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. 1998 Feb 15;330(Pt 1):35–40. doi: 10.1042/bj3300035

Mutagenesis of the aspartic acid ligands in human serum transferrin: lobe-lobe interaction and conformation as revealed by antibody, receptor-binding and iron-release studies.

A Mason 1, Q Y He 1, B Tam 1, R A MacGillivray 1, R Woodworth 1
PMCID: PMC1219104  PMID: 9461487

Abstract

Recombinant non-glycosylated human serum transferrin and mutants in which the liganding aspartic acid (D) in one or both lobes was changed to a serine residue (S) were produced in a mammalian cell system and purified from the tissue culture media. Significant downfield shifts of 20, 30, and 45 nm in the absorption maxima were found for the D63S-hTF, D392S-hTF and the double mutant, D63S/D392S-hTF when compared to wild-type hTF. A monoclonal antibody to a sequential epitope in the C-lobe of hTF reported affinity differences between the apo- and iron-forms of each mutant and the control. Cell-binding studies performed under the same buffer conditions used for the antibody work clearly showed that the mutated lobe(s) had an open cleft. It is not clear whether the receptor itself may play a role in promoting the open conformation or whether the iron remains in the cleft.

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Selected References

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  1. Anderson B. F., Baker H. M., Dodson E. J., Norris G. E., Rumball S. V., Waters J. M., Baker E. N. Structure of human lactoferrin at 3.2-A resolution. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1769–1773. doi: 10.1073/pnas.84.7.1769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bailey S., Evans R. W., Garratt R. C., Gorinsky B., Hasnain S., Horsburgh C., Jhoti H., Lindley P. F., Mydin A., Sarra R. Molecular structure of serum transferrin at 3.3-A resolution. Biochemistry. 1988 Jul 26;27(15):5804–5812. doi: 10.1021/bi00415a061. [DOI] [PubMed] [Google Scholar]
  3. Baker E. N., Baker H. M., Smith C. A., Stebbins M. R., Kahn M., Hellström K. E., Hellström I. Human melanotransferrin (p97) has only one functional iron-binding site. FEBS Lett. 1992 Feb 24;298(2-3):215–218. doi: 10.1016/0014-5793(92)80060-t. [DOI] [PubMed] [Google Scholar]
  4. Baker E. N., Lindley P. F. New perspectives on the structure and function of transferrins. J Inorg Biochem. 1992 Aug 15;47(3-4):147–160. doi: 10.1016/0162-0134(92)84061-q. [DOI] [PubMed] [Google Scholar]
  5. Bali P. K., Aisen P. Receptor-induced switch in site-site cooperativity during iron release by transferrin. Biochemistry. 1992 Apr 28;31(16):3963–3967. doi: 10.1021/bi00131a011. [DOI] [PubMed] [Google Scholar]
  6. Beatty E. J., Cox M. C., Frenkiel T. A., Tam B. M., Mason A. B., MacGillivray R. T., Sadler P. J., Woodworth R. C. Interlobe communication in 13C-methionine-labeled human transferrin. Biochemistry. 1996 Jun 18;35(24):7635–7642. doi: 10.1021/bi960684g. [DOI] [PubMed] [Google Scholar]
  7. Brown-Mason A., Brown S. A., Butcher N. D., Woodworth R. C. Reversible association of half-molecules of ovotransferrin in solution. Basis of co-operative binding to reticulocytes. Biochem J. 1987 Jul 1;245(1):103–109. doi: 10.1042/bj2450103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Egan T. J., Zak O., Aisen P. The anion requirement for iron release from transferrin is preserved in the receptor-transferrin complex. Biochemistry. 1993 Aug 17;32(32):8162–8167. doi: 10.1021/bi00083a016. [DOI] [PubMed] [Google Scholar]
  9. Evans R. W., Crawley J. B., Garratt R. C., Grossmann J. G., Neu M., Aitken A., Patel K. J., Meilak A., Wong C., Singh J. Characterization and structural analysis of a functional human serum transferrin variant and implications for receptor recognition. Biochemistry. 1994 Oct 18;33(41):12512–12520. doi: 10.1021/bi00207a019. [DOI] [PubMed] [Google Scholar]
  10. Evans R. W., Williams J., Moreton K. A variant of human transferrin with abnormal properties. Biochem J. 1982 Jan 1;201(1):19–26. doi: 10.1042/bj2010019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Faber H. R., Bland T., Day C. L., Norris G. E., Tweedie J. W., Baker E. N. Altered domain closure and iron binding in transferrins: the crystal structure of the Asp60Ser mutant of the amino-terminal half-molecule of human lactoferrin. J Mol Biol. 1996 Feb 23;256(2):352–363. doi: 10.1006/jmbi.1996.0091. [DOI] [PubMed] [Google Scholar]
  12. Gerstein M., Anderson B. F., Norris G. E., Baker E. N., Lesk A. M., Chothia C. Domain closure in lactoferrin. Two hinges produce a see-saw motion between alternative close-packed interfaces. J Mol Biol. 1993 Nov 20;234(2):357–372. doi: 10.1006/jmbi.1993.1592. [DOI] [PubMed] [Google Scholar]
  13. Grossmann J. G., Mason A. B., Woodworth R. C., Neu M., Lindley P. F., Hasnain S. S. Asp ligand provides the trigger for closure of transferrin molecules. Direct evidence from X-ray scattering studies of site-specific mutants of the N-terminal half-molecule of human transferrin. J Mol Biol. 1993 Jun 5;231(3):554–558. doi: 10.1006/jmbi.1993.1308. [DOI] [PubMed] [Google Scholar]
  14. He Q. Y., Mason A. B., Woodworth R. C. Spectrophotometric titration with cobalt(III) for the determination of accurate absorption coefficients of transferrins. Biochem J. 1996 Aug 15;318(Pt 1):145–148. doi: 10.1042/bj3180145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. He Q. Y., Mason A. B., Woodworth R. C., Tam B. M., Wadsworth T., MacGillivray R. T. Effects of mutations of aspartic acid 63 on the metal-binding properties of the recombinant N-lobe of human serum transferrin. Biochemistry. 1997 May 6;36(18):5522–5528. doi: 10.1021/bi963028p. [DOI] [PubMed] [Google Scholar]
  16. Klausner R. D., Ashwell G., van Renswoude J., Harford J. B., Bridges K. R. Binding of apotransferrin to K562 cells: explanation of the transferrin cycle. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2263–2266. doi: 10.1073/pnas.80.8.2263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kurokawa H., Mikami B., Hirose M. Crystal structure of diferric hen ovotransferrin at 2.4 A resolution. J Mol Biol. 1995 Nov 24;254(2):196–207. doi: 10.1006/jmbi.1995.0611. [DOI] [PubMed] [Google Scholar]
  18. Lin L. N., Mason A. B., Woodworth R. C., Brandts J. F. Calorimetric studies of the N-terminal half-molecule of transferrin and mutant forms modified near the Fe(3+)-binding site. Biochem J. 1993 Jul 15;293(Pt 2):517–522. doi: 10.1042/bj2930517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lin L. N., Mason A. B., Woodworth R. C., Brandts J. F. Calorimetric studies of the binding of ferric ions to ovotransferrin and interactions between binding sites. Biochemistry. 1991 Dec 17;30(50):11660–11669. doi: 10.1021/bi00114a008. [DOI] [PubMed] [Google Scholar]
  20. Mason A. B., Brown S. A. Differential effect of iodination of ovotransferrin and its two half-molecule domains on binding to transferrin receptors on chick embryo red blood cells. Biochem J. 1987 Oct 15;247(2):417–425. doi: 10.1042/bj2470417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mason A. B., Funk W. D., MacGillivray R. T., Woodworth R. C. Efficient production and isolation of recombinant amino-terminal half-molecule of human serum transferrin from baby hamster kidney cells. Protein Expr Purif. 1991 Apr-Jun;2(2-3):214–220. doi: 10.1016/1046-5928(91)90074-s. [DOI] [PubMed] [Google Scholar]
  22. Mason A. B., Miller M. K., Funk W. D., Banfield D. K., Savage K. J., Oliver R. W., Green B. N., MacGillivray R. T., Woodworth R. C. Expression of glycosylated and nonglycosylated human transferrin in mammalian cells. Characterization of the recombinant proteins with comparison to three commercially available transferrins. Biochemistry. 1993 May 25;32(20):5472–5479. doi: 10.1021/bi00071a025. [DOI] [PubMed] [Google Scholar]
  23. Mason A. B., Tam B. M., Woodworth R. C., Oliver R. W., Green B. N., Lin L. N., Brandts J. F., Savage K. J., Lineback J. A., MacGillivray R. T. Receptor recognition sites reside in both lobes of human serum transferrin. Biochem J. 1997 Aug 15;326(Pt 1):77–85. doi: 10.1042/bj3260077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mason A. B., Woodworth R. C. Monoclonal antibodies to the amino- and carboxyl-terminal domains of human transferrin. Hybridoma. 1991 Oct;10(5):611–623. doi: 10.1089/hyb.1991.10.611. [DOI] [PubMed] [Google Scholar]
  25. Mason A. B., Woodworth R. C., Oliver R. W., Green B. N., Lin L. N., Brandts J. F., Savage K. J., Tam B. M., MacGillivray R. T. Association of the two lobes of ovotransferrin is a prerequisite for receptor recognition. Studies with recombinant ovotransferrins. Biochem J. 1996 Oct 15;319(Pt 2):361–368. doi: 10.1042/bj3190361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nelson R. M., Long G. L. A general method of site-specific mutagenesis using a modification of the Thermus aquaticus polymerase chain reaction. Anal Biochem. 1989 Jul;180(1):147–151. doi: 10.1016/0003-2697(89)90103-6. [DOI] [PubMed] [Google Scholar]
  27. Oe H., Doi E., Hirose M. Amino-terminal and carboxyl-terminal half-molecules of ovotransferrin: preparation by a novel procedure and their interactions. J Biochem. 1988 Jun;103(6):1066–1072. doi: 10.1093/oxfordjournals.jbchem.a122381. [DOI] [PubMed] [Google Scholar]
  28. Pace C. N., Vajdos F., Fee L., Grimsley G., Gray T. How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 1995 Nov;4(11):2411–2423. doi: 10.1002/pro.5560041120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ward P. P., Zhou X., Conneely O. M. Cooperative interactions between the amino- and carboxyl-terminal lobes contribute to the unique iron-binding stability of lactoferrin. J Biol Chem. 1996 May 31;271(22):12790–12794. doi: 10.1074/jbc.271.22.12790. [DOI] [PubMed] [Google Scholar]
  30. Williams J., Chasteen N. D., Moreton K. The effect of salt concentration on the iron-binding properties of human transferrin. Biochem J. 1982 Mar 1;201(3):527–532. doi: 10.1042/bj2010527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Woodworth R. C., Mason A. B., Funk W. D., MacGillivray R. T. Expression and initial characterization of five site-directed mutants of the N-terminal half-molecule of human transferrin. Biochemistry. 1991 Nov 12;30(45):10824–10829. doi: 10.1021/bi00109a002. [DOI] [PubMed] [Google Scholar]
  32. Young S. P., Aisen P. Transferrin receptors and the uptake and release of iron by isolated hepatocytes. Hepatology. 1981 Mar-Apr;1(2):114–119. doi: 10.1002/hep.1840010205. [DOI] [PubMed] [Google Scholar]
  33. Young S. P., Bomford A., Madden A. D., Garratt R. C., Williams R., Evans R. W. Abnormal in vitro function of a variant human transferrin. Br J Haematol. 1984 Apr;56(4):581–587. doi: 10.1111/j.1365-2141.1984.tb02183.x. [DOI] [PubMed] [Google Scholar]
  34. Young S. P., Bomford A., Williams R. The effect of the iron saturation of transferrin on its binding and uptake by rabbit reticulocytes. Biochem J. 1984 Apr 15;219(2):505–510. doi: 10.1042/bj2190505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zak O., Trinder D., Aisen P. Primary receptor-recognition site of human transferrin is in the C-terminal lobe. J Biol Chem. 1994 Mar 11;269(10):7110–7114. [PubMed] [Google Scholar]

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