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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.
R K Holmes, and
K Harutyunyan, and
M Shah, and
H Joenje, and
H Youssoufian
November 1997,
Blood,
R K Holmes, and
K Harutyunyan, and
M Shah, and
H Joenje, and
H Youssoufian
January 1999,
Blood,
R K Holmes, and
K Harutyunyan, and
M Shah, and
H Joenje, and
H Youssoufian
January 2010,
Anemia,
R K Holmes, and
K Harutyunyan, and
M Shah, and
H Joenje, and
H Youssoufian
December 2001,
Experimental hematology,
R K Holmes, and
K Harutyunyan, and
M Shah, and
H Joenje, and
H Youssoufian
November 2012,
Bioorganic & medicinal chemistry,
R K Holmes, and
K Harutyunyan, and
M Shah, and
H Joenje, and
H Youssoufian
January 2000,
Blood,
R K Holmes, and
K Harutyunyan, and
M Shah, and
H Joenje, and
H Youssoufian
January 1998,
Blood,
R K Holmes, and
K Harutyunyan, and
M Shah, and
H Joenje, and
H Youssoufian
September 2011,
The journal of gene medicine,
R K Holmes, and
K Harutyunyan, and
M Shah, and
H Joenje, and
H Youssoufian
October 2003,
Molecular therapy : the journal of the American Society of Gene Therapy,
R K Holmes, and
K Harutyunyan, and
M Shah, and
H Joenje, and
H Youssoufian
September 1996,
Blood,
