Abstract
The characterization of proteins in their native state is essential for the understanding of pathogenic isoforms. A variant of the cysteine protease inhibitor cystatin C is the major constituent of the amyloid deposited in the cerebral vasculature of patients with the Icelandic form of hereditary cerebral hemorrhage with amyloidosis (HCHWA-I) (1,2). In order to study the nature of the biophysical changes owing to the Leu68Gln substitution in cystatin C, we have developed a purification procedure of human cystatin C in its native state. The protein is isolated from media of stably transfected tissue culture cells using physiological conditions that preclude protein denaturation. The importance of mild purification conditions is underscored by the finding that denaturation of the wild-type and variant proteins facilitates a similar folding of both molecules, diminishing their differences in structure and biophysical properties. Following native purification conditions, variant cystatin C has a distinct structure compared to the wild-type protein.
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References
Cohen, D. H., Feiner, H., Jensson, O., and Frangione, B. (1983) Amyloid fibril in hereditary cerebral hemorrhage with amyloidosis (HCHWA) is related to the gastroentero-pancreatic neuroendocrine protein, β trace. J. Exp. Med. 158, 623–628.
Ghiso, J., Jensson, O., and Frangione, B. (1986) Amyloid fibrils in hereditary cerebral hemorrhage with amyloidosis of Icelandic type is a variant of β trace basic protein (cystatin C). Proc. Natl. Acad. Sci. USA 83, 2974–2978.
Wei, L., Berman, Y., Castano, E. M., et al. (1998) Instability of the amyloidogenic cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type. J. Biol. Chem. 273, 11806–11814.
Abrahamson, M. and Grubb, A. (1994) Increased body temperature accelerates aggregation of the Leu-68 βGln mutant cystatin C, the amyloid-forming protein in hereditary cystatin C amyloid angiopathy. Proc. Natl. Acad. Sci. USA 91,1416–1420.
Calero, M., Pawlik, M., Soto, C., et al. (2001) Distinct properties of wild-type and the amyloidogenic human cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type. J. Neurochem. 77, 628–637.
Hall, A., Dalboge, H., Grubb, A. O., and Abrahamson, M. (1993) Importance of the evolutionarily conserved glycine residue in the N-terminal region of human cystatin C (Gly-11) for cysteine endopeptidase inhibition. Biochem. J. 291, 123–129.
Abrahamson, M., Mason, R. W., Hansson, H., Buttle, D. J., Grubb, A., and Ohlsson, K. (1991) Human cystatin C. Role of the N-terminal segment in the inhibition of human cysteine proteinases and its inactivation by leukocyte elastase. Biochem. J. 273, 621–626.
Grubb, A. and Lofberg, H. (1982) Human β-trace, a basic microprotein: amino acid sequence and presence in the adenohypophysis. Proc. Natl. Acad. Sci. USA 79, 3024–3027.
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Prelli, F., Pawlik, M., Frangione, B., Levy, E. (2005). Purification of Human Wild-Type or Variant Cystatin C From Conditioned Media of Transfected Cells. In: Sigurdsson, E.M. (eds) Amyloid Proteins. Methods in Molecular Biology™, vol 299. Humana Press. https://doi.org/10.1385/1-59259-874-9:221
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DOI: https://doi.org/10.1385/1-59259-874-9:221
Publisher Name: Humana Press
Print ISBN: 978-1-58829-337-4
Online ISBN: 978-1-59259-874-8
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