000378:metabolism; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D000998:Antiviral Agents; D004268:DNA-Binding Proteins; D000922:Immunotoxins; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D022622:Shiga Toxin 1; D014281:Trihexosylceramides; D014760:Viral Fusion Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D020126:Brefeldin A; C018549:globotriaosylceramide; D005372:Filipin; D004247:DNA