title: Stabilization, characterization, and selective removal of cystatin C amyloid oligomers.
abstract: The pathophysiological process in amyloid disorders usually involves the transformation of a functional monomeric protein via potentially toxic oligomers into amyloid fibrils. The structure and properties of the intermediary oligomers have been difficult to study due to their instability and dynamic equilibrium with smaller and larger species. In hereditary cystatin C amyloid angiopathy, a cystatin C variant is deposited in arterial walls and cause brain hemorrhage in young adults. In the present investigation, we use redox experiments of monomeric cystatin C, stabilized against domain swapping by an intramolecular disulfide bond, to generate stable oligomers (dimers, trimers, tetramers, decamers, and high molecular weight oligomers). These oligomers were characterized concerning size by gel filtration, polyacrylamide gel electrophoresis, and mass spectrometry, shape by electron and atomic force microscopy, and, function by assays of their capacity to inhibit proteases. The results showed the oligomers to be highly ordered, domain-swapped assemblies of cystatin C and that the oligomers could not build larger oligomers, or fibrils, without domain swapping. The stabilized oligomers were used to induce antibody formation in rabbits. After immunosorption, using immobilized monomeric cystatin C, and elution from columns with immobilized cystatin C oligomers, oligomer-specific antibodies were obtained. These could be used to selectively remove cystatin C dimers from biological fluids containing both dimers and monomers.
authors: Department of Clinical Chemistry, Lund University Hospital, S-22185 Lund, Sweden.
authors: Gustav
authors: G
authors: Östner
authors: Department of Clinical Chemistry, Lund University Hospital, S-22185 Lund, Sweden.
authors: Veronica
authors: V
authors: Lindström
authors: Dako Denmark A/S, DK-2600 Glostrup, Denmark.
authors: Per
authors: P
authors: Hjort Christensen
authors: Department of Macromolecular Physics, Adam Mickiewicz University in Poznań, 61-614 Poznań, Poland.
authors: Maciej
authors: M
authors: Kozak
authors: Department of Clinical Chemistry, Lund University Hospital, S-22185 Lund, Sweden.
authors: Magnus
authors: M
authors: Abrahamson
authors: Department of Clinical Chemistry, Lund University Hospital, S-22185 Lund, Sweden. Electronic address: anders.grubb@med.lu.se.
authors: Anders
authors: A
authors: Grubb
mesh_terms: D000328:Adult; D000682:Amyloid / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D000686:Amyloidosis / Q000276:immunology / Q000378:metabolism / Q000628:therapy; D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000276:immunology; D055316:Cystatin C / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D006801:Humans; D055503:Protein Multimerization; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D011817:Rabbits
chemical_list: D000682:Amyloid; D000906:Antibodies; C525030:CST3 protein, human; D055316:Cystatin C
keywords: Amyloid; Cystatin C; Domain Swapping; Protease Inhibitor; Protein Aggregation; Protein Conformation; Protein Domains; Protein Misfolding
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