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. 1994 Feb 15;91(4):1416–1420. doi: 10.1073/pnas.91.4.1416

Increased body temperature accelerates aggregation of the Leu-68-->Gln mutant cystatin C, the amyloid-forming protein in hereditary cystatin C amyloid angiopathy.

M Abrahamson 1, A Grubb 1
PMCID: PMC43169  PMID: 8108423

Abstract

Hereditary cystatin C amyloid angiopathy is a dominantly inherited disorder, characterized by dementia, paralysis, and death from cerebral hemorrhage in early adult life. A variant of the cysteine proteinase inhibitor, cystatin C, is deposited as amyloid in the tissues of the patients and their spinal-fluid level of cystatin C is abnormally low. The disease-associated Leu-68-->Gln mutant (L68Q) cystatin C has been produced in an Escherichia coli expression system and isolated by use of denaturing buffers, immunosorption, and gel filtration. Parallel physicochemical and functional investigations of L68Q-cystatin C and wild-type cystatin C revealed that both proteins effectively inhibit the cysteine proteinase cathepsin B (equilibrium constants for dissociation, 0.4 and 0.5 nM, respectively) but differ considerably in their tendency to dimerize and form aggregates. While wild-type cystatin C is monomeric and functionally active even after prolonged storage at elevated temperatures, L68Q-cystatin C starts to dimerize and lose biological activity immediately after it is transferred to a nondenaturing buffer. The dimerization of L68Q-cystatin C is highly temperature-dependent, with a rise in incubation temperature from 37 to 40 degrees C resulting in a 150% increase in dimerization rate. The aggregation at physiological concentrations is likewise increased at 40 compared to 37 degrees C, by approximately 60%. These properties of L68Q-cystatin C have bearing upon our understanding of the pathophysiological process of hereditary cystatin C amyloid angiopathy. They might also be of clinical relevance, since medical intervention to abort febrile periods of carriers of the disease trait may reduce the in vivo formation of L68Q-cystatin C aggregates.

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Selected References

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  1. Abrahamson M., Barrett A. J., Salvesen G., Grubb A. Isolation of six cysteine proteinase inhibitors from human urine. Their physicochemical and enzyme kinetic properties and concentrations in biological fluids. J Biol Chem. 1986 Aug 25;261(24):11282–11289. [PubMed] [Google Scholar]
  2. Abrahamson M., Dalbøge H., Olafsson I., Carlsen S., Grubb A. Efficient production of native, biologically active human cystatin C by Escherichia coli. FEBS Lett. 1988 Aug 15;236(1):14–18. doi: 10.1016/0014-5793(88)80276-x. [DOI] [PubMed] [Google Scholar]
  3. Abrahamson M., Grubb A., Olafsson I., Lundwall A. Molecular cloning and sequence analysis of cDNA coding for the precursor of the human cysteine proteinase inhibitor cystatin C. FEBS Lett. 1987 Jun 1;216(2):229–233. doi: 10.1016/0014-5793(87)80695-6. [DOI] [PubMed] [Google Scholar]
  4. Abrahamson M., Jonsdottir S., Olafsson I., Jensson O., Grubb A. Hereditary cystatin C amyloid angiopathy: identification of the disease-causing mutation and specific diagnosis by polymerase chain reaction based analysis. Hum Genet. 1992 Jun;89(4):377–380. doi: 10.1007/BF00194306. [DOI] [PubMed] [Google Scholar]
  5. Abrahamson M., Olafsson I., Palsdottir A., Ulvsbäck M., Lundwall A., Jensson O., Grubb A. Structure and expression of the human cystatin C gene. Biochem J. 1990 Jun 1;268(2):287–294. doi: 10.1042/bj2680287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Barrett A. J., Kembhavi A. A., Brown M. A., Kirschke H., Knight C. G., Tamai M., Hanada K. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L. Biochem J. 1982 Jan 1;201(1):189–198. doi: 10.1042/bj2010189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Benedikz E., Blöndal H., Gudmundsson G. Skin deposits in hereditary cystatin C amyloidosis. Virchows Arch A Pathol Anat Histopathol. 1990;417(4):325–331. doi: 10.1007/BF01605784. [DOI] [PubMed] [Google Scholar]
  8. Cohen D. H., Feiner H., Jensson O., Frangione B. Amyloid fibril in hereditary cerebral hemorrhage with amyloidosis (HCHWA) is related to the gastroentero-pancreatic neuroendocrine protein, gamma trace. J Exp Med. 1983 Aug 1;158(2):623–628. doi: 10.1084/jem.158.2.623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dalbøge H., Jensen E. B., Tøttrup H., Grubb A., Abrahamson M., Olafsson I., Carlsen S. High-level expression of active human cystatin C in Escherichia coli. Gene. 1989 Jul 15;79(2):325–332. doi: 10.1016/0378-1119(89)90214-x. [DOI] [PubMed] [Google Scholar]
  10. Denning G. M., Anderson M. P., Amara J. F., Marshall J., Smith A. E., Welsh M. J. Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive. Nature. 1992 Aug 27;358(6389):761–764. doi: 10.1038/358761a0. [DOI] [PubMed] [Google Scholar]
  11. Funk M. O., Nakagawa Y., Skochdopole J., Kaiser E. T. Affinity chromatographic purification of papain. Int J Pept Protein Res. 1979 Mar;13(3):296–303. doi: 10.1111/j.1399-3011.1979.tb01883.x. [DOI] [PubMed] [Google Scholar]
  12. Ghiso J., Jensson O., Frangione B. Amyloid fibrils in hereditary cerebral hemorrhage with amyloidosis of Icelandic type is a variant of gamma-trace basic protein (cystatin C). Proc Natl Acad Sci U S A. 1986 May;83(9):2974–2978. doi: 10.1073/pnas.83.9.2974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Grubb A., Jensson O., Gudmundsson G., Arnason A., Löfberg H., Malm J. Abnormal metabolism of gamma-trace alkaline microprotein. The basic defect in hereditary cerebral hemorrhage with amyloidosis. N Engl J Med. 1984 Dec 13;311(24):1547–1549. doi: 10.1056/NEJM198412133112406. [DOI] [PubMed] [Google Scholar]
  14. Grubb A., Löfberg H. Human gamma-trace, a basic microprotein: amino acid sequence and presence in the adenohypophysis. Proc Natl Acad Sci U S A. 1982 May;79(9):3024–3027. doi: 10.1073/pnas.79.9.3024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hall A., Dalbøge H., Grubb A., Abrahamson M. Importance of the evolutionarily conserved glycine residue in the N-terminal region of human cystatin C (Gly-11) for cysteine endopeptidase inhibition. Biochem J. 1993 Apr 1;291(Pt 1):123–129. doi: 10.1042/bj2910123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Henderson P. J. A linear equation that describes the steady-state kinetics of enzymes and subcellular particles interacting with tightly bound inhibitors. Biochem J. 1972 Apr;127(2):321–333. doi: 10.1042/bj1270321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jensson O., Gudmundsson G., Arnason A., Blöndal H., Petursdottir I., Thorsteinsson L., Grubb A., Löfberg H., Cohen D., Frangione B. Hereditary cystatin C (gamma-trace) amyloid angiopathy of the CNS causing cerebral hemorrhage. Acta Neurol Scand. 1987 Aug;76(2):102–114. doi: 10.1111/j.1600-0404.1987.tb03553.x. [DOI] [PubMed] [Google Scholar]
  18. Jeppsson J. O., Laurell C. B., Franzén B. Agarose gel electrophoresis. Clin Chem. 1979 Apr;25(4):629–638. [PubMed] [Google Scholar]
  19. Lomas D. A., Evans D. L., Finch J. T., Carrell R. W. The mechanism of Z alpha 1-antitrypsin accumulation in the liver. Nature. 1992 Jun 18;357(6379):605–607. doi: 10.1038/357605a0. [DOI] [PubMed] [Google Scholar]
  20. Löfberg H., Grubb A. O., Nilsson E. K., Jensson O., Gudmundsson G., Blöndal H., Arnason A., Thorsteinsson L. Immunohistochemical characterization of the amyloid deposits and quantitation of pertinent cerebrospinal fluid proteins in hereditary cerebral hemorrhage with amyloidosis. Stroke. 1987 Mar-Apr;18(2):431–440. doi: 10.1161/01.str.18.2.431. [DOI] [PubMed] [Google Scholar]
  21. Löfberg H., Grubb A. O. Quantitation of gamma-trace in human biological fluids: indications for production in the central nervous system. Scand J Clin Lab Invest. 1979 Nov;39(7):619–626. doi: 10.3109/00365517909108866. [DOI] [PubMed] [Google Scholar]
  22. Mancini G., Carbonara A. O., Heremans J. F. Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry. 1965 Sep;2(3):235–254. doi: 10.1016/0019-2791(65)90004-2. [DOI] [PubMed] [Google Scholar]
  23. Nicklin M. J., Barrett A. J. Inhibition of cysteine proteinases and dipeptidyl peptidase I by egg-white cystatin. Biochem J. 1984 Oct 1;223(1):245–253. doi: 10.1042/bj2230245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Olafsson I., Gudmundsson G., Abrahamson M., Jensson O., Grubb A. The amino terminal portion of cerebrospinal fluid cystatin C in hereditary cystatin C amyloid angiopathy is not truncated: direct sequence analysis from agarose gel electropherograms. Scand J Clin Lab Invest. 1990 Feb;50(1):85–93. doi: 10.1080/00365519009091569. [DOI] [PubMed] [Google Scholar]
  25. Olafsson I., Löfberg H., Abrahamson M., Grubb A. Production, characterization and use of monoclonal antibodies against the major extracellular human cysteine proteinase inhibitors cystatin C and kininogen. Scand J Clin Lab Invest. 1988 Oct;48(6):573–582. doi: 10.3109/00365518809085775. [DOI] [PubMed] [Google Scholar]
  26. Palsdottir A., Abrahamson M., Thorsteinsson L., Arnason A., Olafsson I., Grubb A., Jensson O. Mutation in cystatin C gene causes hereditary brain haemorrhage. Lancet. 1988 Sep 10;2(8611):603–604. doi: 10.1016/s0140-6736(88)90641-1. [DOI] [PubMed] [Google Scholar]
  27. Rawlings N. D., Barrett A. J. FLUSYS: a software package for the collection and analysis of kinetic and scanning data from Perkin-Elmer fluorimeters. Comput Appl Biosci. 1990 Apr;6(2):118–119. doi: 10.1093/bioinformatics/6.2.118. [DOI] [PubMed] [Google Scholar]
  28. Rich D. H., Brown M. A., Barrett A. J. Purification of cathepsin B by a new form of affinity chromatography. Biochem J. 1986 May 1;235(3):731–734. doi: 10.1042/bj2350731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. SCHEIDEGGER J. J. Une micro-méthode de l'immuno-electrophorèse. Int Arch Allergy Appl Immunol. 1955;7(2):103–110. [PubMed] [Google Scholar]
  30. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  31. Thorsteinsson L., Georgsson G., Asgeirsson B., Bjarnadóttir M., Olafsson I., Jensson O., Gudmundsson G. On the role of monocytes/macrophages in the pathogenesis of central nervous system lesions in hereditary cystatin C amyloid angiopathy. J Neurol Sci. 1992 Apr;108(2):121–128. doi: 10.1016/0022-510x(92)90042-j. [DOI] [PubMed] [Google Scholar]
  32. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res. 1982 Oct 25;10(20):6487–6500. doi: 10.1093/nar/10.20.6487. [DOI] [PMC free article] [PubMed] [Google Scholar]

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