Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1996 Nov 15;320(Pt 1):39–47. doi: 10.1042/bj3200039

Physical and kinetic characterization of recombinant human cholesteryl ester transfer protein.

D T Connolly 1, J McIntyre 1, D Heuvelman 1, E E Remsen 1, R E McKinnie 1, L Vu 1, M Melton 1, R Monsell 1, E S Krul 1, K Glenn 1
PMCID: PMC1217895  PMID: 8947465

Abstract

Cholesteryl ester transfer protein (CETP) mediates the exchange of triglycerides (TGs), cholesteryl esters (CEs) and phospholipids (PLs) between lipoproteins in the plasma. In order to better understand the lipid transfer process, we have used recombinant human CETP expressed in cultured mammalian cells, purified to homogeneity by immunoaffinity chromatography. Purified recombinant CETP had a weight-average relative molecular mass (MW) of 69561, determined by sedimentation equilibrium, and a specific absorption coefficient of 0.83 litre.g-1.cm-1. The corresponding hydrodynamic diameter (Dh) of the protein, determined by dynamic light scattering, was 14 nm, which is nearly twice the expected value for a spheroidal protein of this molecular mass. These data suggest that CETP has a non-spheroidal shape in solution. The secondary structure of CETP was estimated by CD to contain 32% alpha-helix, 35% beta-sheet, 17% turn and 16% random coil. Like the natural protein from plasma, the recombinant protein consisted of several glycoforms that could be only partially deglycosylated using N-glycosidase F. Organic extraction of CETP followed by TLC showed that CE, unesterified cholesterol (UC), PL, TG and fatty acids (FA) were associated with the pure protein. Quantitative analyses verified that each mol of CETP contained 1.0 mol of cholesterol, 0.5 mol of TG and 1.3 mol of PL. CETP mediated the transfer of CE, TG, PL, and UC between lipoproteins, or between protein-free liposomes. In dual-label transfer experiments, the transfer rates for CE or TG from HDL to LDL were found to be proportional to the initial concentrations of the respective ligands in the donor HDL particles. Kinetic analysis of CE transfer was consistent with a carrier mechanism, having a Km of 700 nM for LDL particles and of 2000 nM for HDL particles, and a kcat of 2 s-1. The Km values were thus in the low range of the normal physiological concentration for each substrate. The carrier mechanism was verified independently for CE, TG, PL and UC in 'half-reaction' experiments.

Full Text

The Full Text of this article is available as a PDF (580.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andrews P. The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem J. 1965 Sep;96(3):595–606. doi: 10.1042/bj0960595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arakawa T., Yphantis D. A., Lary J. W., Narhi L. O., Lu H. S., Prestrelski S. J., Clogston C. L., Zsebo K. M., Mendiaz E. A., Wypych J. Glycosylated and unglycosylated recombinant-derived human stem cell factors are dimeric and have extensive regular secondary structure. J Biol Chem. 1991 Oct 5;266(28):18942–18948. [PubMed] [Google Scholar]
  3. Atzel A., Wetterau J. R. Mechanism of microsomal triglyceride transfer protein catalyzed lipid transport. Biochemistry. 1993 Oct 5;32(39):10444–10450. doi: 10.1021/bi00090a021. [DOI] [PubMed] [Google Scholar]
  4. Barter P. J., Jones M. E. Kinetic studies of the transfer of esterified cholesterol between human plasma low and high density lipoproteins. J Lipid Res. 1980 Feb;21(2):238–249. [PubMed] [Google Scholar]
  5. Bock P. E., Halvorson H. R. Molecular weight of human high-molecular-weight kininogen light chain by equilibrium sedimentation in an air-driven ultracentrifuge. Anal Biochem. 1983 Nov;135(1):172–179. doi: 10.1016/0003-2697(83)90747-9. [DOI] [PubMed] [Google Scholar]
  6. Bruce C., Davidson W. S., Kussie P., Lund-Katz S., Phillips M. C., Ghosh R., Tall A. R. Molecular determinants of plasma cholesteryl ester transfer protein binding to high density lipoproteins. J Biol Chem. 1995 May 12;270(19):11532–11542. doi: 10.1074/jbc.270.19.11532. [DOI] [PubMed] [Google Scholar]
  7. Chen Y. H., Yang J. T., Martinez H. M. Determination of the secondary structures of proteins by circular dichroism and optical rotatory dispersion. Biochemistry. 1972 Oct 24;11(22):4120–4131. doi: 10.1021/bi00772a015. [DOI] [PubMed] [Google Scholar]
  8. Drayna D., Jarnagin A. S., McLean J., Henzel W., Kohr W., Fielding C., Lawn R. Cloning and sequencing of human cholesteryl ester transfer protein cDNA. Nature. 1987 Jun 18;327(6123):632–634. doi: 10.1038/327632a0. [DOI] [PubMed] [Google Scholar]
  9. Epps D. E., Greenlee K. A., Harris J. S., Thomas E. W., Castle C. K., Fisher J. F., Hozak R. R., Marschke C. K., Melchior G. W., Kézdy F. J. Kinetics and inhibition of lipid exchange catalyzed by plasma cholesteryl ester transfer protein (lipid transfer protein). Biochemistry. 1995 Oct 3;34(39):12560–12569. doi: 10.1021/bi00039a010. [DOI] [PubMed] [Google Scholar]
  10. Glenn K. C., Melton M. A. Quantification of cholesteryl ester transfer protein: activity and immunochemical assay. Methods Enzymol. 1996;263:339–351. doi: 10.1016/s0076-6879(96)63026-2. [DOI] [PubMed] [Google Scholar]
  11. Hesler C. B., Swenson T. L., Tall A. R. Purification and characterization of a human plasma cholesteryl ester transfer protein. J Biol Chem. 1987 Feb 15;262(5):2275–2282. [PubMed] [Google Scholar]
  12. Hippenmeyer P., Highkin M. High level, stable production of recombinant proteins in mammalian cell culture using the herpesvirus VP16 transactivator. Biotechnology (N Y) 1993 Sep;11(9):1037–1041. doi: 10.1038/nbt0993-1037. [DOI] [PubMed] [Google Scholar]
  13. Ihm J., Ellsworth J. L., Chataing B., Harmony J. A. Plasma protein-facilitated coupled exchange of phosphatidylcholine and cholesteryl ester in the absence of cholesterol esterification. J Biol Chem. 1982 May 10;257(9):4818–4827. [PubMed] [Google Scholar]
  14. Ihm J., Quinn D. M., Busch S. J., Chataing B., Harmony J. A. Kinetics of plasma protein-catalyzed exchange of phosphatidylcholine and cholesteryl ester between plasma lipoproteins. J Lipid Res. 1982 Dec;23(9):1328–1341. [PubMed] [Google Scholar]
  15. Johnson W. C., Jr Protein secondary structure and circular dichroism: a practical guide. Proteins. 1990;7(3):205–214. doi: 10.1002/prot.340070302. [DOI] [PubMed] [Google Scholar]
  16. Ko K. W., Ohnishi T., Yokoyama S. Triglyceride transfer is required for net cholesteryl ester transfer between lipoproteins in plasma by lipid transfer protein. Evidence for a hetero-exchange transfer mechanism demonstrated by using novel monoclonal antibodies. J Biol Chem. 1994 Nov 11;269(45):28206–28213. [PubMed] [Google Scholar]
  17. Ko K. W., Oikawa K., Ohnishi T., Kay C. M., Yokoyama S. Purification, characterization, and conformational analysis of rabbit plasma lipid transfer protein. Biochemistry. 1993 Jul 6;32(26):6729–6736. doi: 10.1021/bi00077a028. [DOI] [PubMed] [Google Scholar]
  18. Lagrost L., Athias A., Gambert P., Lallemant C. Comparative study of phospholipid transfer activities mediated by cholesteryl ester transfer protein and phospholipid transfer protein. J Lipid Res. 1994 May;35(5):825–835. [PubMed] [Google Scholar]
  19. Lagrost L. Regulation of cholesteryl ester transfer protein (CETP) activity: review of in vitro and in vivo studies. Biochim Biophys Acta. 1994 Dec 8;1215(3):209–236. doi: 10.1016/0005-2760(94)90047-7. [DOI] [PubMed] [Google Scholar]
  20. McKinney M. M., Parkinson A. A simple, non-chromatographic procedure to purify immunoglobulins from serum and ascites fluid. J Immunol Methods. 1987 Feb 11;96(2):271–278. doi: 10.1016/0022-1759(87)90324-3. [DOI] [PubMed] [Google Scholar]
  21. Morton R. E. Free cholesterol is a potent regulator of lipid transfer protein function. J Biol Chem. 1988 Sep 5;263(25):12235–12241. [PubMed] [Google Scholar]
  22. Morton R. E., Greene D. J. Enhanced detection of lipid transfer inhibitor protein activity by an assay involving only low density lipoprotein. J Lipid Res. 1994 Nov;35(11):2094–2099. [PubMed] [Google Scholar]
  23. Morton R. E. Interaction of lipid transfer protein with plasma lipoproteins and cell membranes. Experientia. 1990 Jun 15;46(6):552–560. doi: 10.1007/BF01939693. [DOI] [PubMed] [Google Scholar]
  24. Morton R. E. Specificity of lipid transfer protein for molecular species of cholesteryl ester. J Lipid Res. 1986 May;27(5):523–529. [PubMed] [Google Scholar]
  25. Morton R. E., Zilversmit D. B. Inter-relationship of lipids transferred by the lipid-transfer protein isolated from human lipoprotein-deficient plasma. J Biol Chem. 1983 Oct 10;258(19):11751–11757. [PubMed] [Google Scholar]
  26. Morton R. E., Zilversmit D. B. Purification and characterization of lipid transfer protein(s) from human lipoprotein-deficient plasma. J Lipid Res. 1982 Sep;23(7):1058–1067. [PubMed] [Google Scholar]
  27. Ohnishi T., Hicks L. D., Oikawa K., Kay C. M., Yokoyama S. Properties of human plasma lipid transfer protein in aqueous solution and at interfaces. Biochemistry. 1994 May 24;33(20):6093–6099. doi: 10.1021/bi00186a008. [DOI] [PubMed] [Google Scholar]
  28. Ohnishi T., Tan C., Yokoyama S. Selective transfer of cholesteryl ester over triglyceride by human plasma lipid transfer protein between apolipoprotein-activated lipid microemulsions. Biochemistry. 1994 Apr 19;33(15):4533–4542. doi: 10.1021/bi00181a014. [DOI] [PubMed] [Google Scholar]
  29. Ohnishi T., Yokoyama S., Yamamoto A. Rapid purification of human plasma lipid transfer proteins. J Lipid Res. 1990 Mar;31(3):397–406. [PubMed] [Google Scholar]
  30. Pollet R. J. Characterization of macromolecules by sedimentation equilibrium in the air-turbine ultracentrifuge. Methods Enzymol. 1985;117:3–27. doi: 10.1016/s0076-6879(85)17003-5. [DOI] [PubMed] [Google Scholar]
  31. Pollet R. J., Haase B. A., Standaert M. L. Macromolecular characterization by sedimentation equilibrium in the preparative ultracentrifuge. J Biol Chem. 1979 Jan 10;254(1):30–33. [PubMed] [Google Scholar]
  32. Puett D., Holladay L. A., Ford J. D., Cunningham L. W. Circular dichroism of glycopeptide fractions from alpha1-acid glycoprotein, thyroglobulin, and ovalbumin. Biochim Biophys Acta. 1977 Mar 28;491(1):129–136. doi: 10.1016/0005-2795(77)90048-4. [DOI] [PubMed] [Google Scholar]
  33. Rajaram O. V., Chan R. Y., Sawyer W. H. Effect of unesterified cholesterol on the activity of cholesteryl ester transfer protein. Biochem J. 1994 Dec 1;304(Pt 2):423–430. doi: 10.1042/bj3040423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rehberg E. F., Greenlee K. A., Melchior G. W., Marotti K. R. Purification of human cholesteryl ester transfer protein by affinity chromatography on immobilized triazine dyes. Protein Expr Purif. 1994 Jun;5(3):285–290. doi: 10.1006/prep.1994.1043. [DOI] [PubMed] [Google Scholar]
  35. Sarcich J. L., Fischer H. D., Babcock M. S., Leone J. W., Tomasselli A. G. Expression and purification of recombinant cynomolgus monkey cholesteryl ester transfer protein from Chinese hamster ovary cells. J Protein Chem. 1995 Feb;14(2):73–80. doi: 10.1007/BF01888364. [DOI] [PubMed] [Google Scholar]
  36. Stevenson S. C., Wang S., Deng L., Tall A. R. Human plasma cholesteryl ester transfer protein consists of a mixture of two forms reflecting variable glycosylation at asparagine 341. Biochemistry. 1993 May 18;32(19):5121–5126. doi: 10.1021/bi00070a021. [DOI] [PubMed] [Google Scholar]
  37. Swenson T. L., Brocia R. W., Tall A. R. Plasma cholesteryl ester transfer protein has binding sites for neutral lipids and phospholipids. J Biol Chem. 1988 Apr 15;263(11):5150–5157. [PubMed] [Google Scholar]
  38. Swenson T. L., Hesler C. B., Brown M. L., Quinet E., Trotta P. P., Haslanger M. F., Gaeta F. C., Marcel Y. L., Milne R. W., Tall A. R. Mechanism of cholesteryl ester transfer protein inhibition by a neutralizing monoclonal antibody and mapping of the monoclonal antibody epitope. J Biol Chem. 1989 Aug 25;264(24):14318–14326. [PubMed] [Google Scholar]
  39. Takahashi K., Jiang X. C., Sakai N., Yamashita S., Hirano K., Bujo H., Yamazaki H., Kusunoki J., Miura T., Kussie P. A missense mutation in the cholesteryl ester transfer protein gene with possible dominant effects on plasma high density lipoproteins. J Clin Invest. 1993 Oct;92(4):2060–2064. doi: 10.1172/JCI116802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tall A. R. Plasma cholesteryl ester transfer protein. J Lipid Res. 1993 Aug;34(8):1255–1274. [PubMed] [Google Scholar]
  41. Walsh M. T., Watzlawick H., Putnam F. W., Schmid K., Brossmer R. Effect of the carbohydrate moiety on the secondary structure of beta 2-glycoprotein. I. Implications for the biosynthesis and folding of glycoproteins. Biochemistry. 1990 Jul 3;29(26):6250–6257. doi: 10.1021/bi00478a020. [DOI] [PubMed] [Google Scholar]
  42. Wang S., Kussie P., Deng L., Tall A. Defective binding of neutral lipids by a carboxyl-terminal deletion mutant of cholesteryl ester transfer protein. Evidence for a carboxyl-terminal cholesteryl ester binding site essential for neutral lipid transfer activity. J Biol Chem. 1995 Jan 13;270(2):612–618. doi: 10.1074/jbc.270.2.612. [DOI] [PubMed] [Google Scholar]
  43. Weinberg R. B., Cook V. R., Jones J. B., Kussie P., Tall A. R. Interfacial properties of recombinant human cholesterol ester transfer protein. J Biol Chem. 1994 Nov 25;269(47):29588–29591. [PubMed] [Google Scholar]
  44. Yang J. T., Wu C. S., Martinez H. M. Calculation of protein conformation from circular dichroism. Methods Enzymol. 1986;130:208–269. doi: 10.1016/0076-6879(86)30013-2. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES