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. 2002 Jul 1;365(Pt 1):311–316. doi: 10.1042/BJ20020014

Loop variants of the serpin thyroxine-binding globulin: implications for hormone release upon limited proteolysis.

Helmut Grasberger 1, Henriette M B Golcher 1, Anja Fingerhut 1, Onno E Janssen 1
PMCID: PMC1222644  PMID: 11931635

Abstract

Thyroxine-binding globulin (TBG) and corticosteroid-binding globulin are unique among non-inhibitory members of the superfamily of serine-proteinase inhibitors (serpins) in undergoing a dramatic increase in stability [stressed-to-relaxed (S-->R) transition] after proteolytic cleavage within their exposed reactive-site-loop (RSL) equivalent. This structural rearrangement involves the insertion of the cleaved loop as a new strand into the beta-sheet A and is accompanied by a decrease in hormone binding. To define the mechanism that leads to disruption of hormone binding of TBG after proteolytic cleavage, the effect of partial loop deletions and replacements by the alpha(1)-proteinase inhibitor homologues of TBG were evaluated. Unexpectedly, deletion of the loop's C-terminus, thought to be important for thyroxine binding, improved the binding affinity over that of normal TBG. Proteolytic cleavage of this variant revealed an intact S-->R transition and reduced its binding activity to that of cleaved TBG. In contrast, a chimaera with C-terminal loop extension mimicked the decreased binding affinity of cleaved TBG and had a thermal stability intermediate between that of native and cleaved serpins. This variant was still susceptible to loop cleavage and underwent an S-->R transition, yet without changing its binding affinity. Our data exclude a direct involvement of loop residues in thyroxine binding of native TBG. Limited insertion of the RSL into beta-sheet A appears to trigger hormone release after proteolytic cleavage. In support of this concept, residues within the hinge region of the TBG loop are phylogenetically highly conserved, suggestive of their physiological role as a functional switch in vivo.

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Selected References

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  1. Afandi B., Schussler G. C., Arafeh A. H., Boutros A., Yap M. G., Finkelstein A. Selective consumption of thyroxine-binding globulin during cardiac bypass surgery. Metabolism. 2000 Feb;49(2):270–274. doi: 10.1016/s0026-0495(00)91611-1. [DOI] [PubMed] [Google Scholar]
  2. Becerra S. P., Sagasti A., Spinella P., Notario V. Pigment epithelium-derived factor behaves like a noninhibitory serpin. Neurotrophic activity does not require the serpin reactive loop. J Biol Chem. 1995 Oct 27;270(43):25992–25999. doi: 10.1074/jbc.270.43.25992. [DOI] [PubMed] [Google Scholar]
  3. Bottomley S. P., Chang W. S. The effects of reactive centre loop length upon serpin polymerisation. Biochem Biophys Res Commun. 1997 Dec 18;241(2):264–269. doi: 10.1006/bbrc.1997.7805. [DOI] [PubMed] [Google Scholar]
  4. Bruch M., Weiss V., Engel J. Plasma serine proteinase inhibitors (serpins) exhibit major conformational changes and a large increase in conformational stability upon cleavage at their reactive sites. J Biol Chem. 1988 Nov 15;263(32):16626–16630. [PubMed] [Google Scholar]
  5. Buettner C., Grasberger H., Hermansdorfer K., Chen B., Treske B., Janssen O. E. Characterization of the thyroxine-binding site of thyroxine-binding globulin by site-directed mutagenesis. Mol Endocrinol. 1999 Nov;13(11):1864–1872. doi: 10.1210/mend.13.11.0367. [DOI] [PubMed] [Google Scholar]
  6. Carrell R. W., Owen M. C. Plakalbumin, alpha 1-antitrypsin, antithrombin and the mechanism of inflammatory thrombosis. Nature. 1985 Oct 24;317(6039):730–732. doi: 10.1038/317730a0. [DOI] [PubMed] [Google Scholar]
  7. Chang W. S., Whisstock J., Hopkins P. C., Lesk A. M., Carrell R. W., Wardell M. R. Importance of the release of strand 1C to the polymerization mechanism of inhibitory serpins. Protein Sci. 1997 Jan;6(1):89–98. doi: 10.1002/pro.5560060110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ciliberto G., Dente L., Cortese R. Cell-specific expression of a transfected human alpha 1-antitrypsin gene. Cell. 1985 Jun;41(2):531–540. doi: 10.1016/s0092-8674(85)80026-x. [DOI] [PubMed] [Google Scholar]
  9. Evans D. L., McGrogan M., Scott R. W., Carrell R. W. Protease specificity and heparin binding and activation of recombinant protease nexin I. J Biol Chem. 1991 Nov 25;266(33):22307–22312. [PubMed] [Google Scholar]
  10. Flink I. L., Bailey T. J., Gustafson T. A., Markham B. E., Morkin E. Complete amino acid sequence of human thyroxine-binding globulin deduced from cloned DNA: close homology to the serine antiproteases. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7708–7712. doi: 10.1073/pnas.83.20.7708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gettins P. Absence of large-scale conformational change upon limited proteolysis of ovalbumin, the prototypic serpin. J Biol Chem. 1989 Mar 5;264(7):3781–3785. [PubMed] [Google Scholar]
  12. Grasberger H., Buettner C., Janssen O. E. Modularity of serpins. A bifunctional chimera possessing alpha1-proteinase inhibitor and thyroxine-binding globulin properties. J Biol Chem. 1999 May 21;274(21):15046–15051. doi: 10.1074/jbc.274.21.15046. [DOI] [PubMed] [Google Scholar]
  13. Horton R. M., Hunt H. D., Ho S. N., Pullen J. K., Pease L. R. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene. 1989 Apr 15;77(1):61–68. doi: 10.1016/0378-1119(89)90359-4. [DOI] [PubMed] [Google Scholar]
  14. Huber R., Carrell R. W. Implications of the three-dimensional structure of alpha 1-antitrypsin for structure and function of serpins. Biochemistry. 1989 Nov 14;28(23):8951–8966. doi: 10.1021/bi00449a001. [DOI] [PubMed] [Google Scholar]
  15. Janssen O. E., Refetoff S. In vitro expression of thyroxine-binding globulin (TBG) variants. Impaired secretion of TBGPRO-227 but not TBGPRO-113. J Biol Chem. 1992 Jul 15;267(20):13998–14004. [PubMed] [Google Scholar]
  16. Janssen Onno E., Golcher Henriette M. B., Grasberger Helmut, Saller Bernhard, Mann Klaus, Refetoff Samuel. Characterization of T(4)-binding globulin cleaved by human leukocyte elastase. J Clin Endocrinol Metab. 2002 Mar;87(3):1217–1222. doi: 10.1210/jcem.87.3.8332. [DOI] [PubMed] [Google Scholar]
  17. Jirasakuldech B., Schussler G. C., Yap M. G., Drew H., Josephson A., Michl J. A characteristic serpin cleavage product of thyroxine-binding globulin appears in sepsis sera. J Clin Endocrinol Metab. 2000 Nov;85(11):3996–3999. doi: 10.1210/jcem.85.11.6966. [DOI] [PubMed] [Google Scholar]
  18. Jirasakuldech B., Schussler G. C., Yap M. G., Zirkind R., Afandi B., Michl J. Cleavage of thyroxine-binding globulin during cardiopulmonary bypass. Metabolism. 2001 Sep;50(9):1113–1116. doi: 10.1053/meta.2001.24881. [DOI] [PubMed] [Google Scholar]
  19. Kvassman J. O., Lawrence D. A., Shore J. D. The acid stabilization of plasminogen activator inhibitor-1 depends on protonation of a single group that affects loop insertion into beta-sheet A. J Biol Chem. 1995 Nov 17;270(46):27942–27947. doi: 10.1074/jbc.270.46.27942. [DOI] [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Mottonen J., Strand A., Symersky J., Sweet R. M., Danley D. E., Geoghegan K. F., Gerard R. D., Goldsmith E. J. Structural basis of latency in plasminogen activator inhibitor-1. Nature. 1992 Jan 16;355(6357):270–273. doi: 10.1038/355270a0. [DOI] [PubMed] [Google Scholar]
  22. Murata Y., Refetoff S., Sarne D. H., Dick M., Watson F. Variant thyroxine-binding globulin in serum of Australian aborigines: its physical, chemical and biological properties. J Endocrinol Invest. 1985 Jun;8(3):225–232. doi: 10.1007/BF03348482. [DOI] [PubMed] [Google Scholar]
  23. Pemberton P. A., Stein P. E., Pepys M. B., Potter J. M., Carrell R. W. Hormone binding globulins undergo serpin conformational change in inflammation. Nature. 1988 Nov 17;336(6196):257–258. doi: 10.1038/336257a0. [DOI] [PubMed] [Google Scholar]
  24. Pemberton P. A., Wong D. T., Gibson H. L., Kiefer M. C., Fitzpatrick P. A., Sager R., Barr P. J. The tumor suppressor maspin does not undergo the stressed to relaxed transition or inhibit trypsin-like serine proteases. Evidence that maspin is not a protease inhibitory serpin. J Biol Chem. 1995 Jun 30;270(26):15832–15837. doi: 10.1074/jbc.270.26.15832. [DOI] [PubMed] [Google Scholar]
  25. Potempa J., Korzus E., Travis J. The serpin superfamily of proteinase inhibitors: structure, function, and regulation. J Biol Chem. 1994 Jun 10;269(23):15957–15960. [PubMed] [Google Scholar]
  26. Refetoff S. Inherited thyroxine-binding globulin abnormalities in man. Endocr Rev. 1989 Aug;10(3):275–293. doi: 10.1210/edrv-10-3-275. [DOI] [PubMed] [Google Scholar]
  27. Schechter I., Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun. 1967 Apr 20;27(2):157–162. doi: 10.1016/s0006-291x(67)80055-x. [DOI] [PubMed] [Google Scholar]
  28. Schussler G. C. The thyroxine-binding proteins. Thyroid. 2000 Feb;10(2):141–149. doi: 10.1089/thy.2000.10.141. [DOI] [PubMed] [Google Scholar]
  29. Stein P. E., Tewkesbury D. A., Carrell R. W. Ovalbumin and angiotensinogen lack serpin S-R conformational change. Biochem J. 1989 Aug 15;262(1):103–107. doi: 10.1042/bj2620103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Stein P., Chothia C. Serpin tertiary structure transformation. J Mol Biol. 1991 Sep 20;221(2):615–621. doi: 10.1016/0022-2836(91)80076-7. [DOI] [PubMed] [Google Scholar]
  31. Suda S. A., Gettins P. G., Patston P. A. Linkage between the hormone binding site and the reactive center loop of thyroxine binding globulin. Arch Biochem Biophys. 2000 Dec 1;384(1):31–36. doi: 10.1006/abbi.2000.2110. [DOI] [PubMed] [Google Scholar]
  32. Takeda K., Mori Y., Sobieszczyk S., Seo H., Dick M., Watson F., Flink I. L., Seino S., Bell G. I., Refetoff S. Sequence of the variant thyroxine-binding globulin of Australian aborigines. Only one of two amino acid replacements is responsible for its altered properties. J Clin Invest. 1989 Apr;83(4):1344–1348. doi: 10.1172/JCI114021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Terry C. J., Blake C. C. Comparison of the modelled thyroxine binding site in TBG with the experimentally determined site in transthyretin. Protein Eng. 1992 Sep;5(6):505–510. doi: 10.1093/protein/5.6.505. [DOI] [PubMed] [Google Scholar]
  34. Whisstock J., Skinner R., Lesk A. M. An atlas of serpin conformations. Trends Biochem Sci. 1998 Feb;23(2):63–67. doi: 10.1016/s0968-0004(97)01172-9. [DOI] [PubMed] [Google Scholar]

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