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. 1994 Jun 1;300(Pt 2):437–442. doi: 10.1042/bj3000437

Structure-function studies on human retinol-binding protein using site-directed mutagenesis.

A Sivaprasadarao 1, J B Findlay 1
PMCID: PMC1138181  PMID: 8002949

Abstract

Retinol-binding protein (RBP) transports vitamin A in the plasma. It consists of eight anti-parallel beta-strands (A to H) that fold to form an orthogonal barrel. The loops connecting the strands A and B, C and D, and E and F form the entrance to the binding site in the barrel. The retinol molecule is found deep inside this barrel. Apart from its specific interaction with retinol, RBP is involved in two other molecular-recognition properties, that is it binds to transthyretin (TTR), another serum protein, and to a cell-surface receptor. Using site-directed mutagenesis, specific changes were made to the loop regions of human RBP and the resultant mutant proteins were tested for their ability to bind to retinol, to TTR and to the RBP receptor. While all the variants retained their ability to bind retinol, that in which residues 92 to 98 of the loop E-F were deleted completely lost its ability to interact with TTR, but retained some binding activity for the receptor. In contrast, the double mutant in which leucine residues at positions 63 and 64 of the loop C-D were changed to arginine and serine respectively partially retained its TTR-binding ability, but completely lost its affinity for the RBP receptor. Mutation of Leu-35 of loop A-B to valine revealed no apparent effect on any of the binding activities of RBP. However, substitution of leucine for proline at position 35 markedly reduced the affinity of the protein for TTR, but showed no apparent change in its receptor-binding activity. These results demonstrate that RBP interacts with both TTR and the receptor via loops C-D and E-F. The binding sites, however, are overlapping rather than identical. RBP also appears to make an additional contact with TTR via its loop A-B. A further implication of these results is that RBP, when bound to TTR, cannot bind simultaneously to the receptor. This observation is consistent with our previously proposed mechanism for delivery of retinol to target tissues [Sivaprasadarao and Findlay (1988) Biochem. J. 255, 571-579], according to which retinol delivery involves specific binding of RBP to the cell-surface receptor, an interaction that triggers release of retinol from RBP to the bound cell rather than internalization of retinol-RBP complex.

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Selected References

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  1. Aqvist J., Sandblom P., Jones T. A., Newcomer M. E., van Gunsteren W. F., Tapia O. Molecular dynamics simulations of the holo and apo forms of retinol binding protein. Structural and dynamical changes induced by retinol removal. J Mol Biol. 1986 Dec 5;192(3):593–603. doi: 10.1016/0022-2836(86)90279-2. [DOI] [PubMed] [Google Scholar]
  2. Aqvist J., Tapia O. A molecular model for the retinol binding protein-transthyretin complex. J Mol Graph. 1992 Jun;10(2):120-3, 111. doi: 10.1016/0263-7855(92)80065-l. [DOI] [PubMed] [Google Scholar]
  3. Berman P., Gray P., Chen E., Keyser K., Ehrlich D., Karten H., LaCorbiere M., Esch F., Schubert D. Sequence analysis, cellular localization, and expression of a neuroretina adhesion and cell survival molecule. Cell. 1987 Oct 9;51(1):135–142. doi: 10.1016/0092-8674(87)90018-3. [DOI] [PubMed] [Google Scholar]
  4. Blaner W. S. Radioimmunoassays for retinol-binding protein, cellular retinol-binding protein, and cellular retinoic acid-binding protein. Methods Enzymol. 1990;189:270–281. doi: 10.1016/0076-6879(90)89298-v. [DOI] [PubMed] [Google Scholar]
  5. Blomhoff R., Green M. H., Berg T., Norum K. R. Transport and storage of vitamin A. Science. 1990 Oct 19;250(4979):399–404. doi: 10.1126/science.2218545. [DOI] [PubMed] [Google Scholar]
  6. Båvik C. O., Busch C., Eriksson U. Characterization of a plasma retinol-binding protein membrane receptor expressed in the retinal pigment epithelium. J Biol Chem. 1992 Nov 15;267(32):23035–23042. [PubMed] [Google Scholar]
  7. Båvik C. O., Eriksson U., Allen R. A., Peterson P. A. Identification and partial characterization of a retinal pigment epithelial membrane receptor for plasma retinol-binding protein. J Biol Chem. 1991 Aug 15;266(23):14978–14985. [PubMed] [Google Scholar]
  8. Båvik C. O., Lévy F., Hellman U., Wernstedt C., Eriksson U. The retinal pigment epithelial membrane receptor for plasma retinol-binding protein. Isolation and cDNA cloning of the 63-kDa protein. J Biol Chem. 1993 Sep 25;268(27):20540–20546. [PubMed] [Google Scholar]
  9. Chen C. C., Heller J. Uptake of retinol and retinoic acid from serum retinol-binding protein by retinal pigment epithelial cells. J Biol Chem. 1977 Aug 10;252(15):5216–5221. [PubMed] [Google Scholar]
  10. Cowan S. W., Newcomer M. E., Jones T. A. Crystallographic refinement of human serum retinol binding protein at 2A resolution. Proteins. 1990;8(1):44–61. doi: 10.1002/prot.340080108. [DOI] [PubMed] [Google Scholar]
  11. Fex G., Johannesson G. Retinol transfer across and between phospholipid bilayer membranes. Biochim Biophys Acta. 1988 Oct 6;944(2):249–255. doi: 10.1016/0005-2736(88)90438-5. [DOI] [PubMed] [Google Scholar]
  12. Heller J. Interactions of plasma retinol-binding protein with its receptor. Specific binding of bovine and human retinol-binding protein to pigment epithelium cells from bovine eyes. J Biol Chem. 1975 May 25;250(10):3613–3619. [PubMed] [Google Scholar]
  13. Horwitz J., Heller J. Modification of tryptophan residues in retinol-binding protein and prealbumin with 2-hydroxy-5-nitrobenzyl bromide. Effects of the modification of the protein-retinol and protein-protein interaction. J Biol Chem. 1974 Nov 25;249(22):7181–7185. [PubMed] [Google Scholar]
  14. Landt O., Grunert H. P., Hahn U. A general method for rapid site-directed mutagenesis using the polymerase chain reaction. Gene. 1990 Nov 30;96(1):125–128. doi: 10.1016/0378-1119(90)90351-q. [DOI] [PubMed] [Google Scholar]
  15. Matarese V., Lodish H. F. Specific uptake of retinol-binding protein by variant F9 cell lines. J Biol Chem. 1993 Sep 5;268(25):18859–18865. [PubMed] [Google Scholar]
  16. Newcomer M. E., Jones T. A., Aqvist J., Sundelin J., Eriksson U., Rask L., Peterson P. A. The three-dimensional structure of retinol-binding protein. EMBO J. 1984 Jul;3(7):1451–1454. doi: 10.1002/j.1460-2075.1984.tb01995.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Noy N., Xu Z. J. Interactions of retinol with binding proteins: implications for the mechanism of uptake by cells. Biochemistry. 1990 Apr 24;29(16):3878–3883. doi: 10.1021/bi00468a012. [DOI] [PubMed] [Google Scholar]
  18. Papiz M. Z., Sawyer L., Eliopoulos E. E., North A. C., Findlay J. B., Sivaprasadarao R., Jones T. A., Newcomer M. E., Kraulis P. J. The structure of beta-lactoglobulin and its similarity to plasma retinol-binding protein. 1986 Nov 27-Dec 3Nature. 324(6095):383–385. doi: 10.1038/324383a0. [DOI] [PubMed] [Google Scholar]
  19. Peterson P. A. Characteristics of a vitamin A-transporting protein complex occurring in human serum. J Biol Chem. 1971 Jan 10;246(1):34–43. [PubMed] [Google Scholar]
  20. Rask L., Peterson P. A. In vitro uptake of vitamin A from the retinol-binding plasma protein to mucosal epithelial cells from the monkey's small intestine. J Biol Chem. 1976 Oct 25;251(20):6360–6366. [PubMed] [Google Scholar]
  21. Raz A., Shiratori T., Goodman D. S. Studies on the protein-protein and protein-ligand interactions involved in retinol transport in plasma. J Biol Chem. 1970 Apr 25;245(8):1903–1912. [PubMed] [Google Scholar]
  22. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Senoo H., Stang E., Nilsson A., Kindberg G. M., Berg T., Roos N., Norum K. R., Blomhoff R. Internalization of retinol-binding protein in parenchymal and stellate cells of rat liver. J Lipid Res. 1990 Jul;31(7):1229–1239. [PubMed] [Google Scholar]
  24. Sivaprasadarao A., Boudjelal M., Findlay J. B. Lipocalin structure and function. Biochem Soc Trans. 1993 Aug;21(3):619–622. doi: 10.1042/bst0210619. [DOI] [PubMed] [Google Scholar]
  25. Sivaprasadarao A., Findlay J. B. Expression of functional human retinol-binding protein in Escherichia coli using a secretion vector. Biochem J. 1993 Nov 15;296(Pt 1):209–215. doi: 10.1042/bj2960209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sivaprasadarao A., Findlay J. B. The interaction of retinol-binding protein with its plasma-membrane receptor. Biochem J. 1988 Oct 15;255(2):561–569. [PMC free article] [PubMed] [Google Scholar]
  27. Sivaprasadarao A., Findlay J. B. The mechanism of uptake of retinol by plasma-membrane vesicles. Biochem J. 1988 Oct 15;255(2):571–579. [PMC free article] [PubMed] [Google Scholar]
  28. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  29. Vahlquist A., Nilsson S. F., Peterson P. A. Isolation of the human retinol binding protein by affinity chromatography. Eur J Biochem. 1971 May 28;20(2):160–168. doi: 10.1111/j.1432-1033.1971.tb01374.x. [DOI] [PubMed] [Google Scholar]
  30. Zanotti G., Berni R., Monaco H. L. Crystal structure of liganded and unliganded forms of bovine plasma retinol-binding protein. J Biol Chem. 1993 May 25;268(15):10728–10738. [PubMed] [Google Scholar]
  31. Zanotti G., Ottonello S., Berni R., Monaco H. L. Crystal structure of the trigonal form of human plasma retinol-binding protein at 2.5 A resolution. J Mol Biol. 1993 Mar 20;230(2):613–624. doi: 10.1006/jmbi.1993.1173. [DOI] [PubMed] [Google Scholar]

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