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. 1998 Dec;51(6):317–326. doi: 10.1136/mp.51.6.317

Intracellular accumulation of the amyloidogenic L68Q variant of human cystatin C in NIH/3T3 cells.

M Bjarnadottir 1, B S Wulff 1, M Sameni 1, B F Sloane 1, D Keppler 1, A Grubb 1, M Abrahamson 1
PMCID: PMC395658  PMID: 10193512

Abstract

AIM: To study the cellular transport of L68Q cystatin C, the cystatin variant causing amyloidosis and brain haemorrhage in patients suffering from hereditary cystatin C amyloid angiopathy (HCCAA). METHODS: Expression vectors for wild-type and L68Q cystatin C were constructed and used to transfect mouse NIH/3T3 cells. Stable cell clones were isolated after cotransfection with pSV2neo. Clones expressing human wild-type and L68Q cystatin C were compared with respect to secreted cystatin C by enzyme linked immunosorbent assay (ELISA), and for intracellular cystatin C by western blotting and immunofluorescence cytochemistry. Colocalisation studies in cells were performed by double staining with antibodies against human cystatin C and marker proteins for lysosomes, the Golgi apparatus, or the endoplasmic reticulum, and evaluated by confocal microscopy. RESULTS: Concentrations of human cystatin C secreted from transfected NIH/3T3 cells were similar to those secreted from human cells in culture. In general, clones expressing the gene encoding L68Q cystatin C secreted slightly lower amounts of the protein than clones expressing wild-type human cystatin C. Both immunofluorescence cytochemistry and western blotting experiments showed an increased accumulation of cystatin C in cells expressing the gene encoding L68Q cystatin C compared with cells expressing the gene for the wild-type protein. The intracellularly accumulating L68Q cystatin C was insoluble and located mainly in the endoplasmic reticulum. CONCLUSIONS: The cellular transport of human cystatin C is impeded by the pathogenic amino acid substitution Leu68-->Gln. The resulting intracellular accumulation and increased localised concentration of L68Q cystatin C might be an important event in the molecular pathophysiology of amyloid formation and brain haemorrhage in patients with HCCAA.

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Selected References

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  1. 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]
  2. Abrahamson M., Grubb A. Increased body temperature accelerates aggregation of the Leu-68-->Gln mutant cystatin C, the amyloid-forming protein in hereditary cystatin C amyloid angiopathy. Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1416–1420. doi: 10.1073/pnas.91.4.1416. [DOI] [PMC free article] [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. Human cysteine proteinase inhibitors. Isolation, physiological importance, inhibitory mechanism, gene structure and relation to hereditary cerebral hemorrhage. Scand J Clin Lab Invest Suppl. 1988;191:21–31. [PubMed] [Google Scholar]
  5. Abrahamson M., Islam M. Q., Szpirer J., Szpirer C., Levan G. The human cystatin C gene (CST3), mutated in hereditary cystatin C amyloid angiopathy, is located on chromosome 20. Hum Genet. 1989 Jun;82(3):223–226. doi: 10.1007/BF00291159. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Asgeirsson B., Haebel S., Thorsteinsson L., Helgason E., Gudmundsson K. O., Gudmundsson G., Roepstorff P. Hereditary cystatin C amyloid angiopathy: monitoring the presence of the Leu-68-->Gln cystatin C variant in cerebrospinal fluids and monocyte cultures by MS. Biochem J. 1998 Feb 1;329(Pt 3):497–503. doi: 10.1042/bj3290497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Barrett A. J., Davies M. E., Grubb A. The place of human gamma-trace (cystatin C) amongst the cysteine proteinase inhibitors. Biochem Biophys Res Commun. 1984 Apr 30;120(2):631–636. doi: 10.1016/0006-291x(84)91302-0. [DOI] [PubMed] [Google Scholar]
  9. Barrett A. J., Fritz H., Grubb A., Isemura S., Järvinen M., Katunuma N., Machleidt W., Müller-Esterl W., Sasaki M., Turk V. Nomenclature and classification of the proteins homologous with the cysteine-proteinase inhibitor chicken cystatin. Biochem J. 1986 May 15;236(1):312–312. doi: 10.1042/bj2360312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bjarnadóttir M., Grubb A., Olafsson I. Promoter-mediated, dexamethasone-induced increase in cystatin C production by HeLa cells. Scand J Clin Lab Invest. 1995 Nov;55(7):617–623. doi: 10.3109/00365519509110261. [DOI] [PubMed] [Google Scholar]
  11. Bloom G. S., Brashear T. A. A novel 58-kDa protein associates with the Golgi apparatus and microtubules. J Biol Chem. 1989 Sep 25;264(27):16083–16092. [PubMed] [Google Scholar]
  12. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Corticchiato O., Cajot J. F., Abrahamson M., Chan S. J., Keppler D., Sordat B. Cystatin C and cathepsin B in human colon carcinoma: expression by cell lines and matrix degradation. Int J Cancer. 1992 Oct 21;52(4):645–652. doi: 10.1002/ijc.2910520425. [DOI] [PubMed] [Google Scholar]
  15. Curiel D. T., Holmes M. D., Okayama H., Brantly M. L., Vogelmeier C., Travis W. D., Stier L. E., Perks W. H., Crystal R. G. Molecular basis of the liver and lung disease associated with the alpha 1-antitrypsin deficiency allele Mmalton. J Biol Chem. 1989 Aug 15;264(23):13938–13945. [PubMed] [Google Scholar]
  16. Ekiel I., Abrahamson M. Folding-related dimerization of human cystatin C. J Biol Chem. 1996 Jan 19;271(3):1314–1321. doi: 10.1074/jbc.271.3.1314. [DOI] [PubMed] [Google Scholar]
  17. Ekiel I., Abrahamson M., Fulton D. B., Lindahl P., Storer A. C., Levadoux W., Lafrance M., Labelle S., Pomerleau Y., Groleau D. NMR structural studies of human cystatin C dimers and monomers. J Mol Biol. 1997 Aug 15;271(2):266–277. doi: 10.1006/jmbi.1997.1150. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Gudmundsson G., Hallgrímsson J., Jónasson T. A., Bjarnason O. Hereditary cerebral haemorrhage with amyloidosis. Brain. 1972;95(2):387–404. doi: 10.1093/brain/95.2.387. [DOI] [PubMed] [Google Scholar]
  23. Gudmundsson G., Hallgrímsson J., Jónasson T. A., Bjarnason O. Hereditary cerebral haemorrhage with amyloidosis. Brain. 1972;95(2):387–404. doi: 10.1093/brain/95.2.387. [DOI] [PubMed] [Google Scholar]
  24. Heidtmann H. H., Salge U., Abrahamson M., Bencina M., Kastelic L., Kopitar-Jerala N., Turk V., Lah T. T. Cathepsin B and cysteine proteinase inhibitors in human lung cancer cell lines. Clin Exp Metastasis. 1997 Jul;15(4):368–381. doi: 10.1023/a:1018494020001. [DOI] [PubMed] [Google Scholar]
  25. Huovila A. P., Eder A. M., Fuller S. D. Hepatitis B surface antigen assembles in a post-ER, pre-Golgi compartment. J Cell Biol. 1992 Sep;118(6):1305–1320. doi: 10.1083/jcb.118.6.1305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Håkansson K., Huh C., Grubb A., Karlsson S., Abrahamson M. Mouse and rat cystatin C: Escherichia coli production, characterization and tissue distribution. Comp Biochem Physiol B Biochem Mol Biol. 1996 Jul;114(3):303–311. doi: 10.1016/0305-0491(96)00025-9. [DOI] [PubMed] [Google Scholar]
  27. Isidoro C., Baccino F. M., Hasilik A. Mis-sorting of procathepsin D in metastogenic tumor cells is not due to impaired synthesis of the phosphomannosyl signal. Int J Cancer. 1997 Mar 4;70(5):561–566. doi: 10.1002/(sici)1097-0215(19970304)70:5<561::aid-ijc12>3.0.co;2-g. [DOI] [PubMed] [Google Scholar]
  28. Jarrett J. T., Lansbury P. T., Jr Seeding "one-dimensional crystallization" of amyloid: a pathogenic mechanism in Alzheimer's disease and scrapie? Cell. 1993 Jun 18;73(6):1055–1058. doi: 10.1016/0092-8674(93)90635-4. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Johansen T. E., Schifter S., Vogel C. K., Tolstoy S., Schwartz T. W. Processing of pro-CGRP in a rat medullary thyroid carcinoma cell line transfected with protease inhibitors. Mol Cell Endocrinol. 1991 Nov;82(1):51–60. doi: 10.1016/0303-7207(91)90008-g. [DOI] [PubMed] [Google Scholar]
  31. Johansen T. E., Schøller M. S., Tolstoy S., Schwartz T. W. Biosynthesis of peptide precursors and protease inhibitors using new constitutive and inducible eukaryotic expression vectors. FEBS Lett. 1990 Jul 16;267(2):289–294. doi: 10.1016/0014-5793(90)80947-h. [DOI] [PubMed] [Google Scholar]
  32. Johansen T. E., Vogel C. K., Schwartz T. W. C-terminal KDEL-modified cystatin C is retained in transfected CHO cells. Biochem Biophys Res Commun. 1990 Nov 15;172(3):1384–1391. doi: 10.1016/0006-291x(90)91603-p. [DOI] [PubMed] [Google Scholar]
  33. Keppler D., Waridel P., Abrahamson M., Bachmann D., Berdoz J., Sordat B. Latency of cathepsin B secreted by human colon carcinoma cells is not linked to secretion of cystatin C and is relieved by neutrophil elastase. Biochim Biophys Acta. 1994 May 25;1226(2):117–125. doi: 10.1016/0925-4439(94)90018-3. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Lippincott-Schwartz J., Bonifacino J. S., Yuan L. C., Klausner R. D. Degradation from the endoplasmic reticulum: disposing of newly synthesized proteins. Cell. 1988 Jul 15;54(2):209–220. doi: 10.1016/0092-8674(88)90553-3. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. Moin K., Day N. A., Sameni M., Hasnain S., Hirama T., Sloane B. F. Human tumour cathepsin B. Comparison with normal liver cathepsin B. Biochem J. 1992 Jul 15;285(Pt 2):427–434. doi: 10.1042/bj2850427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. North M. J., Mottram J. C., Coombs G. H. Cysteine proteinases of parasitic protozoa. Parasitol Today. 1990 Aug;6(8):270–275. doi: 10.1016/0169-4758(90)90189-b. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Otsu M., Urade R., Kito M., Omura F., Kikuchi M. A possible role of ER-60 protease in the degradation of misfolded proteins in the endoplasmic reticulum. J Biol Chem. 1995 Jun 23;270(25):14958–14961. doi: 10.1074/jbc.270.25.14958. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Saitoh E., Sabatini L. M., Eddy R. L., Shows T. B., Azen E. A., Isemura S., Sanada K. The human cystatin C gene (CST3) is a member of the cystatin gene family which is localized on chromosome 20. Biochem Biophys Res Commun. 1989 Aug 15;162(3):1324–1331. doi: 10.1016/0006-291x(89)90818-8. [DOI] [PubMed] [Google Scholar]
  44. Schnittger S., Rao V. V., Abrahamson M., Hansmann I. Cystatin C (CST3), the candidate gene for hereditary cystatin C amyloid angiopathy (HCCAA), and other members of the cystatin gene family are clustered on chromosome 20p11.2. Genomics. 1993 Apr;16(1):50–55. doi: 10.1006/geno.1993.1139. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Vaux D., Tooze J., Fuller S. Identification by anti-idiotype antibodies of an intracellular membrane protein that recognizes a mammalian endoplasmic reticulum retention signal. Nature. 1990 Jun 7;345(6275):495–502. doi: 10.1038/345495a0. [DOI] [PubMed] [Google Scholar]
  47. Vogel L. K., Spiess M., Sjöström H., Norén O. Evidence for an apical sorting signal on the ectodomain of human aminopeptidase N. J Biol Chem. 1992 Feb 5;267(4):2794–2797. [PubMed] [Google Scholar]
  48. Wulff B. S., Johansen T. E., Dalbøge H., O'Hare M. M., Schwartz T. W. Processing of two homologous precursors, pro-neuropeptide Y and pro-pancreatic polypeptide, in transfected cell lines expressing different precursor convertases. J Biol Chem. 1993 Jun 25;268(18):13327–13335. [PubMed] [Google Scholar]

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