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. 2002 Aug 15;366(Pt 1):245–253. doi: 10.1042/BJ20020058

Size is a major determinant of dissociation and denaturation behaviour of reconstituted high-density lipoproteins.

Elisabetta Gianazza 1, Ivano Eberini 1, Cesare R Sirtori 1, Guido Franceschini 1, Laura Calabresi 1
PMCID: PMC1222753  PMID: 11996671

Abstract

Lipid-free apolipoprotein A-I (apoA-I) and A-I(Milano) (A-I(M)) were compared for their denaturation behaviour by running across transverse gradients of a chaotrope, urea, and of a ionic detergent, SDS. For both apo A-I and monomeric apoA-I(M) in the presence of increasing concentrations of urea the transition from high to low mobility had a sigmoidal course, whereas for dimeric A-I(M)/A-I(M) a non-sigmoidal shape was observed. The co-operativity of the unfolding process was lower for dimeric A-I(M)/A-I(M) than for apoA-I or for monomeric apoA-I(M). A slightly higher susceptibility to denaturation was observed for dimeric A-I(M)/A-I(M) than for monomeric apoA-I(M). A similar behaviour of A-I(M)/A-IM versus apoA-I(M) was observed in CD experiments. Large- (12.7/12.5 nm) and small- (7.8 nm) sized reconstituted high-density lipoproteins (rHDL) containing either apoA-I or A-I(M)/A-I(M) were compared with respect to their protein-lipid dissociation behaviour by subjecting them to electrophoresis in the presence of urea, of SDS and of a non-ionic detergent, Nonidet P40. A higher susceptibility to dissociation of small-sized versus large-sized rHDL, regardless of the apolipoprotein component, was observed in all three instances. Our data demonstrate that the differential plasticity of the various classes of rHDL is a function of their size; the higher stability of 12.5/12.7 nm rHDL is likely connected to the higher number of protein-lipid and lipid-lipid interactions in larger as compared with smaller rHDL.

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Selected References

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  1. Attie A. D., Kastelein J. P., Hayden M. R. Pivotal role of ABCA1 in reverse cholesterol transport influencing HDL levels and susceptibility to atherosclerosis. J Lipid Res. 2001 Nov;42(11):1717–1726. [PubMed] [Google Scholar]
  2. Calabresi L., Tedeschi G., Treu C., Ronchi S., Galbiati D., Airoldi S., Sirtori C. R., Marcel Y., Franceschini G. Limited proteolysis of a disulfide-linked apoA-I dimer in reconstituted HDL. J Lipid Res. 2001 Jun;42(6):935–942. [PubMed] [Google Scholar]
  3. Calabresi L., Vecchio G., Frigerio F., Vavassori L., Sirtori C. R., Franceschini G. Reconstituted high-density lipoproteins with a disulfide-linked apolipoprotein A-I dimer: evidence for restricted particle size heterogeneity. Biochemistry. 1997 Oct 14;36(41):12428–12433. doi: 10.1021/bi970505a. [DOI] [PubMed] [Google Scholar]
  4. Calabresi L., Vecchio G., Longhi R., Gianazza E., Palm G., Wadensten H., Hammarström A., Olsson A., Karlström A., Sejlitz T. Molecular characterization of native and recombinant apolipoprotein A-IMilano dimer. The introduction of an interchain disulfide bridge remarkably alters the physicochemical properties of apolipoprotein A-I. J Biol Chem. 1994 Dec 23;269(51):32168–32174. [PubMed] [Google Scholar]
  5. Creighton T. E. Detection of folding intermediates using urea-gradient electrophoresis. Methods Enzymol. 1986;131:156–172. doi: 10.1016/0076-6879(86)31040-1. [DOI] [PubMed] [Google Scholar]
  6. Creighton T. E. Electrophoretic analysis of the unfolding of proteins by urea. J Mol Biol. 1979 Apr 5;129(2):235–264. doi: 10.1016/0022-2836(79)90279-1. [DOI] [PubMed] [Google Scholar]
  7. Creighton T. E. Kinetic study of protein unfolding and refolding using urea gradient electrophoresis. J Mol Biol. 1980 Feb 15;137(1):61–80. doi: 10.1016/0022-2836(80)90157-6. [DOI] [PubMed] [Google Scholar]
  8. Evans R. W., Williams J. The electrophoresis of transferrins in urea/polyacrylamide gels. Biochem J. 1980 Sep 1;189(3):541–546. doi: 10.1042/bj1890541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ewbank J. J., Creighton T. E. Structural characterization of the disulfide folding intermediates of bovine alpha-lactalbumin. Biochemistry. 1993 Apr 13;32(14):3694–3707. doi: 10.1021/bi00065a023. [DOI] [PubMed] [Google Scholar]
  10. FERGUSON K. A. STARCH-GEL ELECTROPHORESIS--APPLICATION TO THE CLASSIFICATION OF PITUITARY PROTEINS AND POLYPEPTIDES. Metabolism. 1964 Oct;13:SUPPL–SUPPL1002. doi: 10.1016/s0026-0495(64)80018-4. [DOI] [PubMed] [Google Scholar]
  11. Franceschini G., Vecchio G., Gianfranceschi G., Magani D., Sirtori C. R. Apolipoprotein AIMilano. Accelerated binding and dissociation from lipids of a human apolipoprotein variant. J Biol Chem. 1985 Dec 25;260(30):16321–16325. [PubMed] [Google Scholar]
  12. Gianazza E., Calabresi L., Santi O., Sirtori C. R., Franceschini G. Denaturation and self-association of apolipoprotein A-I investigated by electrophoretic techniques. Biochemistry. 1997 Jun 24;36(25):7898–7905. doi: 10.1021/bi962600+. [DOI] [PubMed] [Google Scholar]
  13. Gianazza E., Galliano M., Miller I. Structural transitions of human serum albumin: an investigation using electrophoretic techniques. Electrophoresis. 1997 May;18(5):695–700. doi: 10.1002/elps.1150180507. [DOI] [PubMed] [Google Scholar]
  14. Gianazza E., Sirtori C. R., Castiglioni S., Eberini I., Chrambach A., Rondanini A., Vecchio G. Interactions between carbonic anhydrase and its inhibitors revealed by gel electrophoresis and circular dichroism. Electrophoresis. 2000 May;21(8):1435–1445. doi: 10.1002/(SICI)1522-2683(20000501)21:8<1435::AID-ELPS1435>3.0.CO;2-E. [DOI] [PubMed] [Google Scholar]
  15. Gianazza E., Vignati M., Santi O., Vecchio G. Electrophoresis of proteins across a transverse sodium dodecyl sulfate gradient. Electrophoresis. 1998 Jul;19(10):1631–1641. doi: 10.1002/elps.1150191019. [DOI] [PubMed] [Google Scholar]
  16. Ji Y., Jian B., Wang N., Sun Y., Moya M. L., Phillips M. C., Rothblat G. H., Swaney J. B., Tall A. R. Scavenger receptor BI promotes high density lipoprotein-mediated cellular cholesterol efflux. J Biol Chem. 1997 Aug 22;272(34):20982–20985. doi: 10.1074/jbc.272.34.20982. [DOI] [PubMed] [Google Scholar]
  17. Jonas A. Reconstitution of high-density lipoproteins. Methods Enzymol. 1986;128:553–582. doi: 10.1016/0076-6879(86)28092-1. [DOI] [PubMed] [Google Scholar]
  18. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  19. Masson P., Goasdoue J. L. Evidence that the conformational stability of 'aged' organophosphate-inhibited cholinesterase is altered. Biochim Biophys Acta. 1986 Feb 14;869(3):304–313. doi: 10.1016/0167-4838(86)90070-1. [DOI] [PubMed] [Google Scholar]
  20. Narayanaswami V., Ryan R. O. Molecular basis of exchangeable apolipoprotein function. Biochim Biophys Acta. 2000 Jan 3;1483(1):15–36. doi: 10.1016/s1388-1981(99)00176-6. [DOI] [PubMed] [Google Scholar]
  21. Nolte R. T., Atkinson D. Conformational analysis of apolipoprotein A-I and E-3 based on primary sequence and circular dichroism. Biophys J. 1992 Nov;63(5):1221–1239. doi: 10.1016/S0006-3495(92)81698-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Phillips J. C., Wriggers W., Li Z., Jonas A., Schulten K. Predicting the structure of apolipoprotein A-I in reconstituted high-density lipoprotein disks. Biophys J. 1997 Nov;73(5):2337–2346. doi: 10.1016/S0006-3495(97)78264-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rothblat G. H., de la Llera-Moya M., Atger V., Kellner-Weibel G., Williams D. L., Phillips M. C. Cell cholesterol efflux: integration of old and new observations provides new insights. J Lipid Res. 1999 May;40(5):781–796. [PubMed] [Google Scholar]
  24. Segrest J. P., Jones M. K., De Loof H., Brouillette C. G., Venkatachalapathi Y. V., Anantharamaiah G. M. The amphipathic helix in the exchangeable apolipoproteins: a review of secondary structure and function. J Lipid Res. 1992 Feb;33(2):141–166. [PubMed] [Google Scholar]
  25. Segrest J. P., Jones M. K., Klon A. E., Sheldahl C. J., Hellinger M., De Loof H., Harvey S. C. A detailed molecular belt model for apolipoprotein A-I in discoidal high density lipoprotein. J Biol Chem. 1999 Nov 5;274(45):31755–31758. doi: 10.1074/jbc.274.45.31755. [DOI] [PubMed] [Google Scholar]
  26. Sparks D. L., Lund-Katz S., Phillips M. C. The charge and structural stability of apolipoprotein A-I in discoidal and spherical recombinant high density lipoprotein particles. J Biol Chem. 1992 Dec 25;267(36):25839–25847. [PubMed] [Google Scholar]
  27. Takayama M., Itoh S., Nagasaki T., Tanimizu I. A new enzymatic method for determination of serum choline-containing phospholipids. Clin Chim Acta. 1977 Aug 15;79(1):93–98. doi: 10.1016/0009-8981(77)90465-x. [DOI] [PubMed] [Google Scholar]
  28. Tricerri M. A., Behling Agree A. K., Sanchez S. A., Bronski J., Jonas A. Arrangement of apolipoprotein A-I in reconstituted high-density lipoprotein disks: an alternative model based on fluorescence resonance energy transfer experiments. Biochemistry. 2001 Apr 24;40(16):5065–5074. doi: 10.1021/bi002815q. [DOI] [PubMed] [Google Scholar]
  29. Wald J. H., Krul E. S., Jonas A. Structure of apolipoprotein A-I in three homogeneous, reconstituted high density lipoprotein particles. J Biol Chem. 1990 Nov 15;265(32):20037–20043. [PubMed] [Google Scholar]
  30. Weisgraber K. H., Rall S. C., Jr, Bersot T. P., Mahley R. W., Franceschini G., Sirtori C. R. Apolipoprotein A-IMilano. Detection of normal A-I in affected subjects and evidence for a cysteine for arginine substitution in the variant A-I. J Biol Chem. 1983 Feb 25;258(4):2508–2513. [PubMed] [Google Scholar]
  31. Yamashita H., Nakatsuka T., Hirose M. Structural and functional characteristics of partially disulfide-reduced intermediates of ovotransferrin N lobe. Cystine localization by indirect end-labeling approach and implications for the reduction pathway. J Biol Chem. 1995 Dec 15;270(50):29806–29812. doi: 10.1074/jbc.270.50.29806. [DOI] [PubMed] [Google Scholar]
  32. Yokoyama S. Release of cellular cholesterol: molecular mechanism for cholesterol homeostasis in cells and in the body. Biochim Biophys Acta. 2000 Dec 15;1529(1-3):231–244. doi: 10.1016/s1388-1981(00)00152-9. [DOI] [PubMed] [Google Scholar]

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