Blood, 15 December 2001, Vol. 98, No. 13, pp. 3817-3822
RED CELLS
Correction of cross-linker sensitivity of Fanconi anemia group F
cells by CD33-mediated protein transfer
Rebecca K. Holmes,
Karine Harutyunyan,
Maulik Shah,
Hans Joenje, and
Hagop Youssoufian
From the Departments of Molecular and Human Genetics,
Pediatrics, and Medicine, Baylor College of Medicine, Houston,
TX; and Department of Clinical and Human Genetics, Free University of
Amsterdam, The Netherlands.
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.
