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J Biol Chem, Vol. 274, Issue 3, 1814-1820, January 15, 1999
Molecular Cloning and Expression of Lipid Transfer Inhibitor
Protein Reveals Its Identity with Apolipoprotein F
Xinxing
Wang,
Donna M.
Driscoll, and
Richard E.
Morton
From the Department of Cell Biology, Lerner Research Institute,
Cleveland Clinic Foundation, Cleveland, Ohio 44195
Published studies demonstrate that
lipid transfer inhibitor protein (LTIP) is an important regulator of
cholesteryl ester transfer protein (CETP) activity. Although LTIP
inhibits CETP activity among different lipoprotein classes, it
preferentially suppresses transfer events involving low density
lipoprotein (LDL), whereas transfers involving high density lipoprotein
as donor are less affected. In this study, we report the purification
of LTIP and the expression of its cDNA in cultured cells.
Purification of LTIP, in contrast to other published protocols, took
advantage of the tight association of this protein with LDL.
Ultracentrifugally isolated LDL was further purified on anti-apoE and
apoA-I affinity columns. Affinity purified LDL was delipidated by
tetramethylurea, and the tetramethylurea-soluble proteins were
separated by SDS-polyacrylamide gel electrophoresis. The protein
migrating at a molecular mass of ~33 kDa was excised from the gel and
its N-terminal amino acid sequence determined. The 14-amino acid
sequence obtained showed complete homology with the sequence deduced
for apolipoprotein F (apoF) cDNA isolated from Hep G2 cells. On
Western blots, peptide-specific antibodies raised against synthetic
fragments of apoF reacted with the same 33-kDa protein in
LTIP-containing fractions purified from LDL and from
lipoprotein-deficient plasma. In contrast to that previously reported,
apoF was shown to be associated almost exclusively with LDL, identical
to the distribution of LTIP activity. The cDNA for apoF was cloned
from a human liver cDNA library, ligated into a mammalian
expression vector, and transiently transfected into COS-7 cells.
Conditioned media containing secreted apoF demonstrated CETP inhibitor
activity, whereas cells transfected with vector alone did not. This
CETP inhibitor activity was efficiently removed from the media by
nickel-Sepharose, consistent with the 6-His tag incorporated into
recombinant apoF. By Western blot, the 6-His-tagged protein had a
molecular weight slightly larger than native apoF. The CETP inhibitor
activity of recombinant apoF possessed the same LDL specificity, oleate
sensitivity, and dependence on lipoprotein concentration as previously
noted for LTIP. We conclude that LTIP and apoF are identical.
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

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Copyright © 1999 by the American Society for Biochemistry and Molecular Biology.
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