title: Correction of cross-linker sensitivity of Fanconi anemia group F cells by CD33-mediated protein transfer.
abstract: Studies have previously described the feasibility of receptor-mediated protein transfer in a cell culture model of Fanconi anemia (FA) group C. This study explores the versatility of this approach by using an antibody single-chain fusion protein to correct the phenotypic defect in FA group F cells. A 68.5-kd chimeric protein (His-M195FANCF) was expressed, consisting of a His tag, a single-chain antibody to the myeloid antigen CD33, and the FANCF protein, as well as a 43-kd His-FANCF fusion protein lacking the antibody motif, in Escherichia coli. The nickel-agarose-purified His-M195FANCF protein bound specifically to the surface of HeLa cells transfected with CD33 and internalized through vesicular structures. The fusion protein, but not CD33, sorted to the nucleus, consistent with the known nuclear localization of FANCF. No similar binding or internalization was observed with His-FANCF. Pretreatment of the transfected cells with chloroquine abolished nuclear accumulation, but there was little change with brefeldin A, indicating a minimal if any role for the Golgi apparatus in mediating transport from endosomes to the cytosol and the nucleus. The intracellular half-life of His-M195FANCF was approximately 160 minutes. Treatment of CD33-transfected FA group F lymphoblastoid cells with 0.1 mg/mL His-M195FANCF conferred resistance to mitomycin C. No similar protection was noted in CD33(-) parental cells or CD33(+) FA cells belonging to groups A and C. These results demonstrate that antibody-directed, receptor-mediated protein transfer is a versatile method for the delivery of biologically active proteins into hematopoietic cells.
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA.
authors.forename: R K
authors.initials: RK
authors.lastname: Holmes
authors.forename: K
authors.initials: K
authors.lastname: Harutyunyan
authors.forename: M
authors.initials: M
authors.lastname: Shah
authors.forename: H
authors.initials: H
authors.lastname: Joenje
authors.forename: H
authors.initials: H
authors.lastname: Youssoufian
mesh_terms: D015703:Antigens, CD / Q000235:genetics / Q000502:physiology*; D015214:Antigens, Differentiation, Myelomonocytic / Q000235:genetics / Q000502:physiology*; D020126:Brefeldin A / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002738:Chloroquine / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology; D011992:Endosomes / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005199:Fanconi Anemia; D052219:Fanconi Anemia Complementation Group F Protein; D015870:Gene Expression; D018390:Gene Targeting; D006056:Golgi Apparatus / Q000187:drug effects / Q000502:physiology; D006207:Half-Life; D006367:HeLa Cells / Q000378:metabolism / Q000648:ultrastructure; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006639:Histidine / Q000235:genetics; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D063268:Sialic Acid Binding Ig-like Lectin 3; D014162:Transfection
chemical_list: D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; C571163:CD33 protein, human; D003432:Cross-Linking Reagents; C499901:FANCF protein, human; D052219:Fanconi Anemia Complementation Group F Protein; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D063268:Sialic Acid Binding Ig-like Lectin 3; D020126:Brefeldin A; D006639:Histidine; D002738:Chloroquine