Molecular cloning and expression of lipid transfer inhibitor protein reveals its identity with apolipoprotein F

Published studies demonstrate that lipid transfer inhibitor protein (LTIP) is an important regulator of cholesteryl ester transfer protein (CETP) acti vity. Although LTIP inhibits CETP activity among different lipoprotein clas ses, it preferentially suppresses transfer events involving low density lip oprotein (LDL), whereas transfers involving high density lipoprotein as don or 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 con trast to other published protocols, took advantage of the tight association of this protein with LDL. Ultracentrifugally isolated LDL was further puri fied on anti-apoE and apoA-I affinity columns. Affinity purified LDL was de lipidated by tetramethylurea, and the tetramethylurea-soluble proteins were separated by SDS-polyacrylamide gel electrophoresis, The protein migrating at a molecular mass of similar to 33 kDa was excised from the gel and its N-terminal amino acid sequence determined. The 14-amino acid sequence obtai ned 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 sam e 33 kDa protein in LTIP-containing fractions purified from LDL and from li poprotein-deficient plasma. In contrast to that previously reported, apoF w as shown to be associated almost exclusively with LDL, identical to the dis tribution 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 de monstrated CETP inhibitor activity, whereas cells transfected with vector a lone 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 molecula r weight slightly larger than native apoF, The CETP inhibitor activity of r ecombinant apoF possessed the same LDL specificity, oleate sensitivity, and dependence on lipoprotein concentration as previously noted for LTIP, We c onclude that LTIP and apoF are identical.